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What happens to IP3 molecules after release from IP3 receptors?


IP3 molecules bind to IP3 receptors and open up the calcium channels on the endoplasmic reticulum. I am wondering what happens to IP3 molecules after they have been released from the IP3 receptor? Do they remain as IP3 molecules, or do they break down? Do they have any purpose after they have been released, and perhaps after been broken down?


All biomolecules are eventually broken down. This process is called turnover. Since inositol is a signalling molecule it is necessary to remove in order to terminate the signal, despite adaptive mechanism in the ER calcium channels.

IP3 is generally dephosphorylated by a family of phosphatase enzymes called inositol polyphosphate phosphatases. After dephosphorylation, the inositol can be recycled into the membrane by phosphatidyl-inositol synthase.

Reference:

The IUPHAR/BPS Guide to PHARMACOLOGY 1


1 I'm citing this website instead of a research article because apart from citing the original research articles, it also has other informative links about the enzymes and the substrate molecules. It is also from a very reliable source.


What happens to IP3 molecules after release from IP3 receptors? - Biology

C2006/F2402 '11 Outline for Lecture 19 -- (c) 2011 D. Mowshowitz -- Lecture updated 04/05/11 . (Minor corrections made see Note on refractory periods.)

Handouts: Extra copies of paper handouts are in boxes outside Dr. M's office 7th floor Mudd.

Note on refractory periods: T here is some disagreement between authorities on the timing of the refractory periods. According to Dr. Firestein, Becker, and most texts, the absolute refractory period comes right after the spike of the action potential. According to some, the absolute refractory period coincides more or less with the spike, and the relative refractory period follows after the spike. All agree about the underlying mechanism. The absolute refractory period corresponds to the time when the Na + channels are inactivated (so depolarization is not possible). The relative refractory period corresponds to the time when the Na + channels can be activated, but the voltage gated K + channels are still open (so depolarization to threshold requires a larger stimulus).

I. Nerve-Nerve Synapses, cont. -- What determines if a nerve impulse will be passed on to the next neuron? For nice overall pictures see Sadava fig. 45.13 (44.130 or Becker fig. 13-19. Some of this is review, but is included for clarity.

A. Presynaptic Side -- Transmitters
-- See Sadava Table 44.1 (8th ed.) & Becker fig. 13-18 (13-20).

1. One major type of transmitter (NT) per synapse (released from pre-synaptic side)

a. In CNS , many diff. transmitters. Usually amino acids or their derivatives. Major ones are glutamate (excitatory) & GABA (inhibitory).

b. In PNS usually norepinephrine (NE) or acetyl choline (AcCh).

2. One transmitter per Neuron. Usually only one major transmitter released by any neuron. Therefore one major transmitter released -- the same one -- at all synapses made by that neuron.

3. Release of Transmitter:

a. Location of Transmitter: Neurotransmitters are in vesicles (except for gaseous NTs)

b . Trigger for release of NT

(1). Role of AP: Action potential (AP) stimulates Ca ++ channel opening in plasma membrane, raising intracellular Ca ++

Note that Ca ++ is the same as Ca 2+ . Chemists tend to use Ca 2+ and biologists tend to use Ca ++ .

(2). Role of Ca ++: High intracellular Ca ++ promotes release of transmitter (& exocytosis in general).

c. General Case -- how is exoctyosis of secretory vesicles triggered by high Ca ++ ?

(1). Source of Calcium:Ca ++ can come from outside the cell or be released from ER.

(2). How Calcium enters cytoplasm: Channels open -- in plasma membrane or ER membrane.

(3). Which channels open: depends on signal. Signal can be NT, hormone, etc.

4. Effects of transmitter -- depends both on NT and receptors present

a. Role of NT: Most NTs always have the same effect on next neuron (or other target cell) -- either NT is always excitatory or always inhibitory.

b. Role of Receptor (on post synaptic side): Some NTs can produce be inhibitory or excitatory depending on the receptor(s) present.

5. Getting rid of transmitters -- How to turn off the signal?

a. Transmitter doesn't remain in synaptic cleft for long.

b. Different methods for getting rid of dif. transmitters

  • Endocytosis recovers membrane.

  • Transporters recover transmitters.

c. Many drugs affect release/fate of transmitters for ex.

(1). Prozacprevents serotonin reuptake -- transmitter stays around longer → more stimulation of target nerves. (Probably has other effects as well.)

(2). Malathion (insecticide) & nerve gas block AcChEsterase → continuous stimulation of muscle target cells→ spasms

d. Question to think about -- how are other signals turned off ? What if the signaling molecule is not an NT? (How do you turn off a signal from EGF? A typical endocrine or paracrine?)

B . Post Synaptic Side -- PSP's = post synaptic potentials = small change in potential due to release of transmitter

1. NT can be inhibitory (NT generates an IPSP) or excitatory (NT generates an EPSP)

a. Inhibitory -- causes hyperpolarization (or stabilizes existing negative polarization) -- IPSP -- due to opening of K + or Cl - channels. Either K + goes out or Cl - comes in.

b. Excitatory -- causes depolarization -- EPSP -- due to opening of cation channels. Allows movement of both Na + & K + . Why does this depolarize, or why is Na + in>> K + out?

c. Terminology -- a single IPSP or EPSP usually refers to the small change in potential due to release of transmitter caused by a single AP. The total PSP depends on the algebraic sum of multiple IPSP's and EPSP's as explained below.

2. Any given synapse is excitatory or inhibitory -- What determines it?

a. Receptor is crucial (See table below)

  • Faster response.

  • Ligand binding always opens channel.

  • Not always excitatory -- depends on type of channel opened.

  • Example -- the nicotinic AcCh receptor (see table below)

  • For pictures see Sadava fig. 45.16 (44.17) or Becker 13-21 (13-23). Also see many pictures in both books of neuromuscular junction, or handout 18.

  • Response is slower.

  • Effects can be more varied & more extensive.

  • Can be used to open or close ion channels.

  • Example: the muscarinic AcCh receptor & all adrenergic receptors (see table)

  • For picture see Sadava fig. 7.19 (15. 17 or 44.16 ).

(4) Agonists and antagonists

  • Agonists

    Mechanism: Bind to receptor & cause same change in receptor conformation as the normal signal molecule.

    Result: Mimic signal molecule and cause same effect as normal ligand.

  • Antagonists

    Mechanism: Bind to receptor, blocking binding of normal signal molecule, but do not cause usual conformational change. Do not activate receptor.

    Result: Block usual effect of ligand.

(c). Terminology: Some receptors named by their common ligand and most common agonist or antagonist. For example, the nicotinic acetyl choline receptor. Ac Ch = NT nicotine = agonist.

b. Overall: One major receptor/neurotransmitter pair per synapse.

3. Major Types of Receptors in the PNS -- Reference & summary

*Both alpha and beta have subtypes that differ in location, mechanism & effect same receptors used for epinephrine acting as a hormone or neurotransmitter.

** This is the receptor at the neuromuscular junction. See Becker fig. 13-21 (13-23). This is the AcCh receptor people mean when they talk about "THE" acetyl choline receptor.

Look at problem 8-16, part C. This problem as written does not refer to a standard synapse between two nerves. However, suppose you are looking at a standard synapse between two nerves that uses AcCh as a transmitter. What effects will each of the treatments listed (1-5) have on transmission at a standard synapse? (Note -- The answers in the back of the book are correct for a standard synapse except for part 4. What is the right answer to part 4 for a standard synapse?)

4. Features of Total PSP's (To compare to AP's)

a. Total PSP's are graded -- size is proportional to stimulus (as with receptor potentials, see below). Size is not all or none. (Unlike action potentials.)

b. PSP's are local -- die out if don't reach threshold. (Not regenerated like AP's.)

c. PSP's are caused by opening/closing of ligand gated channels. (What kind of channel is needed for AP's?)

To review IPSP's and EPSP's try problem 8-10.

C. Post Synaptic Side -- Summation -- See Handout 19A , bottom, or Sadava fig. fig. 45.15 (44.15) or Becker 13-22 (13-24).

1. Inputs (IPSP's & EPSP's) to cell body/dendrites are summed -- changes spread around cell body to initial segment (or die out).

2. No AP in cell body. No voltage gated channels in cell body so no AP generated there

3. Axon Hillock. Voltage gated channels begin at initial segment (also called the "trigger zone" or axon hillock) so AP starts there. See Becker fig. 13-11 (13-14) or Sadava fig. 45.15 (44.15).

4. Inputs summed over space and/or time -- need to depolarize past threshold at axon hillock to → AP. See handout 19A, bottom (cases a-d)

a. No summation achieved

b. Temporal summation: Multiple EPSP's delivered close enough together in time can add up → AP

c & d. Spatial summation:

Case c: Multiple EPSP's delivered at different spots can add up → AP

Case d: EPSP + IPSP cancel each other out

e. Why you need summation:

(1). A single EPSP is not enough to → AP

(2). IPSP's & EPSP's are summed: net effect depends on both inhibitory and excitatory input. Remember there are about 1000 synapses (inputs) on body & dendrites of average neuron. See Becker fig. 13-22 (13-24). (See circuits next time for how you use this.)

Review Problem 8-8, parts A to H, and recitation problem 8-2.

So far: Have explained how nerve signal is transmitted down an axon and on to the next nerve. How does signal get started? How does it have an effect? These Qs are addressed in the next two sections.

II. Sensors -- How a nerve signal gets started. See handout 19A, top. Sadava has a whole chapter on Sensory Systems. (Chapter # depends on which edition of the text you have.) Only a few general principles discussed here. See Sadava for examples & details.

A. The Problem: How does a small stimulus (from the internal or external environment) reach the nervous system?

1. The Question: Where does input come from, if not from another neuron? How do you get input from the environment -- from sight, sound, etc. -- and send it to the CNS?

2. The Short Ans :

Touch, hearing, etc., produce a small response = change in polarization by opening/closing channels in special cells (receptor cells or sensors).

Change in opening of channels (& therefore change in polarization) is proportional to stimulus.

If the small change in polarization is big enough (over threshold) -- it opens voltage gated channels which generates a big response -- an AP. (The 'big bang' or the 'toilet flush,' so to speak.) See left-most case on the 'big bang' handout.

1. Special (Sensory) cells contain receptor proteins for stimuli (pressure, light, heat, chemicals, etc.).

2. How do protein receptors detect stimuli?

a. Stimuli → Change in conformation of receptor → open or close channels in membrane → change in polarization of membrane.

b. How are channels opened or closed? See Sadava fig. 46.1 (45.1).

(1) Directly -- receptor is part of a channel = an ionotropic receptor. Examples: receptors for mechanical stimuli (touch, hearing, balance), & temperature (heat/cold). Receptor changes shape and channel opens.

(2) Indirectly -- receptor is not part of a channel = a metabotropic receptor. Change in conformation of receptor activates a G protein. G protein or 2nd messenger opens/closes channel. Examples: receptors for chemicals (taste, smell, etc.), electromagnetic radiation (vision).

C. Receptor Potentials

1. Response to stimulus is graded. Stimulus → local graded response. The more stimulus, the more channels open (or close), and the bigger the graded potential (bigger depolarization or bigger hyperpolarization) in the sensory/receptor cell.

2. Terminology -- graded response in receptor/sensory cell is called a generator potential or receptor potential.

D. Receptor or Sensory Cells. Special cells with receptor proteins for detecting stimuli

1. Modified neuron -- sensory cell is a modified neuron capable of generating AP itself. (See Sadava fig. 46.2 (45.2) & Handout 19A.) Examples: sensory cells for detecting stretch or smell.

2. Cell that cannot generate an AP itself .

a. How get an AP? Sensory cell releases transmitter and triggers AP in next cell (a neuron). See Sadava fig. 46.5 (45.5) & handout. Examples: sensory cells for detecting light, taste, sound and balance.

b. Type of cell. This type of sensory cell can be a modified neuron or epithelial cell.

Question to ask yourself: What type of channels does a cell need in order to generate an AP? If you are curious about the details of how the AP is generated, see notes of '08.

E. All stimuli (whatever the modality) give same message to CNS (= AP's). If AP is all or nothing, how do you know which stimulus it was? And how much?

1. Number, frequency of AP's indicate length (duration) and strength (intensity) of stimulus.

2. Wiring (what part of brain is stimulated) = labeled lines = indicates location of stimulus and type (modality) of stimulus -- taste, stretch, etc. If you get a punch in the eye, you set off light receptors. For a less violent example, take a very sharp pencil and tap your upper lip. What sensors did you trip off? (For contrast, tap your arm.)


III. How does a nerve signal produce an effect?

A. General solution - - nerve signal triggers an effect in a target tissue (an effector) -- muscle contracts, gland secretes, etc. Some examples of how this works next time.

B. The Problem -- One signal molecule (hormone, transmitter, etc.) can produce different effects on different target tissues such as different smooth muscles or skeletal muscle vs. smooth muscle.

C. Two Basic Methods -- See Handout 19B.

1. Using the Same Receptor & same 2nd messenger, but different Target Proteins

a. An example:

(1). In skeletal muscle: epinephrine causes glycogen breakdown.

(2). In smooth muscle of lung: epinephrine causes muscle relaxation.

b. Why does this make sense?

(1). Epinephrine (also called adrenaline) is produced in response to stress.

(2). In response to stress, need to "mobilize" glucose-- release it from storage so it can be broken down to provide energy. Therefore need to increase glycogen breakdown (and decrease glycogen synthesis) in muscle (& liver).

(3). In response to stress, need to breathe more deeply.Therefore need smooth muscle around tubes that carry air (bronchioles) to relax.

c. How is this possible? Same receptors, same 2nd messenger (cAMP) are used.

d. The solution: Different target proteins. PKA is activated in both skeletal and smooth muscle. However the target proteins available to be phosphorylated are different in the two tissues. Therefore different proteins are phosphorylated and activated (or inactivated) in the two different tissue types.

(1). In skeletal muscle -- PKA phosphorylates (& activates) the enzyme phosphorylase kinase, which in turn phosphorylates (& activates) the enzyme that breaks down glycogen to release glucose. See texts for more details. (Becker, fig. 14-25 or Sadava fig. 7.20 (15.18)

(2).In smooth muscle surrounding the bronchioles -- PKA phosphorylates a protein (MLCK) needed for contraction, inactivating it. Therefore contraction cannot occur.

2. Using different receptors & second messengers in different cell types

(See Becker fig. 14-24 (14-23). An example -- effects of epinephrine (adrenaline) on smooth muscle. Some smooth muscles relax, and some contract in response to epinephrine. In this case, different receptors & 2nd messengers are involved. How does this work? See below.

Try problem 6-11.

D. Example of Using Different Second Messengers (& Different Receptors). See handout 19B.

1. The phenomenon:

a. Epinephrine (secreted in response to stress) has different effects on different smooth muscles:

(1). On some smooth muscles, epi → contraction

(2). On other smooth muscles, epi → relaxation (as above)

b. How does this make sense?

(1). In peripheral circulation -- smooth muscles around blood vessels (arterioles) contract, diverting blood from peripheral circulation to essential internal organs

(2). In lungs -- smooth muscles around tubes carrying air (bronchioles) relax, so lungs can expand more and you can breathe more deeply.

a. Ca ++ stimulates muscle contraction.

b. To give contraction: Epinephrine binds to receptors on some smooth muscles (ex: around arterioles) → Ca ++ released from ER → intracellular Ca ++ up → stimulates contraction.

c. To give relaxation: Epinephrine binds to receptors on some smooth muscles (ex: around bronchioles) → phosphorylates protein needed for response to Ca ++ , preventing response.

a. Two basic types of epinephrine receptors -- called alpha and beta adrenergic receptors (adrenergic = for adrenaline). The two types are distinguished (primarily) by their relative affinities for epinephrine (adrenaline) and norepinephrine (noradrenaline).

b. Some types of smooth muscle have mostly one type of receptors some the other. (See table below and table above for details of receptor properties.)

c. Two types of receptors activate different G proteins and generate different second messengers . (Details next time.)

(1). Beta receptors → G protein of one type (Gs) → activates enzyme (Adenyl cyclase) → second messenger ( cAMP) → PKA

(2). Alpha 1 receptors → different G protein (Gp) → activate different enzyme (phospholipase C or PLC) → different second messenger (IP3) → binds to receptors on ER membrane → opens Ca ++ channels in ER → Ca ++ release from ER → contraction

4. How does this all work to allow appropriate response to stress (epinephrine)?

a. Beta type receptors. Beta receptors are found in lung tissue in smooth muscle surrounding bronchioles.
Stress (pop quiz, lion in street, etc.) → epinephrine → muscles relax → bronchioles dilate → deeper breathing → more oxygen → energy to cope with stress.

b. Alpha type receptors. Alpha receptors are found in smooth muscle surrounding blood vessels of peripheral circulation.
Stress → epinephrine → muscles contract → constrict peripheral circulation → direct blood to essential organs for responding to stress (heart, lungs, skeletal muscle).

To review effects of different receptors, try problems 6-21 & 6-22. Note that you do not need to know the details of how IP3 is generated. If you are curious, see Becker fig. 14-10 or class handout.

5. Medical Uses of all this.

Epinephrine can be used during an asthmatic attack to relax bronchi and ease breathing. Overuse of this type of broncho-dilator eases breathing temporarily but masks underlying problem (inflammation of lung tissue) and can have additional serious long term effects (from overstimulation of heart which also has beta receptors). Heart and lungs have slightly different types of beta receptors, so drugs (agonists) have been developed that stimulate one and not the other (unlike epinephrine). Many drugs are either agonists or antagonists of signaling molecules such as hormones, transmitters, etc.

Try Problem 6-8 & 6-9 if not yet. (To review agonists & antagonists.)

6. Summary of epinephrine effects on smooth muscle (in lung vs peripheral circulation). See also handout 19B.

Note: There are more than two types of epinephrine receptors on smooth muscle cells, so epinephrine may affect smooth muscle in other tissues in other ways. (There are subtypes of alpha and subtypes of beta.)

* Details of how PLC generates IP3 are in texts and on handout for next time.
** Not all alpha receptors use IP3.

Next time: Details of IP3 pathway How are circuits organized, and how do nerves and muscles work to give contractions?


Flex Your Muscles

The cells in your skeletal muscle contain large amounts of endoplasmic reticulum, which in this type of muscle is called sarcoplasmic reticulum. The sarcoplasmic reticulum stores large amounts of calcium because calcium, when released, aids in muscle contraction. Skeletal muscles release calcium and contract when they receive signals from neurons, or nerve cells, that control muscle movement. Your brain sends electrical signals through neurons to your skeletal muscle. When the electric signal reaches the surface of a muscle fiber, which is many muscle cells joined together, it travels along the surface and then through a system of tubes that connects to the sarcoplasmic reticulum. The electrical signal opens a calcium channel on the sarcoplasmic reticulum called the ryanodine receptor, which releases calcium.


Contents

All family members are capable of catalyzing the hydrolysis of PIP2, a phosphatidylinositol at the inner leaflet of the plasma membrane into the two second messengers, inositol trisphosphate (IP3) and diacylglycerol (DAG).

PLCs catalyze the reaction in two sequential steps. The first reaction is a phosphotransferase step that involves an intramolecular attack between the hydroxyl group at the 2' position on the inositol ring and the adjacent phosphate group resulting in a cyclic IP3 intermediate. At this point, DAG is generated. However, in the second phosphodiesterase step, the cyclic intermediate is held within the active site long enough to be attacked by a molecule of water, resulting in a final acyclic IP3 product. It should be mentioned that bacterial forms of the enzyme, which contain only the catalytic lipase domain, produce cyclic intermediates exclusively, whereas the mammalian isoforms generate predominantly the acyclic product. However, it is possible to alter experimental conditions (e.g., temperature, pH) in vitro such that some mammalian isoforms will alter the degree to which they produce mixtures of cyclic/acyclic products along with DAG. [ citation needed ] This catalytic process is tightly regulated by reversible phosphorylation of different phosphoinositides and their affinity for different regulatory proteins. [2] [3] [4]

PLCs perform their catalytic function at the plasma membrane where their substrate PIP2 is present. This membrane docking is mediated mostly by lipid-binding domains (e.g. PH domain and C2 domain) that display affinity for different phospholipid components of the plasma membrane. It is important to note that research has also discovered that, in addition to the plasma membrane, PLCs also exist within other sub-cellular regions such as the cytoplasm and nucleus of the cell. At present, it is unclear exactly what the definitive roles for these enzymes in these cellular compartments are, particularly the nucleus.

Phospholipase C performs a catalytic mechanism, depleting PIP2 and generating inositol trisphosphate (IP3) and diacylglycerol (DAG).

Depletion of PIP2 inactivates numerous effector molecules in the plasma membrane, most notably PIP2 dependent channels and transporters responsible for setting the cell's membrane potential. [5]

The hydrolytic products also go on to modulate the activity of downstream proteins important for cellular signaling. IP3 is soluble, and diffuses through the cytoplasm and interacts with IP3 receptors on the endoplasmic reticulum, causing the release of calcium and raising the level of intracellular calcium.

DAG remains within the inner leaflet of the plasma membrane due to its hydrophobic character, where it recruits protein kinase C (PKC), which becomes activated in conjunction with binding calcium ions. This results in a host of cellular responses through stimulation of calcium-sensitive proteins such as Calmodulin.

In terms of domain organization, all family members possess homologous X and Y catalytic domains in the form of a distorted Triose Phosphate Isomerase (TIM) barrel with a highly disordered, charged, and flexible intervening linker region. Likewise, all isoforms possess four EF hand domains, and a single C2 domain that flank the X and Y catalytic core. An N-terminal PH domain is present in every family except for the sperm-specific ζ isoform.

SH2 (phosphotyrosine binding) and SH3 (proline-rich-binding) domains are found only in the γ form (specifically within the linker region), and only the ε form contains both guanine nucleotide exchange factor (GEF) and RA (Ras Associating) domains. The β subfamily is distinguished from the others by the presence of a long C-terminal extension immediately downstream of the C2 domain, which is required for activation by Gαq subunits, and which plays a role in plasma membrane binding and nuclear localization.

The Phospholipase C family consists of 13 isozymes split between six subfamilies, PLC-δ (1,3 & 4), -β(1-4), -γ(1,2), -ε, -ζ, and the recently discovered -η(1,2) isoform. Depending on the specific subfamily in question, activation can be highly variable. Activation by either Gαq or Gβγ G-protein subunits (making it part of a G protein-coupled receptor signal transduction pathway) or by transmembrane receptors with intrinsic or associated tyrosine kinase activity has been reported. In addition, members of the Ras superfamily of small GTPases (namely the Ras and Rho subfamilies) have also been implicated. It should also be mentioned that all forms of Phospholipase C require calcium for activation, many of them possessing multiple calcium contact sites in the catalytic region. The only isoform that is known to be inactive at basal intracellular calcium levels is the δ subfamily of enzymes suggesting that they function as calcium amplifiers that become activated downstream of other PLC family members.

PLC-β Edit

PLC-β(1-4) (120-155kDa) are activated by Gαq subunits through their C2 domain and long C-terminal extension. Gβγ subunits are known to activate the β2 and β3 isozymes only however, this occurs through the PH domain and/or through interactions with the catalytic domain. The exact mechanism still requires further investigation. The PH domain of β2 and β3 plays a dual role, much like PLC-δ1, by binding to the plasma membrane, as well as being a site of interaction for the catalytic activator. However, PLC-β binds to the lipid surface independent of PIP2 with all isozymes preferring phosphoinositol-3-phosphate or neutral membranes.

Members of the Rho GTPase family (e.g., Rac1, Rac2, Rac3, and cdc42) have been implicated in their activation by binding to an alternate site on the N-terminal PH domain followed by subsequent recruitment to the plasma membrane. A crystal structure of Rac1 bound to the PH domain of PLCβ2 has been solved. Like PLC-δ1, many PLC-β isoforms (in particular, PLC-β1) have been found to take up residence in the nuclear compartment. A basic amino acid region within the enzyme's long C-terminal tail appears to function as a Nuclear Localization Signal for import into the nucleus. PLC-β1 seems to play unspecified roles in cellular proliferation and differentiation.

PLC-γ Edit

PLC-γ (120-155kDa) is activated by receptor and non-receptor tyrosine kinases due to the presence of two SH2 and a single SH3 domain situated between a split PH domain within the linker region. Although this particular isoform does not contain classic nuclear export or localization sequences, it has been found within the nucleus of certain cell lines. [ citation needed ] There are two main isoforms of PLCγ expressed in human specimens, PLC-γ1 and PLC-γ2. [6]

PLC-Y2 Edit

PLC-γ2 plays a major role in BCR signal transduction. Absence of this enzyme in knockout specimens severely inhibits the development of B cells because the same signaling pathways necessary for antigen mediated B cell activation are necessary for B cell development from CLPs. [6]

In B cell signaling, PI 3-kinase is recruited to the BCR early in the signal transduction pathway. PI-3K phosphorylates PIP2 (Phosphatidylinositol 4,5-bisphosphate) into PIP3 (Phosphatidylinositol 3,4,5-trisphosphate). The increase in concentration of PIP3 recruits PLC-γ2 to the BCR complex which binds to BLNK on the BCR scaffold and membrane PIP3. PLC-γ2 is then phosphorylated by Syk on one site and Btk on two sites. PLC-γ2 then competes with PI-3K for PIP2 which it hydrolyzes into IP3 (inositol 1,4,5-trisphosphate), which ultimately raises intercellular calcium, and diacylglycerol (DAG), which activates portions of the PKC family. Because PLC-γ2 competes for PIP2 with the original signaling molecule PI3K, it serves as a negative feedback mechanism. [6]

PLC-δ Edit

The PLC-δ subfamily consists of three family members, δ1, 2, and 3. PLC-δ1 (85kDa) is the most well understood of the three. The enzyme is activated by high calcium levels generated by other PLC family members, and therefore functions as a calcium amplifier within the cell. Binding of its substrate PIP2 to the N-terminal PH domain is highly specific and functions to promote activation of the catalytic core. In addition, this specificity helps tether the enzyme tightly to the plasma membrane in order to access substrate through ionic interactions between the phosphate groups of PIP2 and charged residues in the PH domain. While the catalytic core does possess a weak affinity for PIP2, the C2 domain has been shown to mediate calcium-dependent phospholipid binding as well. In this model, the PH and C2 domains operate in concert as a "tether and fix" apparatus necessary for processive catalysis by the enzyme.

PLC-δ1 also possesses a classical leucine-rich nuclear export signal (NES) in its EF hand motif, as well as a Nuclear localization signal within its linker region. These two elements combined allow PLC-δ1 to actively translocate into and out of the nucleus. However, its function in the nucleus remains unclear.

The widely expressed PLC-δ1 isoform is the best-characterized phospholipase family member, as it was the first to have high-resolution X-ray crystal structures available for analysis. In terms of domain architecture, all of the enzymes are built upon a common PLC-δ backbone, wherein each family displays similarities, as well as obvious distinctions, that contribute to unique regulatory properties within the cell. Because it is the only family found expressed in lower eukaryotic organisms such as yeast and slime molds, it is considered the prototypical PLC isoform. The other family members more than likely evolved from PLC-δ as their domain architecture and mechanism of activation were expanded. Although a full crystal structure has not been obtained, high-resolution X-ray crystallography has yielded the molecular structure of the N-terminal PH domain complexed with its product IP3, as well as the remainder of the enzyme with the PH domain ablated. These structures have provided researchers with the necessary information to begin speculating about other family members such as PLCβ2.


What happens to IP3 molecules after release from IP3 receptors? - Biology

Protein Info:

Ensembl Gene:

UniGene (transcript info):

SNP (polymorphism info):

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IP3 Receptors, Mitochondria, and Ca 2+ Signaling: Implications for Aging

The tight interplay between endoplasmic-reticulum-(ER-) and mitochondria-mediated Ca 2+ signaling is a key determinant of cellular health and cellular fate through the control of apoptosis and autophagy. Proteins that prevent or promote apoptosis and autophagy can affect intracellular Ca 2+ dynamics and homeostasis through binding and modulation of the intracellular Ca 2+ -release and Ca 2+ -uptake mechanisms. During aging, oxidative stress becomes an additional factor that affects ER and mitochondrial function and thus their role in Ca 2+ signaling. Importantly, mitochondrial dysfunction and sustained mitochondrial damage are likely to underlie part of the aging process. In this paper, we will discuss the different mechanisms that control intracellular Ca 2+ signaling with respect to apoptosis and autophagy and review how these processes are affected during aging through accumulation of reactive oxygen species.

1. Intracellular Ca 2+ Signaling

Intracellular Ca 2+ signaling is important in the regulation of multiple cellular processes, including development, proliferation, secretion, gene activation, and cell death. The formation of these Ca 2+ signals is dependent on many cellular Ca 2+ -binding and Ca 2+ -transporting proteins, present in the various cell compartments of which the endoplasmic reticulum (ER) forms the main intracellular Ca 2+ store [1]. The resting cytosolic [Ca 2+ ] remains very low (

100 nM), through active extrusion of Ca 2+ by pumps in the plasma membrane or in intracellular organelles, like the sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA) pump in the ER. Due to SERCA activity and intraluminal Ca 2+ -binding proteins, the ER can accumulate Ca 2+ in more than thousandfold excess compared to the cytosol [1, 2]. In the ER lumen, Ca 2+ functions as an important cofactor for ER chaperones, thereby aiding in the proper folding of newly synthesized proteins [3]. Reciprocally, the Ca 2+ -binding chaperones affect the Ca 2+ capacity of the ER by buffering Ca 2+ [2]. In addition, two tetrameric ER Ca 2+ -release channels exist that, upon stimulation, release Ca 2+ into the cytosol, thereby provoking Ca 2+ signaling: the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) and the ryanodine receptor (RyR). They are similar in function and structure but differ in regulation, conductance, and expression profile [4, 5]. The rise in cytosolic [Ca 2+ ] following its release from the ER results in various Ca 2+ -dependent intracellular events. The exact cellular outcome depends on the spatiotemporal characteristics of the generated Ca 2+ signal [6]. Since close contact sites between the ER and the mitochondria, involving direct molecular links with the IP3R, exist (Figure 1), it is clear that ER-originating Ca 2+ signals critically affect the mitochondrial function.


In a healthy cell, ER Ca 2+ -handling components tightly regulate mitochondrial function and bioenergetics, representing the different key players involved in intracellular Ca 2+ signalling with particular emphasis on the ER-mitochondria connections. The ER-Ca 2+ content is regulated by channels and pumps (IP3Rs, RyRs, SERCAs) and by Ca 2+ -binding chaperones (CaBCs). IP3 stimulates ER Ca 2+ release and consequently the transfer of Ca 2+ (red dots) from ER to mitochondria. Mitochondrial Ca 2+ , transported via VDAC, is directly or indirectly involved in cellular energy metabolism and in the secondary production of reactive oxygen species (ROS). It is clear that IP3R-mediated Ca 2+ release ought to be tightly regulated to sustain mitochondrial activity and function. As a consequence, Ca 2+ -flux properties of IP3Rs are tightly and dynamically regulated by accessory proteins involved in cell death and survival, like Bcl-2, Bcl-Xl, PKB/Akt, Sigma-1 receptor (Sig-1R)/Ankyrin B (AnkB), and the recently identified PML. It is important to note that different regulatory mechanisms occur at the IP3R, which may help cell survival (like Bcl-2, Bcl-Xl, PKB/Akt) or help to promote cell death (like PML). The latter is essential to prevent the survival of altered, damaged, or oncogenic cells. Thus, a tight balance between both outcomes is a requisite for cellular health and homeostasis, and a dynamic switch from prosurvival to prodeath is likely essential. In this paradigm, the production of ROS might contribute to the survival of cells by efficient detection of damaged/altered mitochondria and their removal by autophagy, while preventing excessive apoptosis. In addition, controlled apoptosis is likely to be important to eliminate cells, in which the removal of altered mitocondria by autophagy is not sufficient, thereby avoiding tumor genesis. In this process, the recently identified tumor suppressor PML may play a crucial role as it promotes IP3R-mediated Ca 2+ transfer from the ER into the mitochondria by dephosphorylating and suppressing PKB/Akt activity through PP2A. While PKB/Akt is known to suppress IP3R-channel activity by phosphorylation of the IP3R, the recruitment of PP2A via PML at the interorganellar ER/mitochondrial complex dephosphorylates and inactivates PKB/Akt. This suppresses PKB-dependent phosphorylation of IP3R and thus promotes Ca 2+ release through this channel and Ca 2+ transfer into the mitochondria. At the mitochondrial level, the tumor suppressor Fhit has been shown to increase the affinity for the mitochondrial Ca 2+ uniporter (MCU), thereby enhancing the uptake of mitochondrial Ca 2+ at low and physiologically relevant levels of agonist-induced Ca 2+ signals. Green arrows: stimulation red lines: inhibition black arrows: Ca 2+ flux.

During aging, ER Ca 2+ homeostasis alters and becomes dysregulated [7]. Most observations support a decline in ER [Ca 2+ ] and in ER Ca 2+ release (due to lower activity of SERCA, IP3R, and RyR), but contradictory findings have been published, possibly related to the cell type under investigation (Figure 2). In addition, ER Ca 2+ release and subsequent Ca 2+ uptake by mitochondria regulate reactive oxygen species (ROS) production, autophagy, and cell death, processes implicated in aging.


Altered Ca 2+ signaling during aging and in age-related diseases. The Ca 2+ dyshomeostasis during age is dependent on the cell type and the context. Most aged cells display decreased ER Ca 2+ content and release, due to declined IP3R or RyR levels, reduced SERCA activity, and decreased Ca 2+ buffering by intraluminal Ca 2+ -binding chaperones. However, in neurons and rat hearts, an enhanced Ca 2+ signaling is found, caused by increasing IP3R or RyR activity. Age-related diseases (neurodegeneration, cardiac hypertrophy, and chronic heart failure) are also characterized by enhanced Ca 2+ signaling. However, this property may be disease dependent, since a mouse model for Huntington’s disease displayed attenuated IP3R1 activity due to impaired binding of Grp78 to IP3R1. Hence, caution should be taken with general claims.

In a previous review [8], we have focused on mechanisms regulating the Ca 2+ content in the ER and its relevance for the development of physiological versus pathophysiological Ca 2+ signalling. In the present review, we will focus on the subsequent step which is the mechanisms responsible for controlling Ca 2+ transfer from the ER to the mitochondria. The Ca 2+ level in the mitochondrial matrix plays an important role in the progression of apoptosis and autophagy [9, 10]. Here, we will especially analyze how the Ca 2+ transfer to the mitochondria as well as apoptosis and autophagy are affected by the aging process in general and by reactive oxygen species in particular.

2. Mitochondrial Ca 2+ Handling

In contrast with the role of the ER, the role of the mitochondria in physiological Ca 2+ handling was underestimated or even ignored for a long time, but due to the seminal work of Rizzuto and his colleagues [11], this role is now generally accepted.

The electrochemical gradient (

mV) between the inside and outside of energized mitochondria forms the driving force for the Ca 2+ uptake in the mitochondrial matrix, which implies the transfer of Ca 2+ ions over both the outer mitochondrial membrane (OMM) and the inner mitochondrial membrane (IMM).

The Ca 2+ ions taken up into the mitochondrial matrix stimulate the mitochondrial ATP production by regulating the activities of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, and pyruvate dehydrogenase, three dehydrogenases of the Krebs cycle [12, 13]. Also other mitochondrial processes as fatty acid oxidation, amino acid catabolism, aspartate and glutamate carriers, the adenine-nucleotide translocase, Mn-superoxide dismutase, and F1-ATPase activity, are regulated by mitochondrial Ca 2+ [12, 14, 15].

The ATP produced by the mitochondria is subsequently transferred to the cytoplasm it will so especially regulate the activity of ATP-sensitive proteins localized in the close vicinity of the mitochondria. Two major proteins involved in Ca 2+ transport, the SERCA, responsible for loading the ER, and the IP3Rs, responsible for Ca 2+ release from the ER, are stimulated by ATP. The bidirectional relation between Ca 2+ release and ATP production allows for a positive feedback regulation between ER and mitochondria during increased energetic demand [16].

The uptake of Ca 2+ in the mitochondria will also affect Ca 2+ signaling. The local Ca 2+ concentration near the mitochondria will depend on both the amount of Ca 2+ released by the IP3R and that taken up by the mitochondria. This will in turn depend on the efficiency of the coupling between both. Since both the SERCA pumps and the IP3Rs are also regulated by Ca 2+ , the local Ca 2+ concentration in the vicinity of the mitochondria will determine the refilling of the ER and eventually the spatiotemporal characteristics of the subsequent Ca 2+ signals. The way in which the Ca 2+ signals are affected depends on the exact subcellular localization of the mitochondria, the production of ROS, the local Ca 2+ concentration, the IP3R isoform expressed, and may as well involve stimulation as inhibition of the signals [16–19]. Furthermore, the connection between mitochondria and the ER can be highly dynamic as the local Ca 2+ concentration can also affect mitochondrial motility and ER-mitochondria associations in various ways [20].

3. Transport Proteins Involved in the Transfer of Ca 2+ between ER and Mitochondria

3.1. IP3Rs

The first key player is the IP3R, the main Ca 2+ -release channel in the ER of most cell types. The IP3R consists of 4 subunits of about 310 kDa each (i.e., about 2700 a.a.). In mammals, three different IP3R isoforms are expressed (IP3R1, IP3R2, and IP3R3) while diversity is increased by splicing and the formation of both homo- and heteromeric channels [4, 21, 22]. All IP3R isoforms are activated by IP3, though with varying affinity [23]. Low Ca 2+ concentrations stimulate but high Ca 2+ concentrations inhibit the IP3Rs [24–27]. Further modulation of the IP3Rs is performed by ATP, phosphorylation, and protein-protein interactions [4, 28–30].

For efficient Ca 2+ transfer between ER and mitochondria, it is important that IP3Rs are localized very close to the mitochondrial Ca 2+ -uptake sites. As different IP3R isoforms exist, an important point is whether interaction with the mitochondria is isoform specific [31]. In CHO cells, IP3R3 is the least expressed isoform, but it demonstrated the highest degree of colocalization with the mitochondria and consequently its silencing had the most profound effects on mitochondrial Ca 2+ signals [32]. However, this does not represent a general rule as, for example, in astrocytes IP3R2 was found to preferentially colocalize within the mitochondria [33]. These differences in intracellular localization of the IP3R isoforms may be due to differences in relative expression levels of the various IP3R isoforms and in subcellular localization among different cell types [34]. Moreover, the physiological setting [35] and the differentiation status [36] determine the subcellular localization of the various IP3R isoforms in a given cell type.

3.2. Voltage-Dependent Anion Channels: The Main Ca 2+ -Transport System across the OMM

The Ca 2+ fluxes through the OMM are mainly determined by voltage-dependent anion channels (VDAC). Of the 3 existing VDAC isoforms, VDAC1 is the most abundant in most cell types [37]. It was demonstrated that the transient overexpression of VDAC in various cell types led to an increased Ca 2+ concentration in the mitochondria, leading to a higher susceptibility for ceramide-induced cell death [38].

VDAC, however, allows also the transport of other ions and metabolites, including ATP. It has therefore multiple functions in the cell and is a central player in the crosstalk between the cytoplasm and mitochondria. In this manner, VDAC is also implicated in the induction of apoptosis by various stimuli [15].

The permeabilization of the OMM is a crucial step in apoptosis, but how this is exactly performed is not yet clear. Proteins belonging to the B-cell CLL/lymphoma-2 (Bcl-2)-protein family appear anyway to be necessary [39, 40]. Several Bcl-2-family members can affect the permeability of the OMM, for example, by binding to VDAC and regulating its properties or by forming multimeric channel complexes. Independently of the mechanism by which the increase in permeability of the OMM is achieved, it allows the release of the apoptogenic factors present in the intermembrane space to the cytoplasm and the progression of apoptosis [15, 40–42].

3.3. Ca 2+ -Transport Systems across the IMM

In contrast to the Ca 2+ -transport system across the OMM, that of the IMM is not yet well characterized. For a long time, the main IMM Ca 2+ -transport system was named the mitochondrial Ca 2+ uniporter. Additionally, a so-called rapid mode of mitochondrial Ca 2+ uptake was described, but the nature of neither was known [43].

Three different highly Ca 2+ -selective channels that may contribute to this process were meanwhile characterized, that is, MiCa [44], mCa1, and mCa2 [45]. Two of these channels, MiCa and mCa1, have properties compatible with the former uniporter and may represent species- and/or cell-type-dependent variability [43]. At the molecular level, the mitochondrial Ca 2+ -uptake channels are not yet identified, but evidence for a role of a number of proteins has been presented [46, 47]. Recently, a Ca 2+ -binding protein, named MICU1, which appears essential for mitochondrial Ca 2+ uptake, was described [48]. It is, however, not known whether it actually forms (part of) a Ca 2+ channel or functions as Ca 2+ buffer or Ca 2+ sensor. Interestingly, the tumor suppressor protein Fhit (fragile histidine triad) seems to promote mitochondrial Ca 2+ uptake by increasing the affinity of the mitochondrial Ca 2+ uniporter at the ER/mitochondrial microdomain [49].

Finally, the permeabilization transition pore (PTP) is another channel of still unknown nature [50]. It is voltage and Ca 2+ dependent and is sensitive to cyclosporine A. It is not selective for Ca 2+ as the open conformation of the PTP has a high conductance for all ions, including Ca 2+ , and for molecules up to 1500 Da [51]. Its long-time activation leads to the demise of the cell, either by apoptosis or else by necrosis, depending on whether PTP opening occurs in only a small part of the mitochondria or in all of them, respectively [51, 52].

In addition, Ca 2+ /Na + and Ca 2+ /H + exchangers are also present in the IMM. Their main function is probably to export Ca 2+ from the matrix, but they may also contribute to Ca 2+ uptake under certain conditions [43].

4. Structural and Regulatory Proteins Involved in the Control of Ca 2+ Transfer between ER and Mitochondria

Mitochondria-associated ER membranes (MAMs) were originally described as sites for lipid synthesis and lipid transfer between ER and mitochondria [53]. These MAMs are, however, also ideally suited for Ca 2+ exchange [14]. Several proteins may participate in the stabilization of those MAMs and, through this stabilization, affect Ca 2+ transfer between ER and mitochondria. Other proteins may be directly involved in regulating the Ca 2+ -transport proteins described above.

4.1. Glucose-Regulated Protein 75

Glucose-regulated protein 75 (Grp75) belongs to the Hsp70 family of chaperones but is not inducible by heat shock [54, 55]. Importantly, it can couple the IP3R to VDAC1 and allows for a better transfer of the Ca 2+ ions from the ER to the mitochondrial matrix [56]. The increased Ca 2+ signals in the mitochondria were not due to an increased ER-mitochondria contact area. These results indicate that Grp75 is probably not the main determinant for the ER-mitochondrial linkage but regulates the Ca 2+ flux between ER and mitochondria by controlling the interaction between the IP3R and VDAC1.

4.2. Sigma-1 Receptor

The ER chaperone proteins known as sigma receptors are targets for certain neurosteroids. Based on their biochemical and pharmacological properties, two subclasses, sigma-1 and sigma-2 receptors, are distinguished but only the sigma-1 receptor was cloned and properly characterized [57, 58]. The sigma-1 receptor is involved in many physiological functions as well as in several pathological conditions [58].

Sigma-1 receptors are especially enriched at the MAMs [59]. A specific interaction between the Ca 2+ -binding chaperone BiP and the sigma-1 receptor was described [59]. This interaction depends on the ER Ca 2+ concentration: a decrease in ER Ca 2+ concentration leads to their dissociation, whereby both proteins become active chaperones.

The sigma-1 receptor regulates several ion channels, including the IP3Rs [58]. Agonists of sigma-1 receptors could so potentiate agonist-induced Ca 2+ release in NG108 cells [60]. Hereby, an interaction between the sigma-1 receptor, cytoskeletal ankyrin B, and IP3R3 was demonstrated [61]. In CHO cells, the sigma-1 receptor also interacted with IP3R3, but here ankyrin was not observed in the complex. Finally, a specific role was found for the sigma-1 receptor stabilizing the IP3R3 present at the MAMs, and so regulating Ca 2+ transfer between ER and mitochondria [59].

4.3. Mitofusins

Mitofusin 1 and 2 are two dynamin-related GTPases acting on mitochondria. Mitofusin 2 is enriched at MAMs. The absence of mitofusin 2 not only affected ER and mitochondrial morphology but also reduced the number of contact points between ER and mitochondria by about 40% [62]. Mitofusin 2 on the ER appeared necessary for connecting the two organelles by directly interacting with either mitofusin 1 or mitofusin 2 on the OMM. Moreover, the diminished interaction observed in the absence of mitofusin 2 affected Ca 2+ transfer between the ER and the mitochondria. A too strong ER-mitochondria interaction may also be detrimental as overexpression of mitofusin 2 led to apoptosis [63].

4.4. Bcl-2-Family Members

Bcl-2 is the prototype of a large family containing both anti- and proapoptotic proteins. The antiapoptotic members of this family, including Bcl-2 itself, are characterized by the presence of 4 Bcl-2-homology (BH) domains (BH1 to 4). The proapoptotic members either have 3 BH domains (BH1, BH2, and BH3) as, for example, Bax and Bak, or only a single BH3 domain, as for example, Bim, Bid, and Bad (the so-called BH3-only proteins) [39].

The BH1, BH2, and BH3 domains of the antiapoptotic proteins, as Bcl-2 and Bcl-Xl, form together a hydrophobic cleft that can bind the amphipathic α-helical BH3 domain of proapoptotic proteins. In this manner, the antiapoptotic Bcl-2 family members antagonize apoptosis at the level of the mitochondria by binding and neutralizing proapoptotic Bax and Bak [39, 64]. In addition to this mitochondrial function, antiapoptotic Bcl-2 family members also act on the ER Ca 2+ homeostasis [65, 66]. The exact mechanism is, however, not yet clarified, and effects on several Ca 2+ -binding or Ca 2+ -transporting proteins were described, including on the IP3R [67–69].

Although there is an agreement that the antiapoptotic proteins as Bcl-2 bind to the IP3R, there is among the various studies a discrepancy with respect to the exact binding site and to the functional consequences. The results obtained are summarized here below.

Firstly, cells lacking Bax/Bak displayed a decreased ER Ca 2+ -store content, which was associated with an increased (i) amount of Bcl-2 bound to the IP3R, (ii) protein-kinase-A-(PKA-) dependent phosphorylation of the IP3R, and (iii) Ca 2+ leak rate from the ER. Hence, increasing the ratio of antiapoptotic over proapoptotic Bcl-2-family members seemed to decrease the ER Ca 2+ -store content by promoting the Ca 2+ leak via hyperphosphorylation and hyperactivation of the IP3R [70].

Secondly, IP3Rs were described to be activated by Bcl-Xl. Bcl-Xl bound to all three IP3R isoforms, thereby sensitizing them to low IP3 concentrations [71, 72]. The interaction site was demonstrated to be the C-terminal part of IP3R1 [71]. The binding of Bcl-Xl to the IP3Rs is important for the protection of cells against apoptotic stimuli, since the overexpression of Bcl-Xl in IP3R triple-knockout (TKO) cells did not provoke resistance against apoptotic stimuli. By ectopically overexpressing the different IP3R isoforms in the TKO cells, it was found that all IP3R isoforms were sensitized by Bcl-Xl and so conferred resistance against apoptotic stimuli. However, a decline in steady-state ER Ca 2+ levels was only found in TKO cells ectopically expressing IP3R3 [72], suggesting that decreased ER Ca 2+ levels are not a requisite for cellular protection against apoptosis. The antiapoptotic action may therefore be due to the enhanced Ca 2+ -spiking activity resulting from the sensitization of the IP3Rs, and be mediated either by increased mitochondrial bioenergetics or by modulation of transcriptional activity and gene expression [71, 72]. A similar mechanism was recently proposed for Bcl-2 and Mcl-1 [73].

Thirdly, an inhibition of the IP3-induced Ca 2+ release by Bcl-2 was also demonstrated [74]. In contrast to the work discussed above, the interaction site was mapped to the regulatory domain of IP3R1 moreover, the interaction was mediated through the BH4 domain of Bcl-2, a domain which is not involved in the interaction with the C-terminus of the IP3R [73, 75]. A peptide corresponding to the Bcl-2-binding site on IP3R1 specifically disrupted this interaction and in this way counteracted the functional effects of Bcl-2 on the IP3R [75, 76].

4.5. PKB/Akt and Promyelocytic Leukemia Protein

Another regulatory mechanism of the Ca 2+ -flux properties of the IP3R is its phosphorylation via PKB/Akt [29, 77, 78]. Upon prosurvival stimulation of cells, the prosurvival kinase PKB/Akt binds and phosphorylates the IP3R, thereby reducing its Ca 2+ -release activity. This mechanism underpins the increased resistance of cells towards apoptotic stimuli by inhibiting the Ca 2+ flux into the mitochondria and may be perused by tumor cells, yielding a survival advantage. The latter has been shown to occur in glioblastoma cells that display hyperactive PKB/Akt, leading to IP3R hyperphosphorylation and suppression of IP3R-channel activity [77].

Very recently, extranuclear promyelocytic leukemia protein (PML) has been shown to be present at the ER and mitochondrial-associated membranes, thereby promoting ER Ca 2+ release. At these microdomains, PML controls the Ca 2+ -flux properties of the IP3R by recruiting PP2A, which dephosphorylates PKB/Akt. The latter suppresses its kinase activity and thus the PKB/Akt-mediated phosphorylation of the IP3R, resulting in increased IP3R-mediated Ca 2+ transfer into the mitochondria and thus OMM permeabilization [79, 80]. This mechanism supplements the other known functions of PML in the nucleus of higher eukaryotes. PML nuclear bodies seem to contribute to its tumor suppressive action by inhibiting cell cycle progression and promoting cell death [81].

5. The Transfer of Ca 2+ between the IP3R and Mitochondria in Apoptosis and Autophagy

From the previous it is clear that Ca 2+ transfer from the ER to the mitochondrial matrix is crucial for regulating mitochondrial functions, including bioenergetics. The mitochondrial Ca 2+ signal can, however, also control the choice between cell survival and cell death, as it can participate in the induction and progression of apoptosis and autophagy [9, 10].

5.1. IP3Rs and Mitochondrial Ca 2+ in Apoptosis and Necrosis

Different studies have placed the IP3R as central player in the transfer of Ca 2+ into the mitochondria. Many cell types display the propagation of agonist-induced Ca 2+ signals into the interior of the mitochondria [11, 82].

Ca 2+ uptake in the mitochondria is crucial for multiple important cellular functions, but the risk of mitochondrial Ca 2+ overload exists, which may result in the induction of cell death. At a high concentration, mitochondrial Ca 2+ supports opening of the PTP in the IMM [51, 83]. This opening leads to the release of ions (including Ca 2+ ) and molecules (including ATP), mitochondrial depolarization, ROS production, cessation of oxidative phosphorylation followed by ATP hydrolysis, matrix swelling by osmotic forces, remodeling of the IMM, and eventually rupture of the OMM [52]. Subsequently various apoptogenic factors, including cytochrome C (CytC), apoptosis-inducing factor, Smac/Diablo, HtrA2/Omi, and endonuclease G, are released from the mitochondria [40]. These apoptogenic factors will activate effector caspases, as caspase-3 and caspase-7, and lead the cell into the execution phase of apoptosis. Permeabilization of the OMM is therefore considered as the decisive event in the development of cell death [84]. Given the proximity of IP3Rs to the mitochondrial Ca 2+ -entry sites, IP3-induced Ca 2+ spikes appear ideally suited for the stimulation of apoptosis [85], while the knockdown of the IP3R by siRNA led to the suppression of the Ca 2+ transfer to the mitochondria.

In addition to this canonical pathway, the group of Mikoshiba recently showed that not only excessive IP3R-mediated Ca 2+ release and the concomitant mitochondrial Ca 2+ overload but also the loss of IP3R function may lead to apoptosis by lowering the mitochondrial membrane potential [86]. In this study, it was shown that ER stress in neuronal cell leads to attenuation of IP3R function by impairing the positive regulation of IP3R1 by the ER chaperone Grp78, which acts as a major regulator of the unfolded protein response and thus prevents ER stress. The loss of Grp78 binding to the luminal domain of the IP3R1 leads to impaired subunit assembly and thus dysfunctional channels. This property seems selective for IP3R1, since Grp78 knockdown attenuated IP3R1-mediated Ca 2+ release but did not affect IP3R2- or IP3R3-mediated Ca 2+ release. Hence, it is interesting to note that Ca 2+ transfer from the ER to mitochondria requires a fine-tuned regulation, in which both suppressed and excessive Ca 2+ transfer leads to apoptosis.

While a severe impairment of IP3R1 function and attenuated Ca 2+ release lead to mitochondrial apoptosis, low-level Ca 2+ signaling from ER to mitochondria or enhancing ER-originating Ca 2+ oscillations elicits a prosurvival action by stimulating the mitochondrial energy production or by inducing transcription of specific genes [9, 31, 67, 69, 87]. In this paradigm, Bcl-Xl has been proposed to promote cell survival through its direct action on the IP3R by enhancing prosurvival Ca 2+ signaling, increasing mitochondrial bio-energetics and activation of signaling via nuclear factor of activated T cells [71, 72].

Mitochondrial Ca 2+ is a central factor in several neurodegenerative diseases as Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease [88]. The inhibition of cell death by preventing mitochondrial Ca 2+ overload or by preventing the collapse of the mitochondrial membrane potential is likely therapeutically relevant for the treatment of these diseases. In contrast, enhancement of mitochondrial Ca 2+ overload can lead to inhibition of tumor cell growth. Stimulation of the Ca 2+ transfer between ER and mitochondria could lead to increased apoptosis and in this way inhibit uncontrolled cellular proliferation [89]. In this concept, it is not surprising that many tumor suppressor proteins emerge as regulators of the transfer of Ca 2+ from the ER to the mitochondria, like Fhit and PML. Fhit acts at the mitochondrial level by increasing the affinity of the mitochondrial Ca 2+ uniporter, thereby promoting mitochondrial Ca 2+ elevations at low levels of agonist-induced Ca 2+ signaling [49]. PML acts at the level of the ER, where it is recruited by the IP3R via a phosphorylation-dependent process involving Akt and PP2A, thereby promoting Ca 2+ transfer between the ER and the mitochondria and inducing cell death [79, 80]. Mutations or ablation of proteins, like Fhit and PML, which may involve attenuated ER/mitochondrial Ca 2+ transfers, has been associated with the development of tumors.

5.2. IP3Rs and Mitochondrial Ca 2+ in Autophagy

Autophagy is a delivery pathway used for the lysosomal degradation of long-lived proteins, protein aggregates, damaged organelles, and foreign pathogens. In stress situations (e.g., nutrient starvation), this process offers the cell a fresh pool of building blocks and has thus a prosurvival function [90]. Cells in those conditions have to make the decision between survival (autophagy) and death (apoptosis). Important crosstalks exist between these two pathways [91, 92]. Interestingly, Ca 2+ and IP3Rs have been implicated in both apoptosis and autophagy, although the role of Ca 2+ in autophagy only recently emerged [9, 10, 93]. Nonetheless, Ca 2+ /IP3Rs may represent key players in the apoptosis-autophagy decision.

The first results on Ca 2+ in autophagy even appeared contradictory. On the one hand, autophagy was activated by an increase of the cytosolic Ca 2+ concentration [94–96]. On the other hand, autophagy was also activated by conditions that all would lead to a decrease of the IP3R activity and/or cytosolic Ca 2+ concentration and therefore potentially of the mitochondrial Ca 2+ concentration [97–100]. In a recent report, it was shown that IP3R activity is necessary to provide for a basal Ca 2+ signal to the mitochondria, in order to control mitochondrial bioenergetics. IP3R knockdown or inhibition will blunt these Ca 2+ signals, thereby compromising mitochondrial ATP production. The resulting increase in AMP/ATP ratio will subsequently activate autophagy via AMP-activated protein kinase (AMPK) [87].

Other results indicate that IP3Rs could inhibit autophagy through a scaffold function, via binding of both Bcl-2 and Beclin-1 (an essential autophagy protein), thereby promoting the anti-autophagic interaction between these two proteins. Treatment of HeLa cells with the IP3R inhibitor xestospongin B promoted the release of Beclin-1 from the IP3R-Bcl-2 complex, leading to autophagy activation [101].

So far, the data on Ca 2+ -stimulated autophagy concern the Ca 2+ in the cytosol [94–96] or ER [102, 103]. It is not yet clear whether the IP3R is hereby involved, although treatment with an IP3R inhibitor did blunt cadmium-induced autophagy stimulation [95]. The exact mechanism by which Ca 2+ promotes autophagy is also still under debate. AMPK-dependent [94], AMPK-independent [96], or ERK-dependent pathways [95] are all possible.

Taken together, these data indicate that a specific, low-intensity Ca 2+ transfer from ER to mitochondria is necessary to inhibit autophagy, while an increase of the cytosolic Ca 2+ concentration would activate autophagy.

6. Implications of Ca 2+ Signaling in Aging

6.1. Aging: A Process of Disorganization

All biological processes involved in the transformation of a fertilized egg into a mature individual capable of reproduction are driven by a purposeful genetic program. Through evolution, natural selection has favored individuals that are reproductively successful [104, 105]. Biological systems, like everything else in the universe, change as a result of entropic changes. Entropy is the tendency for concentrated energy to disperse when unhindered. Natural selection has resulted in sufficient relative strengths of the chemical bonds in our molecules to prevent entropic changes and also installed repair and replacement mechanisms. Evolution has therefore kept the biomolecules in a functional state until reproductive maturation.

After sexual maturation, there is no longer a species-survival benefit for indefinitely maintaining these energy states and, hence, the fidelity in most molecules. As we grow older, stochastic or random events not driven by a genetic program cause energy loss resulting in biologically inactive or malfunctioning molecules. Aging is therefore characterized by increasing entropy. The intrinsic thermodynamic instability of the molecules whose precise three-dimensional structures are no longer maintained leads to covalent modifications such as glycation, conformational changes, aggregation and precipitation, amyloid formation, altered protein degradation, synthesis rates, and nuclear and mitochondrial DNA damage and alterations. When the loss of structure and, hence, function ultimately exceeds repair and turnover capacity, vulnerability to pathology and age-associated diseases increases. Because of the randomness of the molecular disorder underlying aging, the loss of molecular fidelity varies within the body. The weakest links in this system will be the first that lead to disease, like in the vascular system and in cells with a high tendency for cancer development. The very heterogeneous aging process contrasts with the virtually identical stages of development until adulthood [106]. In this respect, we will here focus on the age-related disorganization in the Ca 2+ signaling machinery, ROS production, and autophagy.

6.2. Mechanism Involved in Aging: ROS, Mitochondria, and Autophagy

The role of ROS accumulation and subsequent macromolecular damage in age-related degeneration has been supported by a plethora of cellular and biological data from various model systems and organisms [107]. Antioxidants act as ROS scavengers and protect against the detrimental effects of cellular ROS exposure. Genetically, genes that extend lifespan were clustered in the IGF-1/insulin-like signaling pathway in a variety of model systems [108]. Nongenetic mechanisms to extend lifespan in different organisms are achieved by caloric restriction and/or by physical activity [109–113]. The composition of the diet during caloric restriction is important addition of antioxidants (like vitamins, flavonoids), minerals (like Zn and Se), and other compounds such as caffeine, omega 3, and fatty acids has been shown to enhance lifespan [114]. It should be noted, however, that most studies concerning these mechanisms were performed in yeast and animal models, but not yet in humans [115].

Here, we will discuss the molecular mechanisms of ROS underlying aging. First, we will discuss the remodeling of Ca 2+ signaling during aging. This is important since the OMM permeabilization is critically controlled by the elevation of the mitochondrial Ca 2+ concentration, thereby serving as a coincidence detector with ROS [116]. Next, we will focus on the signaling cascade involving sirtuins, p66 Shc , and autophagy in the regulation of mitochondrial function. A schematic overview of the interaction between the different molecular key players in aging is provided in Figure 3.


Ca 2+ signalling and key events involved in aging. Aging cells display decreased function or expression of ER proteins (IP3Rs, RyRs, SERCAs, Ca 2+ -binding chaperones (CaBC)), increased cytosolic [Ca 2+ ], suppressed agonist-mediated signaling, and accumulation of damaged mitochondria due to declined autophagic activity. The simultaneous increase in disorganization and dysfunction of the Ca 2+ -handling proteins and the decline in autophagy will result in the exaggerated production and excessive accumulation of ROS. These events may lead to both ER stress and mitochondrial dysfunction, like PTP opening and OMM permeabilization with the consequent release of apoptogenic factors and cell death. p66 Shc and sirtuins take part in this scenario. P66 Shc translocates to mitochondria upon oxidative-stress-induced PKCβ phosphorylation and peptidylprolyl isomerization by Pin1, thereby supporting ROS production. Sirtuins are downregulated and unable to exert its antiaging effect. It is important to note that while p66 Shc ablation leads to lifespan extension, high levels of p66 Shc have been observed in centenarians. While in normal cells, ROS help to detect and remove altered mitochondria through autophagy, thereby maintaining cellular health, the excessive release of ROS in combination with the decline in autophagy observed during aging may underpin the age-related cell-death processes. In this respect, the recently identified inhibitors of EGF-receptor signaling, the high-performance advanced age phenotype proteins (HPA-1 and HPA-2), whose knockdown promotes locomotory health span of C. elegans, may point towards an important role of proper agonist-induced Ca 2+ signaling via the IP3R axis. The relevance of these ligands or of attenuated agonist-induced signaling in humans needs to be established. However, recent evidence indicates that dysfunction of IP3Rs during ER stress promotes cell death and underlies a neurodegenerative disease, like Huntington’s disease. Given the central role of proper IP3R function for mitochondrial bioenergetics and ATP production, the decline of IP3R activity observed during ER stress or attenuated upstream signaling linked to IP3 may be very relevant for age-related apoptosis but require further investigation. Green arrows: stimulation red lines: inhibition black arrows: Ca 2+ flux dashed-green arrow: stimulation/damage.
6.2.1. Ca 2+ Signaling in Aging

Altered intracellular Ca 2+ signaling is a hallmark of neurodegeneration, like in Alzheimer’s and Huntington’s disease [117–120]. Different models have been proposed for familial Alzheimer’s-disease-linked presenilin mutations, including the function of presenilins as Ca 2+ -leak channels [121], an increase in the expression level of IP3Rs [122], or the direct activation of IP3Rs or RyRs [123–125]. In any case, it is clear that exaggerated Ca 2+ signaling is an upstream event in the pathophysiology of Alzheimer’s disease and contributes to the ROS-mediated cell toxicity [126]. However, the changes in Ca 2+ signaling that occur in neurodegenerative diseases may be dependent on the type of disease. For instance, a mouse model for Huntington’s disease revealed dysfunctional IP3R Ca 2+ -release channel activity in the cerebrum and striatum, which was caused by a prominent decline in the association of Grp78, a positive regulator of the IP3R1-channel formation, with the IP3R1 [86].

Other age-related diseases also display altered Ca 2+ signaling. Cardiac hypertrophy, for example, is characterized by enhanced IP3 signaling, leading to spontaneous Ca 2+ -release events that underlie arrhythmias [127]. Also chronic heart failure can be a consequence of excessive phosphorylation of RyR, leading to an increased Ca 2+ leak [128] (Figure 2).

However, the role and mechanism of ER Ca 2+ signaling in aging is less clear [129], although most studies suggest altered Ca 2+ signaling during aging (Figure 2). In most cell types, ER Ca 2+ dyshomeostasis was caused by a decreased ER Ca 2+ content and a decreased Ca 2+ release from the ER, while the cytosolic [Ca 2+ ] was increased. These effects were the result of a decline in SERCA and/or IP3R and/or RyR activity, caused by changes in mRNA or protein levels, phosphorylation events, or oxidative damage to SERCA [7]. In addition, intraluminal Ca 2+ -buffering protein levels often decline during age, in part also through oxidative damage [130] (Figure 2). Also VDAC undergoes posttranslational modifications in aged cells, possibly through oxidative break-up of tryptophan residues, thereby increasing the susceptibility to apoptosis [131]. This is in line with evidence showing that superoxide can lead to mitochondrial permeabilization in a VDAC-dependent manner [132]. In yeast, this phenomenon can be protected by Cu/Zn-superoxide dismutase, a protein known for its protective role against aging [133].

Some cell types, however, display Ca 2+ dyshomeostasis in a different way (Figure 2). Studies in aged rat hearts, for example, showed increased IP3R levels [134]. Also aged neuronal cells displayed reduced sensitivity towards caffeine, which may be caused by a decline in the steady-state ER Ca 2+ levels [135–137]. The latter may be due to a decreased SERCA Ca 2+ -pump activity, a limited supply of ATP or an increased Ca 2+ leak from the ER. Other studies pointed to a prolonged Ca 2+ -induced Ca 2+ release, resulting in an inhibition of synaptic strength and long-term potentiation [138, 139].

Interestingly, IP3R characteristics also appear to be altered in aged brain tissues [140], as IP3R density and IP3 binding to the IP3R were decreased in aged rat cerebellum. The same observation of decreased IP3 binding was made in aged mice cerebellum [141]. However, the cellular IP3 content increased with age [142]. These findings suggest a role for the phosphoinositide/Ca 2+ signaling in the impaired neuronal responsiveness during aging. In this respect, more recent work revealed that stimulation of IP3Rs in old astrocytes increased protection against ROS and subsequently neuroprotection [143].

Moreover, in aged MII-stage eggs, it was found that the IP3R1 was proteolytically cleaved by caspase-3, resulting in a leaky 95-kDa C-terminal IP3R1 fragment containing the channel pore [144, 145]. In contrast, when the C-terminal channel domain was recombinantly expressed in the mouse oocytes, the sperm-factor-induced Ca 2+ oscillations were abolished and the eggs displayed an apoptotic and fragmented phenotype. Previously, we had shown that caspase-3-dependent cleavage of the IP3R augmented the late phase of apoptosis by providing a prolonged ER Ca 2+ leak [146]. However, in healthy cells, the Ca 2+ leak through a recombinantly expressed C-terminal channel domain was very small. Hence, the caspase-3-dependent cleavage of the IP3R may participate in cellular Ca 2+ overload via a second-hit mechanism. In the case of aged oocytes, accumulated ROS may be the second hit. Currently, it is not clear whether IP3R cleavage contributes to the aging process by overloading the mitochondria with Ca 2+ and sensitizing them towards ROS accumulation. In addition, ROS may also directly regulate IP3R activity, since it is known that oxidizing agents like thimerosal sensitize IP3Rs by stimulating intramolecular interactions between the suppressor and ligand-binding domain [147]. Taken together, IP3R/Ca 2+ signaling appears to be affected in aged cells. Abnormal Ca 2+ signals may then affect many processes (ROS production/protection, autophagy, apoptosis, synaptic transmission, etc.) that are altered during aging (summarized in Figure 5). Nevertheless, the overall changes in ER Ca 2+ handling observed during aging seem relatively small compared to the changes found in Alzheimer’s disease [129].

Recently, an elegant study on Caenorhabditis elegans re-enforced the paradigm that the activation of IP3R pathways may be considered in therapeutic applications for treating age-related decline in skeletal muscle function (sarcopenia) [148]. Indeed, using an RNAi screen, the authors identified two critical factors that delayed the age-associated decline in locomotory health span of C. elegans in a high-performance advanced age phenotype (HPA-1 and HPA-2). The concept underpinning this study was that locomotory decline in humans contributes to frailty and loss of independence. Although the exact mechanism is not yet known, it is clear that HPA-1 and HPA-2 attenuate epidermal-growth-factor-(EGF-) dependent signaling via the EGF receptor [148]. When HPA-1 and HPA-2 are disrupted, EGF signaling via the EGF receptor will increase. The activation of the EGF-signaling pathway normally leads to cell proliferation, survival, integrity, and differentiation. Importantly, phospholipase C-γ (PLC-γ) and IP3Rs were demonstrated to act downstream of EGF-receptor signaling, thereby contributing to prolonged health span in these animals. This is the very first report considering the role of EGF signaling in aging. Therefore, the exact mechanism of how these signaling pathways affect human aging remains to be further clarified, but restoring the attenuated IP3R-mediated Ca 2+ signaling and reestablishing normal mitochondrial function may be an attractive hypothesis in combination with chemical induction of autophagy (Figure 4). Nevertheless, a decline in G-protein-coupled receptor-dependent signaling has been observed in the skeletal muscle and intestine of aged rats [149]. The underlying mechanism involved a prominent decrease in the levels of Gq/11 and Gi protein levels.


A speculative antiaging strategy based on restoring IP3R-mediated Ca 2+ signaling and chemical induction of autophagy. Provided the concept that aging cells are characterized by suppressed IP3 signaling or attenuated IP3R, Ca 2+ -release activity is relevant in humans, and elevating IP3 levels may compensate for the decline in the IP3/IP3R-signaling axis. This may contribute to a decline in the p66 Shc -mediated ROS production, an activation of sirtuin-dependent mitochondrial biogenesis, and the lowering of ROS production. The final step of this compensatory response consists in the autophagic removal of the damaged mitochondria. Hence, chemical induction of autophagy (e.g., by rapamycin or spermidine) is likely critical for successful and healthy aging in human beings. It is important to note that this concept is based on a recent report on C. elegans, in which ablations of inhibitors of EGF signaling enhance IP3R signaling and promote healthy lifespan extension. Green arrows: stimulation red lines: inhibition black arrows: Ca 2+ flux.

(a)
(b)
(a)
(b) Network of interactions between sirtuins, p66 Shc , Ca 2+ , and ROS, which affect mitochondrial function, autophagy, and apoptosis, thereby controlling aging-dependent processes. (a) Ca 2+ signals may increase or prevent aging. Ca 2+ signals are characterized by different spatiotemporal characteristics and subsequently different outcomes on mitochondrial function, autophagy, and apoptosis. For example, a constitutive Ca 2+ transfer from ER to mitochondria would stimulate mitochondrial function and inhibit autophagy and apoptosis, while a mitochondrial Ca 2+ overload would be proapoptotic. The interplay between mitochondrial Ca 2+ elevations and ROS production is a critical determinant in the apoptotic outcome at the level of the mitochondria, which function as co-incidence detectors. Therefore, high mitochondrial Ca 2+ concentrations and ROS act as a double-hit mechanism, triggering mitochondrial-dependent apoptosis. (b) Sirtuins are mainly antiaging genes via the promotion of mitochondrial function and autophagy and inhibition of apoptosis. They also act inhibitingly on ROS. Sirtuin function may be enhanced by restricting caloric intake or increasing physical activity, thereby extending lifespan. Increased ROS activate the Pin1- p66 Shc complex, which, in turn, promotes the production of ROS and subsequently mitochondrial damage. Therefore, p66 Shc may help to target damaged mitochondria and activate cellular processes that deal with dysfunctional mitochondria and oxidative stress. The outcome, however, can be dual: aging may be enhanced via a complete removal of the cell through apoptosis, while the selective removal of the damaged mitochondria through mitophagy, leaving the cell with predominantly healthy mitochondria, may slow down the aging process. Green arrows: stimulation red lines: inhibition black arrows: stimulation or inhibition.
6.2.2. Sirtuins

Sirtuins are a conserved family of proteins that are linked to longevity and stress tolerance in Saccharomyces cerevisiae [150]. Sirtuins have been identified as antiaging genes, since increasing their activity prolonged lifespan not only in yeast, but also in C. elegans and Drosophila melanogaster and is thought to act similarly in mammals [151–153]. In this respect, age is often associated with reduced sirtuin levels. In aged mouse embryonic fibroblasts, progressive loss of the sirtuin-1 protein, but not mRNA, was observed [154]. However, other studies show that this is at least tissue specific sirtuin-1 activity was reduced in rat hearts, but not in adipose tissue [155], and reduced sirtuin-1 expression was found only in distinctive parts of the mouse brain [156]. Sirtuins, which retard aging as a function of their gene dosage, display unique biochemical activities, that is, NAD-dependent protein deacetylase [157, 158]. The subsequent deacetylation of sirtuin substrates alters their activity (activation or inhibition). In mammals, sirtuin-1 deacetylates a variety of key transcription factors and cofactors, like p53 [159], FOXO proteins [160, 161], peroxisome proliferation activating receptor (PPAR)-γ co-activator-1α (PGC-1α) [162], and nuclear factor-κB [163]. The effects of sirtuin-1 on these factors elicit stress tolerance and metabolic changes reminiscent of caloric restriction, while caloric restriction upregulates sirtuin-1 levels, and mice lacking sirtuin-1 did not display phenotypic responses upon caloric restriction [160, 164–166]. Since sirtuins are regulated by NAD + , their activity will be influenced by the NAD + /NADH ratio and thus by the metabolic state of the cell [167]. Hence, sirtuins may be influenced not only by caloric restriction but also by physical activity, both associated with longevity and increased insulin sensitivity [168, 169].

Importantly, sirtuin-1 also regulates mitochondrial biology [150, 167], another key aspect in aging, since the number of functional mitochondria is known to decline during aging. This has been proposed to underlie aging in diseases like type-2 diabetes [170, 171]. In contrast, increasing mitochondrial activity will increase the metabolic rate, enhance glucose metabolism, and improve insulin sensitivity. Even without an increase in the metabolic rate, caloric restriction might be beneficial by inducing mitochondrial biogenesis via sirtuin-1 [165, 172, 173]. Activation of sirtuin-1 has been shown to be involved in mitochondrial biogenesis and improved mitochondrial function by deacetylation of PGC-1α, thereby lowering ROS production [162].

Sirtuin-1 also suppressed stress-induced apoptosis, while the lack of sirtuin-1 inhibited autophagy in vivo [174]. In addition, the extension of lifespan upon caloric restriction was proposed to be dependent on the induction of autophagy by sirtuin-1 [175]. The underlying mechanism probably involves the deacetylation of certain autophagy proteins, such as Atg5, Atg7, and Atg8 [174, 175]. A schematic overview of the role of sirtuins in aging is depicted in Figure 5.

6.2.3. p66 Shc

Recent research revealed the role of p66 Shc , the 66 kDa isoform of the Shc (Src homolog and collagen homolog) family [176]. Although p66 Shc forms stable complexes with Grb2, an adaptor protein for the Ras-exchange factor SOS, it has little effect on Ras-mediated signaling [177].

Nevertheless, p66 Shc is activated by oxidative stress via phosphorylation on Ser36, and this mechanism is indispensable for p66 Shc ’s lifespan regulation [178, 179]. Mice in which p66 Shc has been deleted displayed a prolonged lifespan with a decreased mitochondrial metabolism and ROS production, while lacking pathophysiological characteristics or effects on body size. MEF cells from p66 Shc-/- animals displayed resistance towards oxidative-stress-induced apoptosis in a p53-dependent manner [176].

ROS arise from the mitochondrial electron-transfer chain or from exogenous sources, like UV and ionizing radiations. p66 Shc is involved in mitochondrial ROS production. In basal conditions, about one fifth of p66 Shc is localized to the intermembrane space of the mitochondria, while oxidative stress dramatically increases the mitochondria-associated p66 Shc due to its mitochondrial translocation from the cytosol [180]. In the mitochondria, p66 Shc interacts with CytC, promoting the shuttling of electrons from CytC to molecular oxygen [181]. The latter may underlie the increased ROS production upon p66 Shc overexpression and the decreased ROS production in p66 Shc knockout cells. In addition, p66 Shc knockout cells displayed decreased oxidative capacity, thereby redirecting metabolic energy conversion from oxidative toward glycolytic pathways. Therefore, p66 Shc may provide a molecular switch to oxidative-stress-induced apoptosis by controlling mitochondrial ROS production. It should be noted, however, that studies in yeast correlated higher respiration rates combined with decreased oxidative stress and increased lifespan [182]. This suggests that the respiration rate per se is not the important factor for ROS production, but more likely the electron transmit time and the availability of oxygen [183].

In normal cells, oxidative stress leads to compromised mitochondrial Ca 2+ homeostasis, which is an early event of mitochondrial damage [107, 176]. This is observed as a decreased mitochondrial Ca 2+ signal upon agonist stimulation in cells challenged with H2O2 despite a normal cytosolic Ca 2+ signal. Importantly, cells lacking p66 Shc seemed to be protected against oxidative challenge, since their mitochondrial Ca 2+ signaling upon agonist stimulation was not impaired in the presence of H2O2 [176]. Similar results were found in MEF cells lacking Pin-1, a peptidylprolyl isomerase catalyzing cis/trans isomerization of phosphorylated Ser-Pro bonds, where the reduction of agonist-induced Ca 2+ signals in mitochondria upon oxidative stress was significantly smaller. These findings suggest a phosphorylation-dependent conformational change in Pin-1 targets, like p66 Shc .

Recent work provided important mechanistic insights into the role of p66 Shc in the early mitochondrial response to oxidative stress [178, 179]. ROS are known to activate a variety of kinases, including protein kinase C (PKC) β. The activation of PKCβ will cause the phosphorylation of p66 Shc on Ser36, although other kinases may also participate in this process. Indeed, the mitochondrial fraction of p66 Shc during oxidative challenge was severely reduced after treatment with PKCβ inhibitors. As a result, Ser36-phosphorylated p66 Shc will interact with Pin-1. The catalytic activity of Pin-1 may result in cis/trans isomerization of Ser36-Pro37, thereby triggering the exposure of a mitochondrial targeting sequence or an interaction with mtHsp70, a mitochondrial heat-shock protein. This process may underlie selective targeting of p66 Shc to mitochondria undergoing oxidative challenge. The mitochondrial targeting of p66 Shc involves its protein-phosphatase-(PP-) 2A-mediated dephosphorylation and dissociation from mtHsp70, although the mechanism of their contribution is not fully elucidated. In the intermembrane space, p66 Shc will interact with reduced CytC and enhance intramitochondrial H2O2 production. The latter and its more damaging reaction products, the hydroxyl radicals, have been shown to trigger the opening of the PTP [184]. This will perturb mitochondrial structure and function, resulting in mitochondrial permeabilization, CytC release, and apoptosis induction, and subsequently lead to a coordinated cell-death response and the removal of the cell containing damaged mitochondria. However, in addition to apoptosis, autophagy may be involved in removing the subpopulation of compromised mitochondria suffering from oxidative challenge. Interestingly, this autophagy-mediated removal of damaged mitochondria can be triggered through PTP opening [185]. This will result in the removal of the organelles that are damaged by the oxidative stress (a process termed mitophagy), while maintaining the healthy mitochondria. According to these findings, it is interesting to note that aging has been associated with declined autophagy activity [186], while autophagy activity is a requisite for lifespan extension in C. elegans [187]. In this way, p66 Shc may be important for mitochondrial quality control through the autophagy-mediated removal of damaged mitochondria. However, during aging, the number of mitochondria suffering from oxidative stress may increase, while their cleanup by the autophagic system may become limiting, leading to the accumulation of unprocessed oxidation-damaged mitochondria. Importantly, in mouse models for aging, the levels of p66 Shc seemed to decline, while its phosphorylation at Ser36 was enhanced [188]. This correlated with higher free-radical production and accumulation of damage caused by ROS.

Strikingly, fibroblasts obtained from centenarians displayed elevated levels of p66 Shc [189], indicating that basal mitochondrial p66 Shc plays an important role in normal cell-damage management of stress and in damage repair. Indeed, the selective removal of damaged mitochondria may contribute to lifespan extension. In addition, it is interesting to note that increased physical activity has been associated with lifespan extension and lower mortality, although this is associated with increased mitochondrial ROS production due to an increased metabolic rate. Therefore, it is conceivable that exercise may promote adaptation to ROS by upregulating ROS scavengers, causing a natural resistance against ROS or against cellular damage in general [167]. Hence, it may be worth investigating whether p66 Shc levels are affected by exercise and whether this may contribute to increased cleanup of damaged mitochondria or resistance against ROS. A schematic overview of the role of p66 Shc in aging is depicted in Figure 5.

6.2.4. Autophagy

It has become increasingly clear that autophagy plays a central role in the aging process, in which it is involved in the removal of damaged organelles or of protein aggregates by engulfment in autophagosomes followed by lysosomal degradation. First of all, autophagy was demonstrated to decrease with increasing life time [186]. Caloric restriction slowed down the age-related impairment of autophagy in skeletal muscle of rats [190]. In addition, chemical induction of autophagy by spermidine or by rapamycin prolonged lifespan [191, 192]. In contrast, animals with compromised capacity to perform autophagy were short living and displayed neurodegenerative phenotypes, probably due to the accumulation of deleterious accumulation of protein aggregates [193–195]. Moreover, it is clear that damaged mitochondria ought to be removed, while harboring the healthy mitochondria, which are needed for cell survival. In any case, the accumulation of damaged mitochondria and their impaired removal is a hallmark of aging and will contribute to decreased cell viability. Therefore, mitochondrial quality control is essential for proper cell survival.

The “selective” recognition of damaged mitochondria by autophagosomes without affecting healthy mitochondria remains very poorly understood. However, the first components essential for “selective” mitophagy have been identified in yeast: Uth1, an OMM protein, and Aup1, a mitochondrial phosphatase [196–198]. Additional components of organelle-specific autophagy have been revealed in a systematic screen, including Atg11, Atg20, Atg24, Atg32, and Atg33 [199, 200]. Atg32 is proposed as the receptor for mitophagy via the local recruitment of Atg8, an essential component of the autophagosome formation. NIX/BNIP3L [201, 202], BNIP3 [203], PARKIN [204], and PINK-1 [205–210] were proposed to be involved in mitochondrial degradation in mammalian cells. PARKIN is selectively recruited by dysfunctional mitochondria, thereby mediating the engulfment of these mitochondria by the autophagosomes [204]. A recent study provided clear insights into the underlying mechanism, which required the accumulation of the kinase PINK-1 on damaged mitochondria. In healthy mitochondria, PINK-1 is maintained at a low level by voltage-dependent proteolysis [210]. In mitochondria with sustained damage, PINK-1 levels rapidly accumulated. The latter was required and sufficient to recruit PARKIN to the mitochondria providing a mechanism for the selective removal of damaged mitochondria by autophagy. Importantly, mutations in PINK-1 or PARKIN associated with Parkinson’s disease abolished the recruitment of PARKIN by PINK-1 to the mitochondria, allowing the accumulation of damaged mitochondria. Another recent study revealed the mitochondrial protein NIX as the selective mitophagy receptor for the removal of damaged mitochondria by binding and recruiting LC3/GABARAP proteins [211]. The latter are ubiquitin-like modifiers required for the elongation of autophagosomal membranes.

Besides these mitophagy receptors, mitochondrial proteases and chaperones were needed to prevent the accumulation of misfolded and aggregated proteins within the mitochondria [167].

Finally, various studies point towards a role of ROS upstream of autophagy [212]. Accumulation of ROS directly affects different key players essential for the induction of autophagy, including the activation of the protein kinases AMPK and JNK, the inhibition of other kinases (Akt and TOR), and the inhibition of LC3 delipidation. These processes will stimulate autophagy, thereby alleviating the oxidative stress by removing the ROS-generating mitochondria.

7. Conclusions

Upstream Ca 2+ and ROS signaling tightly control cellular homeostasis by regulating fundamental cell-death and cell-survival processes like apoptosis and autophagy. It is clear that many proteins that mediate apoptosis and autophagy directly affect Ca 2+ signaling through interaction with the ER and mitochondrial Ca 2+ -release and/or Ca 2+ -uptake mechanisms. Furthermore, these Ca 2+ -signaling proteins contribute to the functional and physical linking between ER and mitochondria. Importantly, the interplay between ER and mitochondrial Ca 2+ signaling and ROS signaling mediates the detection, the efficient targeting, and removal of mitochondria with sustained damage. This is the key for cellular homeostasis as well as for homeostasis at the level of the whole organism. In this respect, the efficient and selective removal of damaged mitochondria by autophagy is a crucial element in the maintenance of cellular health, whereby the poisonous accumulation of ROS from dysfunctional mitochondria and eventual cell death via apoptosis are avoided. Recent studies point towards a central role for impaired autophagy and inadequate removal of damaged mitochondria during aging. At the level of the organism, apoptosis will be the ultimate resort to remove seriously damaged cells. This will particularly affect the lifespan of nondividing cells, like neurons, thereby affecting the lifespan of the whole organism.

Acknowledgments

Work performed in the laboratory of the authors in this area was supported by the Research Council of the K.U.Leuven (Concerted Action GOA 04/07 and 09/012 and OT-START research funding STRT1/10/044) and by the Research Foundation Flanders (FWO-Vlaanderen) (Grants G.0604.07, G073109N, and G072409N). J. P. Decuypere and G. Monaco are, respectively, recipients of a Ph.D. fellowship from the Agency for Innovation by Science and Technology (IWT) and the Research Foundation Flanders (FWO-Vlaanderen).

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Copyright

Copyright © 2011 Jean-Paul Decuypere et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


What happens to IP3 molecules after release from IP3 receptors? - Biology

How can one signal molecule (hormone, transmitter, etc.) produce different effects on different tissues?

A . Two Basic Methods

1. Using the Same Receptor & same 2nd messenger, but different Target Proteins

a. An example:

(1). In skeletal muscle: epinephrine causes glycogen breakdown.

(2). In smooth muscle of lung: epinephrine causes muscle relaxation.

b. Why does this make sense?

(1). Epinephrine (also called adrenaline) is produced in response to stress.

(2). In response to stress, need to "mobilize" glucose -- release it from storage so it can be broken down to provide energy. Therefore need to increase glycogen breakdown (and decrease glycogen synthesis) in muscle (& liver).

(3). In response to stress, need to breathe more deeply. Therefore need smooth muscle around tubes that carry air (bronchioles) to relax.

c. How is this possible? Same receptors, same 2nd messenger (cAMP) are used.

2. The solution: PKA is activated in both skeletal and smooth muscle. However the target proteins available to be phosphorylated are different in the two tissues. Therefore different proteins are phosphorylated and activated (or inactivated) in the two different tissue types.

a. In skeletal muscle, PKA phosphorylates phosphorylase kinase, glycogen phosphorylase etc, as on handout 12D.

b, In smooth muscle surrounding the bronchioles, PKA phosphorylates a protein (MLCK) needed for contraction, inactivating it. Therefore contraction cannot occur.

FYI only: For smooth muscle to contract, an active kinase (MLCK) must bind Ca ++ (in the form of a calmodulin /Ca ++ complex). If MLCK is phosphorlyated, the Ca ++ /calmodulin complex cannot bind to it, and contraction does not occur. ( We will discuss the role of calmodulin and the mechanism of smooth muscle contraction later.) If you like to see all the details of the role of MLCK, see Becker fig. 16-24.

2. Using different receptors & second messengers in different cell types

(See Becker fig. 14-23 (10-24). An example -- effects of epinephrine (adrenaline) on smooth muscle. Some smooth muscles relax, and some contract in response to epinephrine. In this case, different receptors & 2nd messengers are involved. How does this work? See below.

Try problem 6-11.

B. Example of Using Different Second Messengers (& Different Receptors)

1. The phenomenon:

a. Epinephrine (secreted in response to stress) has different effects on different smooth muscles:

(1). On some smooth muscles, epi → contraction

(2). On other smooth muscles, epi → relaxation (as above)

b. How does this make sense?

(1). In peripheral circulation, smooth muscles around blood vessels (arterioles) contract, diverting blood from peripheral circulation to essential internal organs

(2). In lungs, smooth muscles around tubes carrying air (bronchioles) relax, so lungs can expand more and you can breathe more deeply.

a. Ca ++ stimulates muscle contraction.

b. Epinephrine binds to receptors on some smooth muscles (ex: around arterioles) → Ca ++ released from ER → intracellular Ca ++ up → stimulates contraction.

c. Epinephrine binds to receptors on some smooth muscles (ex: around bronchioles) → phosphorylates protein needed for response to Ca ++ , preventing response.

a. Two basic types of epinephrine receptors -- called alpha and beta adrenergic receptors (adrenergic = for adrenaline). The two types are distinguished (primarily) by their relative affinities for epinephrine (adrenaline) and norepinephrine (noradrenaline).

b. Some types of smooth muscle have mostly one type of receptors some the other. (See table below and table at end of lecture 15 for details of receptor properties.)

c. Two types of receptors activate different G proteins and generate different second messengers as on handout 12A. (We will go over the details later. What you need to know so far is below.)

(1). Beta receptors → G protein type (Gs) → cAMP response → PKA

(2). Alpha1 receptors → different G protein (Gp) → different second messenger (IP3) → binds to receptors on ER membrane → opens Ca ++ channels in ER → Ca ++ release from ER → contraction

4. How does this all work to allow appropriate response to stress (epinephrine)?

a. Beta type receptors. Beta receptors are found in lung tissue in smooth muscle surrounding bronchioles.
Stress (pop quiz, lion in street, etc.) → epinephrine → muscles relax → bronchioles dilate → deeper breathing → more oxygen → energy to cope with stress.

b. Alpha type receptors. Alpha receptors are found in smooth muscle surrounding blood vessels of peripheral circulation.
Stress → epinephrine → muscles contract → constrict peripheral circulation → direct blood to essential organs for responding to stress (heart, lungs, skeletal muscle).

To review effects of different receptors, try problems 6-20 & 6-21. Note that you do not need to know the details of how IP3 is generated, but you do need to know that PLC is the enzyme responsible for producing IP3. For unknown reasons, the Greek symbols in these problems did not prin t in the latest edition of the problem book, and there are spaces instead. Where it says ' 1' or ' 2' receptors it should say '⓫' or '⓬' receptors. Where it says ' / ' it should say 'β/δ'. (There is a missing β & δ in the description of experiment (4) in the table, and in the sentence after the end of 6-21, part C-3.)

  • Agonist = mimic of hormone or ligand binds to receptor and has same effect as ligand.
  • Antagonist = blocker of effect of hormone or ligand binds to receptor (and prevents binding of normal ligand) but does not activate the receptor.

Try Problem 6-8 & 6-9 if not yet. (To review agonists & antagonists.)

6. Summary of epinephrine effects on smooth muscle (in lung vs peripheral circulation)

Note: There are more than two types of epinephrine receptors on smooth muscle cells, so epinephrine may affect smooth muscle in other tissues in other ways. (There are subtypes of alpha and subtypes of beta.)

* Details of how PLC generates IP3 are on handout 12A. We will go over this later.


Second Messengers inside the cell

Many different kinds of molecules can serve as second messengers. The signal, or ligand, binding to a membrane receptor leads to the production of second messengers inside the cell. The original signal usually doesn't enter the cell. The small molecule "cAMP" was the initial second messenger to be identified. Other examples of second messengers include NO, IP3, and DAG. The figure below shows an example of the production of second messengers.

The figure depicts a system where the signal causes a G-protein to become active,stimulating the membrane enzyme phospholipase C. This enzyme degrades cell membrane phosphatidyl inositol releasing IP3 (inositol triphosphate) and leaving diacyl glycerol (glycerol with two fatty acids, DAG). Both are second messengers, with IP3 causing the endoplasmic reticulum to release Ca ++ (also a second messenger). The DAG activates protein kinase C, a kinase that is dependent on Ca ++ for activity. Note that both second messengers play a role in the activation of protein kinase C. The response made by the cell will depend on what targets for protein kinase C are available


Membrane Transporters in the Pathogenesis of Cardiovascular and Lung Disorders

Lin Zhang , . Vladimir V. Matchkov , in Current Topics in Membranes , 2019

11 Na,K-ATPase scaffolds with several proteins important for [Ca 2 + ]i signaling

The Na,K-ATPase-dependent signal transduction is strongly integrated in [Ca 2 + ]i signaling ( Fig. 1 ). Previous studies showed that Src can modulate [Ca 2 + ]i in isolated vascular smooth muscle cells via phosphorylation and activation of the L-type voltage-gated Ca 2 + channels ( Gui et al., 2006 Wijetunge, Lymn, & Hughes, 2000 ). We suggested that attenuation of the resting and agonist-induced [Ca 2 + ]i in the presence of tyrosine kinase inhibitors might be a result of suppressed tyrosine phosphorylation of voltage-gated Ca 2 + channels ( Bouzinova et al., 2018 ). However, this may also be an effect of membrane hyperpolarization due to activation of the K + channels ( Alioua et al., 2002 ). This suggestion is in accordance with out finding that pNaKtide slightly hyperpolarizes smooth muscle cells ( Hangaard et al., 2017 ).

Notably, well-described ouabain-induced elevation of [Ca 2 + ]i may be meditated not only through the conventional modulation of Na + /Ca 2 + homeostasis but also via physical interactions with several proteins involved in [Ca 2 + ]i signaling ( Fig. 1 ). Thus, fluorescent resonance energy transfer (FRET) measurements in cultured epithelial cells demonstrated a close spatial proximity between the Na,K-ATPase in the plasma membrane and inositol trisphosphate receptor (IP 3R) on the SR. This interaction is further enhanced by ouabain, which induces [Ca 2 + ]i oscillations ( Aizman, Uhlen, Lal, Brismar, & Aperia, 2001 Miyakawa-Naito et al., 2003 ). These oscillations elicit activation of the transcription factor NF-κB and are independent of IP3 generation. The NH2 terminal of the Na,K-ATPase is essential for this interaction with the IP3R ( Miyakawa-Naito et al., 2003 ) and a specific motif is identified ( Zhang et al., 2006 ). It was proposed that binding of ouabain induces E1-to-E2 conformational changes leading to the Na,K-ATPase/IP3R complex formation.

Although these studies ( Aizman et al., 2001 Miyakawa-Naito et al., 2003 ) were originally done in Na,K-ATPase α1 isoform expressing cells, the following co-immunoprecipitation study ( Lencesova, O'Neill, Resneck, Bloch, & Blaustein, 2004 ) demonstrated isoform specificity of the Na-K-ATPase/IP3R complex. This complex was formed with the α1 isoform in epithelial cells and with α3 and α2 isoforms in astrocytes and neurons, respectively. It suggests that proximity of specific isoforms of the Na,K-ATPase to IP3R defines the cell-type-specific [Ca 2 + ]i signal. This protein-protein interaction was not shown in the vasculature. However, a close proximity between the α2 isoform and the SR was reported ( Moore et al., 1993 ) and, therefore, it is likely that similar interactions appear in the vasculature ( Lencesova et al., 2004 ).

A pull-down assay of epithelial cell lysate revealed that the central loop of the Na/K-ATPase α1 isoform interacts with phospholipase Cγ1 (PLCγ1) forming together with the IP3R a Ca 2 + -regulatory complex ( Yuan et al., 2005 ). This suggests that the Na,K-ATPase acts as an important scaffold bringing together IP3Rs to their effector PLCγ1 to facilitate a Ca 2 + response following conformational changes of the Na,K-ATPase. However, the interaction between PLC and the Na,K-ATPase was not shown for the vasculature. This is surprising taking into account the importance of PLC-dependent generation of IP3 for smooth muscle contraction, gene transcription, proliferation and migration ( Narayanan, Adebiyi, & Jaggar, 2012 ). Modulation of this signaling will therefore has important consequences for vascular tone and structure ( Narayanan et al., 2012 ). Notably, both tyrosine kinase activity and ROS are shown to modulate PLC-IP3R signaling in smooth muscle cells. Thus, inhibition of tyrosine kinase phosphorylation reduces PLCγ1 activation and IP3 production in smooth muscle cells ( Marrero, Paxton, Duff, Berk, & Bernstein, 1994 ). Moreover, ROS in smooth muscles suppresses IP3 degradation ( Suzuki & Ford, 1992 ) and increases IP3R affinity in cultured smooth muscle cells ( Bultynck et al., 2004 ) although this is not the case for rabbit mesenteric arteries ( Wada & Okabe, 1997 ). Altogether, this indirectly suggests a possibility for potentiation of PLC-IP3R signaling extending the Na-K-ATPase/IP3R complex to the Na-K-ATPase → Src/ROS → PLC → IP3 → IP3R signal transduction. This hypothesis needs, however, to be validated.


Activation of the Immune System

Darienne R. Myers , Jeroen P. Roose , in Encyclopedia of Immunobiology , 2016

Second Messenger Molecules: Connecting Proximal TCR Signaling to Effector Kinase and Phosphatase Pathways

Many molecular events have been defined to occur when the TCR is ligated by cognate antigen and the IS is formed. In short, the initiation of proximal TCR signaling and the formation of the LAT (linker for activation of T cells) signalosome ultimately lead to the binding and activation of the enzyme phospholipase C gamma 1 (PLCγ1) ( Figure 1 ). The importance of PLCγ1 in a number of thymocyte and T cell processes was demonstrated using mice where PLCγ1 is deleted exclusively in T cells ( Fu et al., 2010 ). Deletion of PLCγ1 results in reduced numbers of thymocytes due to defects in positive and negative selection, and very few T cells populate the secondary lymphoid organs. Those T cells that do make it to lymphoid organs have defects in proliferation and IL-2 production, and the mice develop an autoimmune, inflammatory disease. With respect to biochemical signals, PLCγ1-deficient T cells are impaired in their ability to activate the transcription factors NFAT, NFκB, and AP-1. Of note, PLCγ2 plays a similarly critical role in B lymphocytes ( Wang et al., 2000 ), but we focus on T cells here.

Figure 1 . T cell receptor (TCR) stimulation and proximal signaling events result in the recruitment of PLCγ1 and the generation of second messenger molecules IP3 and DAG. Upon ligation of the TCR by cognate antigen bound to MHC, a number of proximal TCR signaling events (not depicted) are initiated that result in the formation of the LAT signalosome. PLCγ1 can bind to LAT, where it is activated by Tec kinases. Active PLCγ1 hydrolyzes the plasma membrane–associated phospholipid PIP2, leading to the production of two important second messengers: IP3 and DAG. IP3 is a soluble, hydrophilic molecule and diffuses into the cytosol. DAG is a hydrophobic molecule and embeds in the plasma membrane. It is recruited to the IS (immunological synapse), formed at the site of T cell: APC contact.

Figure created by Anna Hupalowska.

How does PLCγ1 connect to activation of NFAT, NFκB, and AP-1? PLCγ1 hydrolyzes the phospholipid PIP2 (phosphatidylinositol 4,5-bisphosphate) at the plasma membrane (PM), generating two important second messenger molecules: the hydrophilic inositol trisphosphate (IP3), which diffuses into the cytosol ( Huang and Sauer, 2010 ), and the hydrophobic diacylglycerol (DAG), which embeds in the PM (and other endomembranes) ( Almena and Merida, 2011 Figure 1 ). IP3 activates calcium (Ca 2+ ) signaling and the transcription factor NFAT, and DAG activates effector proteins such as the protein kinase C family (PKCs) and the Ras guanine nucleotide releasing protein (RasGRP) family members, which can couple to the NFκB and AP-1 pathways, respectively ( Figure 2 ). In the next sections, we will discuss kinase and phosphatase signaling pathways downstream of IP3 and DAG and highlight their role in thymocyte development and T cell activation and function.

Figure 2 . Diacylglycerol (DAG) allows for the recruitment of PKCθ and RasGRP1 via their DAG-binding C1 domains. Depiction of PKCθ and RasGRP1 with their protein domains and highlighting their role in propagating downstream signaling. Both proteins harbor typical C1 domains, which have a conformation that is compatible with binding to DAG. This C1-DAG interaction promotes recruitment of these effectors to the plasma membrane. Membrane-bound PKCθ activates the NFκB signaling pathway. DAG-bound RasGRP1 can interact with Ras and activate Ras-ERK-AP-1 signaling. PKCθ can also phosphorylate RasGRP1 at a threonine residue in the catalytic domain, though the role of this phosphorylation event is still unclear.