G protein coupled receptors that activate phospholipase C

G protein coupled receptors that activate phospholipase C

  • Some G protein coupled receptors activate the inositol phospholipid signaling pathway by activating phospholipase C effector enzyme.
  • Cyclic AMP is not the only second messenger employed by seven transmembrane receptors and the G proteins.
  • The phosphoinositide pathway gives rise to many molecules that have signaling roles.
  • The binding of a hormone such as vasopressin to a 7TM receptor leads to the activation of the β isoform of phospholipase C.
  • Phospholipase C is capable of catalyzing the hydrolysis of PIP2into two important second messenger molecules, which go on to alter cell responses such as proliferation,  differentiation,  apoptosis, cytoskeleton remodeling, vesicular trafficking, ion channel conductance, endocrine function and neurotransmission.

Figure 1: Inositol phospholipid signaling pathway

 Steps involved in the inositol phospholipid signaling pathway

Step 1: The binding of a ligand to GPCR and Receptor activation.

  • The chain of events leading to PIP2 breakdown begins with the binding of a signaling molecule to a G protein linked receptor in the plasma membrane.

Example: Binding of acetylcholine to acetylcholine receptor.

Step 2: Interaction of activated GPCR with G protein and G protein activation

  • There is more than one class of heterotrimeric G proteins that activates PLC enzymes.
  • Most frequently activated receptor stimulates a trimeric G protein called Gq.
  • When GDP is exchanged with GTP, heterotrimeric G protein subunit dissociate.

Step 3: activation of phosphoinositide specific phospholipase C effector enzyme

  • The binding of a G protein brings the enzyme into a catalytically active position.
  • The G protein subunit that activates phospholipase C is called Gαq.

For detailed information on phospholipace C, refer my previous post.



Step 4: hydrolysis of PIP2 to IP3 and DAG by phospholipase C

  • Phospholipase C participates in phosphatidylinositol 4, 5 bisphosphate (PIP2) metabolism and lipid signaling pathways in a calcium dependent manner.
  • Phospholipase C(PLC) is a class of membrane associated enzymes that cleave phospholipids just before the phosphate group.

Cleavage site: hydrolyzes the phosphodiester bond linking the phosphorylated inositol unit to the acylated glycerol moiety.

Figure 3: Phospholipase C cleavage site on PIP2

The cleavage of PIP2 produces two messengers: At this step the signaling pathway splits into two branches.

Figure 3: Synthesis of DAG and IP3 from membrane-bound phosphatidylinositol (PI).

1) DAG (Diacyl glycerol)

  • DAG consists of glycerol and two fatty acids.
  • DAG remains within the inner leaflet of the plasma membrane due to its hydrophobic character, where it recruits protein kinase C (PKC).
  • The principal function of DAG is to activate a family of protein kinases collectively termed protein kinase C (PKC).

DAG has two potential signaling roles.

1) It can be further cleaved to release arachidonic acid which either can act as a messenger in its own right or be used in the synthesis of Eicosanoids.

2) It activates a crucial serine/threonine protein kinase that phosphorylates selected proteins in the target cell.

2) IP3 (Inositol 1,4,5-trisphosphate)

  • IP3 is a negatively charged water soluble molecule that can rapidly diffuse into cytosol to bind with IP3 receptor
  • The production of IP3 is capable of coupling the activated receptor in the plasma membrane to the Ca2+ ions released from an intracellular store.

Step 5: Inositol 1,4,5-trisphosphate opens channels to release calcium Ions from intracellular Stores

  • IP3 is able to increase Ca2+ concentration by associating with a membrane protein called the IP3 -gated channel or IP3 – receptor.
  • This receptor is composed of four large identical subunits which forms an ion channel.
  • Each subunit contains an IP3 binding site in the N-terminal cytosolic domain.
  • IP3 binding induces opening of the channel, allowing Ca2+ ions to exit from the ER into the cytosol.
  • At least three molecules of IP3 must bind to sites on the cytosolic side of the membrane protein to open the channel and release Ca2+
  • The highly cooperative opening of calcium channels by nano molar concentrations of IP3 enables cells to detect and amplify very small changes in the concentration of this messenger.

The basic structure of the IP3 receptor is comprised of three domains:

  1. IP3 binding domain (near the amino terminus)
  2. Coupling domain (in the center of the molecule)
  3. Transmembrane domain (near the carboxyl terminus)
  • Normally cytosolic Ca2+ is kept very low (10 7M) by the action of Ca2+pumps in the ER, mitochondria, and plasma membrane.
  • Hormonal, neural, or other stimuli cause either an influx of Ca2+ into the cell through specific Ca2+ channels in the plasma membrane or the release of sequestered Ca2+ from the ER or mitochondria, in either case raising the cytosolic Ca2+and triggering a cellular response.
  • Ca2+ does not simply rise and then decrease, but rather oscillates with a period of a few seconds even when the extracellular concentration of hormone remains constant.
  • The mechanism underlying Ca2+ oscillations: feedback regulation of either the phospholipase that generates IP3 or the ion channel that regulates Ca2+release from the ER, or both by Ca2+.
  • The IP3 mediated rise in the cytosolic Ca2+ level is only transient because Ca2+ ATPases located in the plasma membrane and ER membrane actively pump Ca2+ from the cytosol to the cell exterior and ER lumen, respectively.
  • Furthermore, within a second of its generation, one specific phosphate on IP3 is hydrolyzed, yielding inositol 1,4 bisphosphate which does not stimulate Ca2+ release from the ER.
  • Higher concentrations of cytosolic Ca2+ inhibit IP3 induced release of Ca2+ from intracellular stores by decreasing the affinity of the receptor for IP3.
  • IP3 gated Ca2+ channels are regulated by positive feedback.
  • Released Ca2+ can bind back to the channels to increase the Ca2+ release, which tends to make the release occur in a sudden, all-or-none fashion.

Step 6: Activation of protein kinase C by DAG and IP3

DAG and IP3 work in tandem: IP3 increases the Ca2+ concentration, and Ca2+ facilitates PKC activation as well as phospholipase C activity.

Figure 4: Activation of Protein kinase C by combined action of DAG and Ca2+

PKC enzyme

  • PKC phosphorylates Ser or Thr residues of specific target proteins, changing their catalytic activities
  • There are a number of isozymes of PKC, each with a characteristic tissue distribution, target protein specificity, and role.
  • In the absence of hormone stimulation, protein kinase C is present as a soluble cytosolic protein that is catalytically inactive.
  • A rise in the cytosolic Ca2+ level causes protein kinase C to bind to the cytosolic leaflet of the plasma membrane, where the membrane associated DAG can activate it.
  • Thus activation of protein kinase C depends on an increase of both Ca2+ ions and DAG, suggesting an interaction between the two branches of the IP3/DAG pathway.
  • The activation of protein kinase C in different cells results in a varied array of cellular responses.
  • It plays a key role in many aspects of cellular growth and metabolism.
  • In liver protein kinase C helps regulate glycogen metabolism by phosphorylating and thus inhibiting glycogen synthase.
  • Protein kinase C also phosphorylates various transcription factors. Depending on the cell type these induce synthesis of mRNAs that trigger cell proliferation.
  • The highest concentrations of PKC are found in the brain, where (among other things) it phosphorylates ion channels in nerve cells, thereby changing their properties and altering the excitability of the nerve cell plasma membrane.
  • In many cells the activation of PKC increases the transcription of specific genes.

Two pathways are there:

First pathway:  PKC activates a protein kinase cascade that leads to the phosphorylation and activation of a DNA-bound gene regulatory protein.

Second pathway: PKC activation leads to the phosphorylation of an inhibitor protein, thereby releasing a cytoplasmic gene regulatory protein so that it can migrate into the nucleus and stimulate the transcription of specific genes.

Activation of PKC occurs in 4 steps: 3 phosphorylation steps+ DAG and Ca2+ binding step

  • Catalytic site on the precursor PKC β is initially accessible to the substrate, but it is inactive.
  • Three phosphorylation steps (Thr 500, Thr 641 and ser 660) make the enzyme catalytically competent but still it remains inactive. Substrate access is prevented by the binding of a pseudo substrate to the catalytic site.
  • Once Ca2+ and DAG binds to the PKC, the enzyme binds firmly to the membrane and the pseudo substrate detaches from the catalytic site.
  • As a result the enzyme becomes competent and accessible to the original substrate.
Figure 5:Protein kinase C activation

Step 7: Termination of IP3 and DAG signal

  • The IP3 signal is terminated by the metabolism of the compound into derivatives lacking second-messenger capabilities.
  • DAG also acts transiently because it is rapidly metabolized. It can be phosphorylated to phosphatidate or it can be hydrolyzed to glycerol and its constituent fatty acids.

Two mechanisms operate to terminate the initial Ca2+ response:  

(1) IP3 is rapidly dephosphorylated (and thereby inactivated) by specific phosphatase.

(2) Ca2+ that enters the cytosol is rapidly pumped out, mainly out of the cell.

How IP3 initiated signal is turned off?

  • IP3 is a short lived messenger because it is rapidly converted into derivatives that do not open the channel
  • Its lifetime in most cells is less than a few
  • IP3 can be degraded to inositol by the sequential action of phosphatases or it can be phosphorylated to inositol 1,3,4,5-tetrakisphosphate, which is then converted into inositol by an alternative route.
  • The enzyme that catalyzes the production of IP4 is activated by the increase in cytosolic Ca2+ induced by IP3 providing a form of negative feedback regulation on IP3

Figure 6: Degradation of IP3 to terminate signaling

Replenishing depleted stores of intracellular Ca2+ ion

  • A plasma membrane Ca2+ channel, called the TRP channel or the store operated channel opens in response to depletion of ER Ca2+
  • When endoplasmic reticulum Ca2+ stores are depleted, the IP3 gated Ca2+ channels binds to and open store operated TRP Ca2+ channels in the plasma membrane allowing influx of extracellular Ca2+.
Figure 7: Replenishing of intracellular Ca2+  ion store by fusion of ion channels.

 Overall decription on inositol phospholipid signaling pathway for better undestanding (Figure 8)

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