Editing G protein-coupled receptor (section)

==Receptor regulation==

GPCRs become desensitized when exposed to their ligand for a long period of time. There are two recognized forms of desensitization: 1) [[homologous desensitization]], in which the activated GPCR is downregulated; and 2) [[heterologous desensitization]], wherein the activated GPCR causes downregulation of a different GPCR. The key reaction of this downregulation is the [[phosphorylation]] of the intracellular (or [[cytoplasm]]ic) receptor domain by [[protein kinase]]s.

===Phosphorylation by cAMP-dependent protein kinases===

Cyclic AMP-dependent protein kinases ([[protein kinase A]]) are activated by the signal chain coming from the G protein (that was activated by the receptor) via [[adenylate cyclase]] and [[cyclic AMP]] (cAMP). In a ''feedback mechanism'', these activated kinases phosphorylate the receptor. The longer the receptor remains active the more kinases are activated and the more receptors are phosphorylated. In [[beta-2 adrenergic receptor|β<sub>2</sub>-adrenoceptor]]s, this phosphorylation results in the switching of the coupling from the G<sub>s</sub> class of G-protein to the [[Gi alpha subunit|G<sub>i</sub>]] class.<ref name="pmid11053129">{{cite journal | vauthors = Chen-Izu Y, Xiao RP, Izu LT, Cheng H, Kuschel M, Spurgeon H, Lakatta EG | title = G(i)-dependent localization of beta(2)-adrenergic receptor signaling to L-type Ca(2+) channels | journal = Biophysical Journal | volume = 79 | issue = 5 | pages = 2547–56 | date = November 2000 | pmid = 11053129 | pmc = 1301137 | doi = 10.1016/S0006-3495(00)76495-2 | bibcode = 2000BpJ....79.2547C }}</ref> cAMP-dependent PKA mediated phosphorylation can cause heterologous desensitisation in receptors other than those activated.<ref name="pmid14744258">{{cite journal | vauthors = Tan CM, Brady AE, Nickols HH, Wang Q, Limbird LE | title = Membrane trafficking of G protein-coupled receptors | journal = Annual Review of Pharmacology and Toxicology | volume = 44 | issue = 1 | pages = 559–609 | year = 2004 | pmid = 14744258 | doi = 10.1146/annurev.pharmtox.44.101802.121558 }}</ref>

===Phosphorylation by GRKs===

The [[G protein-coupled receptor kinases]] (GRKs) are protein kinases that phosphorylate only active GPCRs.<ref name="Trimarco2013">{{cite journal | vauthors = Santulli G, Trimarco B, Iaccarino G | title = G-protein-coupled receptor kinase 2 and hypertension: molecular insights and pathophysiological mechanisms | journal = High Blood Pressure & Cardiovascular Prevention | volume = 20 | issue = 1 | pages = 5–12 | date = March 2013 | pmid = 23532739 | doi = 10.1007/s40292-013-0001-8 | s2cid = 45674941 }}</ref> G-protein-coupled receptor kinases (GRKs) are key modulators of G-protein-coupled receptor (GPCR) signaling. They constitute a family of seven mammalian serine-threonine protein kinases that phosphorylate agonist-bound receptor. GRKs-mediated receptor phosphorylation rapidly initiates profound impairment of receptor signaling and desensitization. Activity of GRKs and subcellular targeting is tightly regulated by interaction with receptor domains, G protein subunits, lipids, anchoring proteins and calcium-sensitive proteins.<ref name="pmid14499340">{{cite journal | vauthors = Penela P, Ribas C, Mayor F | title = Mechanisms of regulation of the expression and function of G protein-coupled receptor kinases | journal = Cellular Signalling | volume = 15 | issue = 11 | pages = 973–81 | date = November 2003 | pmid = 14499340 | doi = 10.1016/S0898-6568(03)00099-8 }}</ref>

Phosphorylation of the receptor can have two consequences:

# ''Translocation'': The receptor is, along with the part of the membrane it is embedded in, brought to the inside of the cell, where it is dephosphorylated within the acidic vesicular environment<ref name="pmid8995214">{{cite journal | vauthors = Krueger KM, Daaka Y, Pitcher JA, Lefkowitz RJ | title = The role of sequestration in G protein-coupled receptor resensitization. Regulation of beta2-adrenergic receptor dephosphorylation by vesicular acidification | journal = The Journal of Biological Chemistry | volume = 272 | issue = 1 | pages = 5–8 | date = January 1997 | pmid = 8995214 | doi = 10.1074/jbc.272.1.5 | doi-access = free }}</ref> and then brought back. This mechanism is used to regulate long-term exposure, for example, to a hormone, by allowing resensitisation to follow desensitisation. Alternatively, the receptor may undergo lysozomal degradation, or remain internalised, where it is thought to participate in the initiation of signalling events, the nature of which depending on the internalised vesicle's subcellular localisation.<ref name="pmid14744258"/>
# ''[[Arrestin]] linking'': The phosphorylated receptor can be linked to ''arrestin'' molecules that prevent it from binding (and activating) G proteins, in effect switching it off for a short period of time. This mechanism is used, for example, with [[rhodopsin]] in [[retina]] cells to compensate for exposure to bright light. In many cases, arrestin's binding to the receptor is a prerequisite for translocation. For example, beta-arrestin bound to β<sub>2</sub>-adrenoreceptors acts as an adaptor for binding with clathrin, and with the beta-subunit of AP2 (clathrin adaptor molecules); thus, the arrestin here acts as a scaffold assembling the components needed for clathrin-mediated endocytosis of β<sub>2</sub>-adrenoreceptors.<ref name="pmid10770944">{{cite journal | vauthors = Laporte SA, Oakley RH, Holt JA, Barak LS, Caron MG | title = The interaction of beta-arrestin with the AP-2 adaptor is required for the clustering of beta 2-adrenergic receptor into clathrin-coated pits | journal = The Journal of Biological Chemistry | volume = 275 | issue = 30 | pages = 23120–6 | date = July 2000 | pmid = 10770944 | doi = 10.1074/jbc.M002581200 | doi-access = free }}</ref><ref name="pmid10097102">{{cite journal | vauthors = Laporte SA, Oakley RH, Zhang J, Holt JA, Ferguson SS, Caron MG, Barak LS | title = The beta2-adrenergic receptor/betaarrestin complex recruits the clathrin adaptor AP-2 during endocytosis | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 96 | issue = 7 | pages = 3712–7 | date = March 1999 | pmid = 10097102 | pmc = 22359 | doi = 10.1073/pnas.96.7.3712 | bibcode = 1999PNAS...96.3712L | doi-access = free }}</ref>

===Mechanisms of GPCR signal termination===

As mentioned above, G-proteins may terminate their own activation due to their intrinsic [[GTPase|GTP→GDP hydrolysis]] capability. However, this reaction proceeds at a slow [[rate constant|rate]] (≈0.02 times/sec) and, thus, it would take around 50 seconds for any single G-protein to deactivate if other factors did not come into play. Indeed, there are around 30 [[protein isoform|isoforms]] of [[regulator of G protein signaling|RGS proteins]] that, when bound to Gα through their [[GTPase activating protein|GAP domain]], accelerate the hydrolysis rate to ≈30 times/sec. This 1500-fold increase in rate allows for the cell to respond to external signals with high speed, as well as spatial [[angular resolution|resolution]] due to limited amount of [[second messenger]] that can be generated and limited distance a G-protein can diffuse in 0.03 seconds. For the most part, the RGS proteins are [[promiscuous]] in their ability to deactivate G-proteins, while which RGS is involved in a given signaling pathway seems more determined by the tissue and GPCR involved than anything else. In addition, RGS proteins have the additional function of increasing the rate of GTP-GDP exchange at GPCRs, (i.e., as a sort of co-GEF) further contributing to the time resolution of GPCR signaling.

In addition, the GPCR may be [[homologous desensitization|desensitized]] itself. This can occur as:

# a direct result of [[receptor theory|ligand occupation]], wherein the change in [[protein conformation|conformation]] allows recruitment of [[G protein-coupled receptor kinase|GPCR-Regulating Kinases]] (GRKs), which go on to [[phosphorylation|phosphorylate]] various [[serine]]/[[threonine]] residues of IL-3 and the [[C-terminal]] tail. Upon GRK phosphorylation, the GPCR's affinity for [[arrestin|β-arrestin]] (β-arrestin-1/2 in most tissues) is increased, at which point β-arrestin may bind and act to both [[sterically]] hinder G-protein coupling as well as initiate the process of [[receptor-mediated endocytosis|receptor internalization]] through [[clathrin-mediated endocytosis]]. Because only the liganded receptor is desensitized by this mechanism, it is called [[homologous desensitization]]
# the affinity for β-arrestin may be increased in a ligand occupation and GRK-independent manner through phosphorylation of different ser/thr sites (but also of IL-3 and the C-terminal tail) by PKC and PKA. These phosphorylations are often sufficient to impair G-protein coupling on their own as well.<ref name="pmid18193069">{{cite journal | vauthors = Tobin AB | title = G-protein-coupled receptor phosphorylation: where, when and by whom | journal = British Journal of Pharmacology | volume = 153 | issue = Suppl 1| pages = S167–76 | date = March 2008 | pmid = 18193069 | pmc = 2268057 | doi = 10.1038/sj.bjp.0707662 }}</ref>
# PKC/PKA may, instead, phosphorylate GRKs, which can also lead to GPCR phosphorylation and β-arrestin binding in an occupation-independent manner. These latter two mechanisms allow for desensitization of one GPCR due to the activities of others, or [[heterologous desensitization]]. GRKs may also have GAP domains and so may contribute to inactivation through non-[[kinase]] mechanisms as well. A combination of these mechanisms may also occur.

Once β-arrestin is bound to a GPCR, it undergoes a conformational change allowing it to serve as a scaffolding protein for an adaptor complex termed [[AP2 adaptors|AP-2]], which in turn recruits another protein called [[clathrin]]. If enough receptors in the local area recruit clathrin in this manner, they aggregate and the [[plasma membrane|membrane]] buds inwardly as a result of interactions between the molecules of clathrin, in a process called [[opsonization]]. Once the pit has been pinched off the [[plasma membrane]] due to the actions of two other proteins called [[amphiphysin]] and [[dynamin]], it is now an [[endocytosis|endocytic]] [[vesicle (biology)|vesicle]]. At this point, the adapter molecules and clathrin have [[dissociated]], and the receptor is either [[protein targeting|trafficked]] back to the plasma membrane or targeted to [[lysosome]]s for [[proteolysis|degradation]].

At any point in this process, the β-arrestins may also recruit other proteins—such as the [[non-receptor tyrosine kinase]] (nRTK), [[Src (gene)|c-SRC]]—which may activate [[extracellular signal-regulated kinases|ERK1/2]], or other [[mitogen-activated protein kinase]] (MAPK) signaling through, for example, phosphorylation of the [[small GTPase]], [[Ras subfamily|Ras]], or recruit the proteins of the [[MAPK/ERK pathway|ERK cascade]] directly (i.e., [[Raf-1]], [[mitogen-activated protein kinase kinase|MEK]], ERK-1/2) at which point signaling is initiated due to their close proximity to one another. Another target of c-SRC are the dynamin molecules involved in endocytosis. Dynamins [[polymerization|polymerize]] around the neck of an incoming vesicle, and their phosphorylation by c-SRC provides the energy necessary for the conformational change allowing the final "pinching off" from the membrane.

===GPCR cellular regulation===
Receptor desensitization is mediated through a combination phosphorylation, β-arr binding, and endocytosis as described above. Downregulation occurs when endocytosed receptor is embedded in an endosome that is trafficked to merge with an organelle called a lysosome. Because lysosomal membranes are rich in proton pumps, their interiors have low pH (≈4.8 vs. the pH≈7.2 cytosol), which acts to denature the GPCRs. In addition, lysosomes contain many [[degradative enzyme]]s, including proteases, which can function only at such low pH, and so the peptide bonds joining the residues of the GPCR together may be cleaved. Whether or not a given receptor is trafficked to a lysosome, detained in endosomes, or trafficked back to the plasma membrane depends on a variety of factors, including receptor type and magnitude of the signal.
GPCR regulation is additionally mediated by gene transcription factors. These factors can increase or decrease gene transcription and thus increase or decrease the generation of new receptors (up- or down-regulation) that travel to the cell membrane.