| GPCR | |||||||||
|---|---|---|---|---|---|---|---|---|---|
 | |||||||||
| Identifiers | |||||||||
| Symbol | 7tm_1 | ||||||||
| Pfam | PF00001 | ||||||||
| Pfam clan | CL0192 | ||||||||
| ECOD | 5001.1.1 | ||||||||
| InterPro | IPR000276 | ||||||||
| PROSITE | PDOC00210 | ||||||||
| TCDB | 9.A.14 | ||||||||
| OPM superfamily | 6 | ||||||||
| OPM protein | 1gzm | ||||||||
| CDD | cd14964 | ||||||||
| |||||||||
G protein-coupled receptors (GPCRs), also known asseven-(pass)-transmembrane domain receptors,7TM receptors,heptahelical receptors,serpentine receptors, andG protein-linked receptors (GPLR), form a largegroup of evolutionarily related proteins that arecell surface receptors that detectmolecules outside thecell and activate cellular responses. They are coupled withG proteins. They pass through thecell membrane seven times in the form of six loops[2] (three extracellular loops interacting withligand molecules, three intracellular loops interacting with G proteins, anN-terminal extracellular region and aC-terminal intracellular region[2]) ofamino acid residues, which is why they are sometimes referred to as seven-transmembrane receptors.[3] Ligands can bind either to the extracellular N-terminus and loops (e.g. glutamate receptors) or to the binding site within transmembrane helices (rhodopsin-like family). They are all activated byagonists, although a spontaneous auto-activation of an empty receptor has also been observed.[3]
G protein-coupled receptors are found only ineukaryotes, includingyeast, andchoanoflagellates.[4] Theligands that bind and activate these receptors include light-sensitive compounds,odors,pheromones,hormones, andneurotransmitters. They vary in size from small molecules topeptides, to largeproteins. G protein-coupled receptors are involved in many diseases.
There are two principal signal transduction pathways involving the G protein-coupled receptors:
When a ligand binds to the GPCR it causes a conformational change in the GPCR, which allows it to act as aguanine nucleotide exchange factor (GEF). The GPCR can then activate an associatedG protein by exchanging theGDP bound to the G protein for aGTP. The G protein's α subunit, together with the bound GTP, can then dissociate from the β and γ subunits to further affect intracellular signaling proteins or target functional proteins directly depending on the α subunit type (Gαs,Gαi/o,Gαq/11,Gα12/13).[6]: 1160
GPCRs are an important drug target, and approximately 34%[7] of all Food and Drug Administration (FDA) approved drugs target 108 members of this family. The global sales volume for these drugs is estimated to be 180 billion US dollars as of 2018[update].[7] It is estimated that GPCRs are targets for about 50% of drugs currently on the market, mainly due to their involvement in signaling pathways related to many diseases i.e. mental, metabolic including endocrinological disorders, immunological including viral infections, cardiovascular, inflammatory, senses disorders, and cancer. The long ago discovered association between GPCRs and many endogenous and exogenous substances, resulting in e.g. analgesia, is another dynamically developing field of the pharmaceutical research.[3]
With the determination of the first structure of the complex between a G-protein coupled receptor (GPCR) and a G-protein trimer (Gαβγ) in 2011 a new chapter of GPCR research was opened for structural investigations of global switches with more than one protein being investigated. The previous breakthroughs involved determination of thecrystal structure of the first GPCR, rhodopsin, in 2000 and the crystal structure of the first GPCR with a diffusible ligand (β2AR) in 2007. The way in which the seven transmembrane helices of a GPCR are arranged into a bundle was suspected based on the low-resolution model of frog rhodopsin fromcryogenic electron microscopy studies of the two-dimensional crystals. The crystal structure of rhodopsin, that came up three years later, was not a surprise apart from the presence of an additional cytoplasmic helix H8 and a precise location of a loop covering retinal binding site. However, it provided a scaffold which was hoped to be a universal template for homology modeling and drug design for other GPCRs – a notion that proved to be too optimistic.[citation needed]
Results 7 years later were surprising because the crystallization of β2-adrenergic receptor (β2AR) with a diffusible ligand revealed quite a different shape of the receptor extracellular side than that of rhodopsin. This area is important because it is responsible for the ligand binding and is targeted by many drugs. Moreover, the ligand binding site was much more spacious than in the rhodopsin structure and was open to the exterior. In the other receptors crystallized shortly afterwards the binding side was even more easily accessible to the ligand. New structures complemented with biochemical investigations uncovered mechanisms of action of molecular switches which modulate the structure of the receptor leading to activation states for agonists or to complete or partial inactivation states for inverse agonists.[3]
The 2012Nobel Prize in Chemistry was awarded toBrian Kobilka andRobert Lefkowitz for their work that was "crucial for understanding how G protein-coupled receptors function".[8] There have been at leastseven other Nobel Prizes awarded for some aspect of G protein–mediated signaling. As of 2012, two of the top ten global best-selling drugs (Advair Diskus andAbilify) act by targeting G protein-coupled receptors.[9]
The exact size of the GPCR superfamily is unknown, but at least 831 differenthumangenes (or about 4% of the entireprotein-codinggenome) have been predicted to code for them from genomesequence analysis.[10][11] Although numerous classification schemes have been proposed, the superfamily was classically divided into three main classes (A, B, and C) with no detectable sharedsequence homology between classes.[citation needed]
The largest class by far is class A, which accounts for nearly 85% of the GPCR genes. Of class A GPCRs, over half of these are predicted to encodeolfactory receptors, while the remaining receptors areliganded by knownendogenouscompounds or are classified asorphan receptors. Despite the lack of sequence homology between classes, all GPCRs have a commonstructure and mechanism ofsignal transduction. The very large rhodopsin A group has been further subdivided into 19 subgroups (A1-A19).[12]
According to the classical A-F system, GPCRs can be grouped into six classes based on sequence homology and functional similarity:[13][14][15][16]
More recently, an alternative classification system calledGRAFS (Glutamate,Rhodopsin,Adhesion,Frizzled/Taste2,Secretin) has been proposed for vertebrate GPCRs.[10] They correspond to classical classes C, A, B2, F, and B.[17]
An early study based on available DNA sequence suggested that the human genome encodes roughly 750 G protein-coupled receptors,[18] about 350 of which detect hormones, growth factors, and other endogenous ligands. Approximately 150 of the GPCRs found in the human genome have unknown functions.
Some web-servers[19] and bioinformatics prediction methods[20][21] have been used for predicting the classification of GPCRs according to their amino acid sequence alone, by means of thepseudo amino acid composition approach.
GPCRs are involved in a wide variety of physiological processes. Some examples of their physiological roles include:
GPCRs areintegral membrane proteins that possess seven membrane-spanning domains ortransmembrane helices.[31][32] The extracellular parts of the receptor can beglycosylated. These extracellular loops also contain two highly conservedcysteine residues that formdisulfide bonds to stabilize the receptor structure. Some seven-transmembrane helix proteins (channelrhodopsin) that resemble GPCRs may contain ion channels, within their protein.[citation needed]
In 2000, the first crystal structure of a mammalian GPCR, that of bovinerhodopsin (1F88), was solved.[33] In 2007, the first structure of a human GPCR was solved[34][1][35] This humanβ2-adrenergic receptor GPCR structure proved highly similar to the bovine rhodopsin. The structures of activated or agonist-bound GPCRs have also been determined.[36][37][38][39] These structures indicate how ligand binding at the extracellular side of a receptor leads to conformational changes in the cytoplasmic side of the receptor. The biggest change is an outward movement of the cytoplasmic part of the 5th and 6th transmembrane helix (TM5 and TM6). The structure of activated beta-2 adrenergic receptor in complex with Gs confirmed that the Gα binds to a cavity created by this movement.[40]
GPCRs exhibit a similar structure to some other proteins with seventransmembrane domains, such asmicrobial rhodopsins and adiponectin receptors 1 and 2 (ADIPOR1 andADIPOR2). However, these 7TMH (7-transmembrane helices) receptors and channels do not associate withG proteins. In addition, ADIPOR1 and ADIPOR2 are oriented oppositely to GPCRs in the membrane (i.e. GPCRs usually have an extracellular N-terminus, cytoplasmic C-terminus, whereas ADIPORs are inverted).[41]
In terms of structure, GPCRs are characterized by an extracellular N-terminus, followed by seventransmembrane (7-TM)α-helices (TM-1 to TM-7) connected by three intracellular (IL-1 to IL-3) and three extracellular loops (EL-1 to EL-3), and finally an intracellular C-terminus. The GPCR arranges itself into atertiary structure resembling a barrel, with the seven transmembrane helices forming a cavity within the plasma membrane that serves aligand-binding domain that is often covered by EL-2. Ligands may also bind elsewhere, however, as is the case for bulkier ligands (e.g.,proteins or largepeptides), which instead interact with the extracellular loops, or, as illustrated by the class Cmetabotropic glutamate receptors (mGluRs), the N-terminal tail. The class C GPCRs are distinguished by their large N-terminal tail, which also contains a ligand-binding domain. Upon glutamate-binding to an mGluR, the N-terminal tail undergoes a conformational change that leads to its interaction with the residues of the extracellular loops and TM domains. The eventual effect of all three types ofagonist-induced activation is a change in the relative orientations of the TM helices (likened to a twisting motion) leading to a wider intracellular surface and "revelation" of residues of the intracellular helices and TM domains crucial to signal transduction function (i.e., G-protein coupling).Inverse agonists andantagonists may also bind to a number of different sites, but the eventual effect must be prevention of this TM helix reorientation.[3]
The structure of the N- and C-terminal tails of GPCRs may also serve important functions beyond ligand-binding. For example, The C-terminus of M3 muscarinic receptors is sufficient, and the six-amino-acid polybasic (KKKRRK) domain in the C-terminus is necessary for its preassembly with Gq proteins.[42] In particular, the C-terminus often containsserine (Ser) orthreonine (Thr) residues that, whenphosphorylated, increase theaffinity of the intracellular surface for the binding of scaffolding proteins called β-arrestins (β-arr).[43] Once bound, β-arrestins bothsterically prevent G-protein coupling and may recruit other proteins, leading to the creation of signaling complexes involved in extracellular-signal regulated kinase (ERK) pathway activation or receptorendocytosis (internalization). As the phosphorylation of these Ser and Thr residues often occurs as a result of GPCR activation, the β-arr-mediated G-protein-decoupling and internalization of GPCRs are important mechanisms ofdesensitization.[44] In addition, internalized "mega-complexes" consisting of a single GPCR, β-arr(in the tail conformation),[45][46] and heterotrimeric G protein exist and may account for protein signaling from endosomes.[47][48]
A final common structural theme among GPCRs ispalmitoylation of one or more sites of the C-terminal tail or the intracellular loops. Palmitoylation is the covalent modification ofcysteine (Cys) residues via addition of hydrophobicacyl groups, and has the effect of targeting the receptor tocholesterol- andsphingolipid-rich microdomains of the plasma membrane calledlipid rafts. As many of the downstream transducer and effector molecules of GPCRs (including those involved innegative feedback pathways) are also targeted to lipid rafts, this has the effect of facilitating rapid receptor signaling.[citation needed]
GPCRs respond to extracellular signals mediated by a huge diversity of agonists, ranging from proteins tobiogenic amines toprotons, but all transduce this signal via a mechanism of G-protein coupling. This is made possible by aguanine-nucleotide exchange factor (GEF) domain primarily formed by a combination of IL-2 and IL-3 along with adjacent residues of the associated TM helices.[citation needed]
The G protein-coupled receptor is activated by an external signal in the form of a ligand or other signal mediator. This creates a conformational change in the receptor, causing activation of aG protein. Further effect depends on the type of G protein. G proteins are subsequently inactivated by GTPase activating proteins, known asRGS proteins.[citation needed]
GPCRs include one or more receptors for the following ligands:sensory signal mediators (e.g., light andolfactory stimulatory molecules);adenosine,bombesin,bradykinin,endothelin, γ-aminobutyric acid (GABA), hepatocyte growth factor (HGF),melanocortins,neuropeptide Y,opioid peptides,opsins,somatostatin,GH,tachykinins, members of thevasoactive intestinal peptide family, andvasopressin;biogenic amines (e.g.,dopamine,epinephrine,norepinephrine,histamine,serotonin, andmelatonin);glutamate (metabotropic effect);glucagon;acetylcholine (muscarinic effect);chemokines;lipid mediators ofinflammation (e.g.,prostaglandins,prostanoids,platelet-activating factor, andleukotrienes);peptide hormones (e.g.,calcitonin, C5aanaphylatoxin,follicle-stimulating hormone [FSH],gonadotropin-releasing hormone [GnRH],neurokinin,thyrotropin-releasing hormone [TRH], andoxytocin);andendocannabinoids.
GPCRs that act as receptors for stimuli that have not yet been identified are known asorphan receptors.[citation needed]
However, in contrast to other types of receptors that have been studied, wherein ligands bind externally to the membrane, theligands of GPCRs typically bind within the transmembrane domain. However,protease-activated receptors are activated by cleavage of part of their extracellular domain.[50]
Thetransduction of the signal through the membrane by the receptor is not completely understood. It is known that in the inactive state, the GPCR is bound to aheterotrimeric G protein complex. Binding of an agonist to the GPCR results in aconformational change in the receptor that is transmitted to the bound Gα subunit of the heterotrimeric G protein viaprotein domain dynamics. The activated Gα subunit exchangesGTP in place ofGDP which in turn triggers the dissociation of Gα subunit from the Gβγ dimer and from the receptor. The dissociated Gα and Gβγ subunits interact with other intracellular proteins to continue the signal transduction cascade while the freed GPCR is able to rebind to another heterotrimeric G protein to form a new complex that is ready to initiate another round of signal transduction.[51]
It is believed that a receptor molecule exists in a conformationalequilibrium between active and inactive biophysical states.[52] The binding of ligands to the receptor may shift the equilibrium toward the active receptor states. Three types of ligands exist: Agonists are ligands that shift the equilibrium in favour of active states;inverse agonists are ligands that shift the equilibrium in favour of inactive states; and neutral antagonists are ligands that do not affect the equilibrium. It is not yet known how exactly the active and inactive states differ from each other.[citation needed]
When the receptor is inactive, theGEF domain may be bound to an also inactive α-subunit of aheterotrimeric G-protein. These "G-proteins" are atrimer of α, β, and γ subunits (known as Gα, Gβ, and Gγ, respectively) that is rendered inactive when reversibly bound toGuanosine diphosphate (GDP) (or, alternatively, no guanine nucleotide) but active when bound toguanosine triphosphate (GTP). Upon receptor activation, the GEF domain, in turn,allosterically activates the G-protein by facilitating the exchange of a molecule of GDP for GTP at the G-protein's α-subunit. The cell maintains a 10:1 ratio of cytosolic GTP:GDP so exchange for GTP is ensured. At this point, the subunits of the G-protein dissociate from the receptor, as well as each other, to yield a Gα-GTPmonomer and a tightly interactingGβγ dimer, which are now free to modulate the activity of other intracellular proteins. The extent to which they maydiffuse, however, is limited due to thepalmitoylation of Gα and the presence of anisoprenoid moiety that has beencovalently added to the C-termini of Gγ.[citation needed]
Because Gα also has slowGTP→GDP hydrolysis capability, the inactive form of the α-subunit (Gα-GDP) is eventually regenerated, thus allowing reassociation with a Gβγ dimer to form the "resting" G-protein, which can again bind to a GPCR and await activation. The rate of GTP hydrolysis is often accelerated due to the actions of another family of allosteric modulating proteins calledregulators of G-protein signaling, or RGS proteins, which are a type ofGTPase-activating protein, or GAP. In fact, many of the primaryeffector proteins (e.g.,adenylate cyclases) that become activated/inactivated upon interaction with Gα-GTP also have GAP activity. Thus, even at this early stage in the process, GPCR-initiated signaling has the capacity for self-termination.[citation needed]
GPCRs downstream signals have been shown to possibly interact withintegrin signals, such asFAK.[53] Integrin signaling will phosphorylate FAK, which can then decrease GPCR Gαs activity.
If a receptor in an active state encounters aG protein, it may activate it. Some evidence suggests that receptors and G proteins are actually pre-coupled.[42] For example, binding of G proteins to receptors affects the receptor's affinity for ligands. Activated G proteins are bound toGTP.
Further signal transduction depends on the type of G protein. The enzymeadenylate cyclase is an example of a cellular protein that can be regulated by a G protein, in this case the G proteinGs. Adenylate cyclase activity is activated when it binds to a subunit of the activated G protein. Activation of adenylate cyclase ends when the G protein returns to theGDP-bound state.
Adenylate cyclases (of which 9 membrane-bound and one cytosolic forms are known in humans) may also be activated or inhibited in other ways (e.g., Ca2+/calmodulin binding), which can modify the activity of these enzymes in an additive or synergistic fashion along with the G proteins.
The signaling pathways activated through a GPCR are limited by theprimary sequence andtertiary structure of the GPCR itself but ultimately determined by the particularconformation stabilized by a particularligand, as well as the availability oftransducer molecules. Currently, GPCRs are considered to utilize two primary types of transducers:G-proteins andβ-arrestins. Because β-arr's have highaffinity only to thephosphorylated form of most GPCRs (see above or below), the majority of signaling is ultimately dependent upon G-protein activation. However, the possibility for interaction does allow for G-protein-independent signaling to occur.
There are three main G-protein-mediated signaling pathways, mediated by foursub-classes of G-proteins distinguished from each other bysequence homology (Gαs,Gαi/o,Gαq/11, andGα12/13). Each sub-class of G-protein consists of multiple proteins, each the product of multiplegenes orsplice variations that may imbue them with differences ranging from subtle to distinct with regard to signaling properties, but in general they appear reasonably grouped into four classes. Because the signal transducing properties of the various possibleβγ combinations do not appear to radically differ from one another, these classes are defined according to the isoform of their α-subunit.[6]: 1163
While most GPCRs are capable of activating more than one Gα-subtype, they also show a preference for one subtype over another. When the subtype activated depends on the ligand that is bound to the GPCR, this is calledfunctional selectivity (also known as agonist-directed trafficking, or conformation-specific agonism). However, the binding of any single particularagonist may also initiate activation of multiple different G-proteins, as it may be capable of stabilizing more than one conformation of the GPCR'sGEF domain, even over the course of a single interaction. In addition, a conformation that preferably activates oneisoform of Gα may activate another if the preferred is less available. Furthermore,feedback pathways may result inreceptor modifications (e.g., phosphorylation) that alter the G-protein preference. Regardless of these various nuances, the GPCR's preferred coupling partner is usually defined according to the G-protein most obviously activated by theendogenous ligand under mostphysiological orexperimental conditions.
The above descriptions ignore the effects ofGβγ–signalling, which can also be important, in particular in the case of activated Gαi/o-coupled GPCRs. The primary effectors of Gβγ are various ion channels, such asG-protein-regulated inwardly rectifying K+ channels (GIRKs),P/Q- andN-typevoltage-gated Ca2+ channels, as well as some isoforms of AC and PLC, along with somephosphoinositide-3-kinase (PI3K) isoforms.
Although they are classically thought of working only together, GPCRs may signal through G-protein-independent mechanisms, and heterotrimeric G-proteins may play functional roles independent of GPCRs. GPCRs may signal independently through many proteins already mentioned for their roles in G-protein-dependent signaling such asβ-arrs,GRKs, andSrcs. Such signaling has been shown to be physiologically relevant, for example,β-arrestin signaling mediated by the chemokine receptorCXCR3 was necessary for full efficacy chemotaxis of activated T cells.[54] In addition, further scaffolding proteins involved insubcellular localization of GPCRs (e.g.,PDZ-domain-containing proteins) may also act as signal transducers. Most often the effector is a member of theMAPK family.
In the late 1990s, evidence began accumulating to suggest that some GPCRs are able to signal without G proteins. TheERK2 mitogen-activated protein kinase, a key signal transduction mediator downstream of receptor activation in many pathways, has been shown to be activated in response to cAMP-mediated receptor activation in theslime moldD. discoideum despite the absence of the associated G protein α- and β-subunits.[55]
In mammalian cells, the much-studied β2-adrenoceptor has been demonstrated to activate the ERK2 pathway after arrestin-mediated uncoupling of G-protein-mediated signaling. Therefore, it seems likely that some mechanisms previously believed related purely to receptor desensitisation are actually examples of receptors switching their signaling pathway, rather than simply being switched off.
In kidney cells, thebradykinin receptor B2 has been shown to interact directly with a protein tyrosine phosphatase. The presence of a tyrosine-phosphorylatedITIM (immunoreceptor tyrosine-based inhibitory motif) sequence in the B2 receptor is necessary to mediate this interaction and subsequently the antiproliferative effect of bradykinin.[56]
Although it is a relatively immature area of research, it appears that heterotrimeric G-proteins may also take part in non-GPCR signaling. There is evidence for roles as signal transducers in nearly all other types of receptor-mediated signaling, includingintegrins,receptor tyrosine kinases (RTKs),cytokine receptors (JAK/STATs), as well as modulation of various other "accessory" proteins such asGEFs,guanine-nucleotide dissociation inhibitors (GDIs) andprotein phosphatases. There may even be specific proteins of these classes whose primary function is as part of GPCR-independent pathways, termed activators of G-protein signalling (AGS). Both the ubiquity of these interactions and the importance of Gα vs. Gβγ subunits to these processes are still unclear.
There are two principal signal transduction pathways involving theG protein-linked receptors: thecAMP signal pathway and thephosphatidylinositol signal pathway.[5]
The cAMP signal transduction contains five main characters: stimulativehormone receptor (Rs) or inhibitoryhormone receptor (Ri); stimulative regulative G-protein (Gs) or inhibitory regulative G-protein (Gi);adenylyl cyclase;protein kinase A (PKA); and cAMPphosphodiesterase.
Stimulative hormone receptor (Rs) is a receptor that can bind with stimulative signal molecules, while inhibitory hormone receptor (Ri) is a receptor that can bind with inhibitory signal molecules.
Stimulative regulative G-protein is a G-protein linked to stimulative hormone receptor (Rs), and its α subunit upon activation could stimulate the activity of an enzyme or other intracellular metabolism. On the contrary, inhibitory regulative G-protein is linked to an inhibitory hormone receptor, and its α subunit upon activation could inhibit the activity of an enzyme or other intracellular metabolism.
Adenylyl cyclase is a 12-transmembrane glycoprotein that catalyzes the conversion of ATP to cAMP with the help of cofactor Mg2+ or Mn2+. The cAMP produced is a second messenger in cellular metabolism and is an allosteric activator of protein kinase A.
Protein kinase A is an important enzyme in cell metabolism due to its ability to regulate cell metabolism by phosphorylating specific committed enzymes in the metabolic pathway. It can also regulate specific gene expression, cellular secretion, and membrane permeability. The protein enzyme contains two catalytic subunits and two regulatory subunits. When there is no cAMP, the complex is inactive. When cAMP binds to the regulatory subunits, their conformation is altered, causing the dissociation of the regulatory subunits, which activates protein kinase A and allows further biological effects.
These signals then can be terminated by cAMP phosphodiesterase, which is an enzyme that degrades cAMP to 5'-AMP and inactivates protein kinase A.
In thephosphatidylinositol signal pathway, the extracellular signal molecule binds with the G-protein receptor (Gq) on the cell surface and activatesphospholipase C, which is located on theplasma membrane. Thelipase hydrolyzesphosphatidylinositol 4,5-bisphosphate (PIP2) into two second messengers:inositol 1,4,5-trisphosphate (IP3) anddiacylglycerol (DAG). IP3 binds with theIP3 receptor in the membrane of the smooth endoplasmic reticulum and mitochondria to open Ca2+ channels. DAG helps activateprotein kinase C (PKC), which phosphorylates many other proteins, changing their catalytic activities, leading to cellular responses.
The effects of Ca2+ are also remarkable: it cooperates with DAG in activating PKC and can activate theCaM kinase pathway, in which calcium-modulated proteincalmodulin (CaM) binds Ca2+, undergoes a change in conformation, and activates CaM kinase II, which has unique ability to increase its binding affinity to CaM by autophosphorylation, making CaM unavailable for the activation of other enzymes. The kinase then phosphorylates target enzymes, regulating their activities. The two signal pathways are connected together by Ca2+-CaM, which is also a regulatory subunit of adenylyl cyclase and phosphodiesterase in the cAMP signal pathway.
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 thephosphorylation of the intracellular (orcytoplasmic) receptor domain byprotein 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) viaadenylate cyclase andcyclic AMP (cAMP). In afeedback mechanism, these activated kinases phosphorylate the receptor. The longer the receptor remains active the more kinases are activated and the more receptors are phosphorylated. Inβ2-adrenoceptors, this phosphorylation results in the switching of the coupling from the Gs class of G-protein to theGi class.[57] cAMP-dependent PKA mediated phosphorylation can cause heterologous desensitisation in receptors other than those activated.[58]
TheG protein-coupled receptor kinases (GRKs) are protein kinases that phosphorylate only active GPCRs.[59] 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.[60]
Phosphorylation of the receptor can have two consequences:
As mentioned above, G-proteins may terminate their own activation due to their intrinsicGTP→GDP hydrolysis capability. However, this reaction proceeds at a slowrate (≈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 30isoforms ofRGS proteins that, when bound to Gα through theirGAP 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 spatialresolution due to limited amount ofsecond messenger that can be generated and limited distance a G-protein can diffuse in 0.03 seconds. For the most part, the RGS proteins arepromiscuous 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 bedesensitized itself. This can occur as:
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 termedAP-2, which in turn recruits another protein calledclathrin. If enough receptors in the local area recruit clathrin in this manner, they aggregate and themembrane buds inwardly as a result of interactions between the molecules of clathrin, in a process calledopsonization. Once the pit has been pinched off theplasma membrane due to the actions of two other proteins calledamphiphysin anddynamin, it is now anendocyticvesicle. At this point, the adapter molecules and clathrin havedissociated, and the receptor is eithertrafficked back to the plasma membrane or targeted tolysosomes fordegradation.
At any point in this process, the β-arrestins may also recruit other proteins—such as thenon-receptor tyrosine kinase (nRTK),c-SRC—which may activateERK1/2, or othermitogen-activated protein kinase (MAPK) signaling through, for example, phosphorylation of thesmall GTPase,Ras, or recruit the proteins of theERK cascade directly (i.e.,Raf-1,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. Dynaminspolymerize 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.
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 manydegradative enzymes, 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.
G-protein-coupled receptor oligomerisation is a widespread phenomenon. One of the best-studied examples is the metabotropicGABAB receptor. This so-called constitutive receptor is formed by heterodimerization ofGABABR1 andGABABR2 subunits. Expression of the GABABR1 without the GABABR2 in heterologous systems leads to retention of the subunit in theendoplasmic reticulum. Expression of the GABABR2 subunit alone, meanwhile, leads to surface expression of the subunit, although with no functional activity (i.e., the receptor does not bind agonist and cannot initiate a response following exposure to agonist). Expression of the two subunits together leads to plasma membrane expression of functional receptor. It has been shown that GABABR2 binding to GABABR1 causes masking of a retention signal[65] of functional receptors.[66]
Signal transduction mediated by the superfamily of GPCRs dates back to the origin ofmulticellularity. Mammalian-like GPCRs are found infungi, and have been classified according to theGRAFS classification system based on GPCR fingerprints.[17] Identification of the superfamily members across theeukaryotic domain, and comparison of the family-specific motifs, have shown that the superfamily of GPCRs have a common origin.[67] Characteristic motifs indicate that three of the five GRAFS families,Rhodopsin,Adhesion, andFrizzled, evolved from theDictyostelium discoideum cAMP receptors before the split ofopisthokonts. Later, theSecretin family evolved from theAdhesion GPCR receptor family before the split ofnematodes.[17] Insect GPCRs appear to be in their own group and Taste2 is identified as descending fromRhodopsin.[67] Note that theSecretin/Adhesion split is based on presumed function rather than signature, as the classical Class B (7tm_2,PfamPF00002) is used to identify both in the studies.
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{{cite journal}}: CS1 maint: article number as page number (link){{cite book}}: CS1 maint: multiple names: authors list (link)Reference for molecular and mathematical models for the initial receptor response
Data, diagrams and web tools for G protein-coupled receptors (GPCRs).;Munk C, Isberg V, Mordalski S, Harpsøe K, Rataj K, Hauser AS, et al. (July 2016)."GPCRdb: the G protein-coupled receptor database - an introduction".British Journal of Pharmacology.173 (14):2195–207.doi:10.1111/bph.13509.PMC 4919580.PMID 27155948.
a classification of GPCRs
a Protein Structure Initiative:Biology Network Center aimed at determining the 3D structures of representative GPCR family proteins