Chrivia GPCR lectures 2 2024.pdf

GPCR lectures Part 2 Heterotrimeric G-proteins G proteins are designated by their alpha chains so a G protein with αs is Called Gs. The alpha chain binds GDP in the inactivated state and GTP in the activated state Gα subunits Response Gs Adenylate cyclase Gq PIP2 (Phosphtidylinositol 4,5 bis phospha...

GPCR lectures Part 2 Heterotrimeric G-proteins G proteins are designated by their alpha chains so a G protein with αs is Called Gs. The alpha chain binds GDP in the inactivated state and GTP in the activated state Gα subunits Response Gs Adenylate cyclase Gq PIP2 (Phosphtidylinositol 4,5 bis phosphate) hydrolysis Gi Inhibits adenylate cyclase Gb and Gg subunits The different alphas (with GTP bound) relay the signal to a different effector to give different responses Synthesis and Hydrolysis of cAMP cAMP is synthesized from ATP by the enzyme adenylyl cyclase. cAMP is broken down to AMP via the enzyme cAMP phosphodiesterase. GPCRs that Regulate Adenylyl Cyclase Adenylyl cyclase is an effector enzyme that synthesizes cAMP. GaGTP subunits bind to the catalytic domains of the cyclase, regulating their activity. Gas-GTP activates the catalytic domains, whereas Gai-GTP inhibits them. A given cell type can express multiple types of GPCRs that all couple to adenylyl cyclase. The net activity of adenylyl cyclase thus depends on the combined level of G protein signaling via the multiple GPCRs. Adenylyl Cyclase Adenylyl cyclase is an integral membrane protein that contains 12 transmembrane segments. It also has 2 cytoplasmic domains that together form the catalytic site for synthesis of cAMP from ATP. Knock out of isoforms of Adenylate cyclase Alters specific physiological functions Table 2. Tissue distribution and physiological functions of individual mammalian isoforms AC isoforms Site of expression Availability of Physiological functions knockout overexpression AC1 Brain, adrenal medulla Yes Learning, memory, synaptic plasticity, opiate withdrawal AC2 Brain, lung, skeletal muscle, heart AC3 Olfactory epithelium, pancreas, brain, heart, lung, testis, BAT Yes Olfaction, sperm function AC4 Widespread AC5 Heart, striatum, kidney, liver, lung, testis, adrenal, BAT Yes Yes1 Cardiac contraction, motor coordination, opiate dependency, pain responses AC6 Heart, kidney, liver, lung, brain, testis, skeletal muscle, adrenal, BAT Yes Yes1 Cardiac contraction and calcium sensitivity AC7 Widespread Yes1 Ethanol dependency AC8 Brain, lung, pancreas, testis, adrenal Yes Yes1 Learning, memory, synaptic plasticity, opiate withdrawal AC9 Widespread Yes2 sAC Testis and detected in all tissues Yes Sperm capacitation, fertilization Sites of expression for all mammalian isoforms of AC have previously been expertly reviewed in detail [57, 176]. Expression pat- Gas and Gai bind at distinct sites on Adenylate cyclase Table 1. Regulatory properties of transmembrane adenylyl cyclase (AC) isoforms AC isoform G protein regulators a Protein kinases stimulatory inhibitory stimulatory Group I AC1 AC8 AC3 Gs! Gs! Gs! G! i, z, o, G"# G"# G"# PKC! (weak) Group II AC2 AC4 AC7 Gs!, G"# Gs!, G"# Gs!, G"# Group III AC5 AC6 Gs!, G"# Gs!, G"# Group IV AC9 Gs! PKC! (weak) PKC! 12 12 12 5 CaMK II RGS2 b Other C1 33 7 1 11 11 2 4 d CaM CaMK IV 1b CC1b Calcium N d CaM d CaM* Gi! f PKC! PKC! G! i, z G! i, z 9 8 10 inhibitory 6 PAM PP2A PAM C2 PKC (!, $) C1 PKA PKA, PKC (%, &) f free Ca2+ f free Ca2+ PKC f via calcineurin f f PAM, Ric8a PAM, Snapin Gs! C2 Low affinity binding of C1to C2 Gives catalytic site with low Fig. 1. Structure of adenylyl cyclase. a Crystal structure of cyto- tor (dark blue). Membrane spans are modeled from the 12-memnearly identical tertiary structures, as predicted from Classification ofGTP Isoforms plasmic domains of AC in complex with "S-Gs!, forskolin brane spanning transporters. b Alternate view from cytoactivity e.g. produces amount their sequence similarities,low despite the fact that these (FSK) and P-site inhibitor, 2!5!-dideoxy-3!ATP. Shown are plasmic side, showing forskolin and catalytic site more clearly. (rust), Gs! (green), FSK (cyan),ACs and P-site inhibiInteraction site of Gi!into with C1four domain is indicated by an arrow. C1 (yellow), C2Membrane-bound structures were solved of witheffector a C1 domain from type 5 AC are often classified cAMP in absence and a C2 domain from type 2 AC. The pseudosymmetry different categories based on regulatory properties. Gas increases affinity of the C2domain interface, a Group I consists of Ca2+-stimulated AC 1, 3 and 8; group creates two related sites along Forskolin also increasesACC1/C2 10x site and acAMP related forskolin 2, 4 and 7;interaction group III is forsubstrate-binding C1 10x increasing made site. Both II consists of G"#-stimulated pockets are well defined and are structurally related. of Gicharacterization. !/Ca 2+together -inhibited AC5 and 6, Topology while and Structure protein comprised for and detailed biochemical The Adenylyl Cyclases: Gas forskolin -100x Gai binds C1 and decrease affinity There are notable differences between the C1a and C2 group IV contains forskolin-insensitive AC9 ( table 1). expression of the two catalytic domains of AC in Esche- Gi binding inhibits despite forskolin activation richia coli largelythat solvedalthough this issue andsignificant resulted in suffi-sequence Mammalian transmembrane structures, particularly comparing the regions that play Note homology ex- ACs share a similar tocient protein for biochemical, kinetic, and structural pology of a variable N-terminus an important role in the binding of Gs! (C2 domain) or ists within members of groups II and III, members of (NT) and two repeats of studies. group I are more distantly related a membrane-spanning domain followed by a cytoplasmic Gbg bind to C2ofdecrease affinity in the formation a P-loop structure that binds pyro. This is reflected made in the identification and for domain. The overall topology is very reminiscent of phosphate in the active site (C1 domain). It is of note thatDespite in the theprogress overall regulatory patterns the various groups and to C1/C2 to increase affinity Activation of Gene Transcription by GPCR Signaling GPCRs regulate gene transcription by cAMP and PKA signaling. cAMP-released PKA catalytic domains enter the nucleus and phosphorylate the CREB (CREbinding) protein, which binds to CRE (cAMP-response element) sequences upstream of cAMPregulated genes. p-CREB interacts with CBP/p300 to help assemble the RNA Pol II transcription machinery at these promoters. CREB, CREM, ATF1 And splice variants bind different promoters GPCRs That Activate Phospholipase C Another common GPCR signaling pathway involves the activation of phospholipase C (PLCb). This enzyme cleaves the membrane lipid, phosphatidylinositol 4,5-bisphosphate (PIP2) to the second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). In this case, the Gaq proteins conducts the signal from the GPCR to PLCb. This is the pathway used in a1-adrenergic GPCR signaling in the liver. * IP3/DAG Signaling Elevates Cytosolic Ca2+ The steps downstream of PLC that make up the IP3/DAG signaling pathway. IP3 diffuses from the cytoplasmic membrane to the smooth ER where it binds to and triggers the opening of IP3-gated Ca2+ channels. Another kinase, protein kinase C (PKC) binds to DAG and calcium in the cytoplasmic membrane and is activated. phosphatidic acid IP2 IP4 FIGURE 2. The cell-surface receptors responsible for InsP3 formation belong to two main classes, the G protein-coupled receptors (GPCRs) and the protein tyrosine kinase-linked receptors (PTKRs) that are coupled to different phospholipase C (PLC) isoforms. The the GPCRs use the PLC! isoforms, whereas the receptor tyrosine kinases (RTKs) are coupled to the PLC-" isoforms. During the transduction process, the precursor lipid PtdIns4,5P2 is hydrolyzed by PLC to produce both InsP3 and diacylglycerol (DAG). The InsP3 released from the membrane diffuses into the cytosol where it engages the InsP3 receptors (InsP3Rs) to release Ca2! from the endoplasmic reticulum. The Ca2!-mobilizing function of InsP3 is terminated through its metabolism by either InsP3 3-kinase or InsP3 5-phosphatase. The resulting InsP2 and InsP4 enter an inositol phosphate metabolic pathway and are recycled back to free inositol. The DAG is recycled back to the precursor CDP-DAG, which then combines with inositol to reform the phosphatidylinositol (PtdIns) that is returned to the plasma membrane to be phosphorylated to the PtdIns4,5P2 precursor to maintain the InsP3 signaling pathway. A key component of this metabolic pathway is the inositol monophosphatase (IMPase) that hydrolyzes InsP1 to free inositol. This IMPase is inhibited by lithium (Li!), which thus acts to reduce the supply of inositol resulting in a decline in the activity of the InsP3/Ca2! signaling pathway. The action of InsP3 is terminated by either by conversion to InsP3 3-kinase or InsP3 5-phosphatase. The resulting InsP2 and InsP4 are recycled back to free inositol. The DAG is recycled back to the precursor CDP-DAG, which then combines with inositol to reform the phosphatidylinositol (PtdIns) that is returned to the plasma membrane to be phosphorylated to the PtdIns 4,5P2 precursor to maintain the InsP3 signaling pathway. Some Galpha subunits activate Rho GEFS which activate RhoA RhoA is a member of the Rho GTPase family and serves as an intracellular molecular switch, cycling between a GTP-bound active form and a GDP-bound inactive form. G12/13 p115 RhoGEF LARG RhoGEF PDZ RhoGEF RhoA Gq p63 RhoGEF RhoA NOTE: you do not need to Memorize these details c-Jun N-terminal kinases (JNKs) Rho-associated protein kinase (ROCK) YAP is a transcriptional coactivator Fig. in Rh Cox-2 is the inducible form of cyclooxygenase and catalyzes the conversion of arachidonic acid to prostaglandins. CCN 1/2 are adaptor molecules connecting the cell surface to extracellular matrix. They bind and they signal thru integrins and proteoglycans. Well-defined G effectors Other G interacting proteins Adenylate cyclase (+), Phospholipase C- (+), Phosphoinositide 3 Btk-family tyrosine kinase, IP3 receptors, Raf kinase, Protein kinase D, Kinases, G protein-coupled receptor kinases, K+ and Ca2+ channels Histone deacetylase 5 (HDAC5), Tubulin, F-actin, Vinculin, ElmoE, Rab11, mitofusinl, Radil, activator protein 1, TFE3, TRPM1 Adenylate cyclase (-) GPCRs that Regulate Ion Channels: Muscarinic Acetylcholine Receptor The binding of ACH to this receptor triggers dissociation of Gai-GTP from Gßg, which in this case, directly binds to and opens a K+ channel. The movement of K+ down its concentration gradient to the outside of the cell, increases the positive charge outside the membrane, hyperpolarizing cells in SA node. This results in the slowing of heart rate. A number of events contribute to the termination of signaling by a GPCR. Example: Galpha s signaling 1. Decrease in cAMP levels a. Decrease AC activity b. Hydrolysis of cAMP via cAMP phosphodiesterase c. Reassembly of PKA holoenzyme-C d. RGS proteins i. Hydrolysis of GTP by Gas enhanced by GAPS ii. GDIs block dissociation of GDP 2. Decrease transcription a. Dephosphorylation of CREB by protein phosphatase 1 b. Dissociation of CBP from CREB and decreased transcription 2. Desensitization of receptor and receptor down regulation a. Phosphorylation of receptor by kinases such as PKA and GRKs This allows binding of b-arrestin and GPCRs can be removed from the membrane by endocytosis 85, 86, 87, 88]. However, the role of the RGS-box in LARG/PDZ-RhoGEF signaling to RhoA activation ptor signaling. non-7TM receptors remains ill-defined. Superfamily of RGS (“regulator Figure 4. Membrane targeting strategies employed by multi-domain RGS proteins. (A) The R7 RGS prot G-protein signaling”) heterodimers with G 5 via a G -like sequence (the “GGL” domain) of N-terminal to the RGS-box. This GG could allow R7 RGS proteins to act as conventional G subunits inproteins coupling G to 7TM receptors, thatsubunits bind Gα subunits RGS-box-mediated GAP activity to particular receptors. The DEP domain of RGS9-1 interacts with a me via a hallmark amino-acid protein (R9AP) ; analogous interactors may exist for the DEP domains of other R7~120 subfamily members [8 domain of RGS12 is able to bind the C-terminus of the IL-8 receptor CXCR2 (at least in vitro). The RGS12 “RGS-box” domain the synprint (“synaptic protein interaction”) region of the N-type calcium channel (Cav2.2); this interaction dramatically accelerating neurotransmitter-mediated phosphorylation of the channel by Src. (C) The AtRGS1 protein oftheir Arabidop cress) has a unique structure for an RGS protein: an N-terminus resembling 7TM receptor and aand C-termin intrinsic aGTPase activity Although a ligand is not known for the 7TM portion of AtRGS1, a simple sugar is most likely. (D) The transm thus attenuating Plexin-B1 couples binding of the membrane-bound semaphorin Sema4D to RhoA activationheterotrimervia an interaction wit of PDZ-RhoGEF (and of the related RGS-RhoGEF LARG). Domain abbreviations : IPT, immunoglobu linked signaling. in plexins, Met and Ron tyrosine kinase receptors, and intracellular transcription factors; PSI, domain found in ple and integrins; Sema, semaphorin domain. were discovered in 1996 in a wide spectrum of species: ast Saccharomyces cerevisiae [5, 19, 20], FlbA in the aspergillus norhabiditis elegans , and RGS1 and RGS2 from human Br, new RGS-box-containing proteins are still being identified plants (Fig. 4C). For example, RGS21 was recently identified that is transacted in lingual epithelium via the T1R2/T1R3 mere 152 amino-acids, RGS21 is the shortest RGS protein -terminal to the central RGS-box. The singular nature of the RGS11 (Figs. 2&3) but is unlike other superfamily R4 subfamilies tandem (i.e., the PKA regulatory subunit binding partner NY2 [a.k.a. RGS22; Willard & Siderovski, unpublished eyond the defining RGS-box (Fig. 2). Several recent findings described below. A. RGS11 Binds Gβ5 via its Gγ-like or “GGL” domain B. RGS12 has tandem Ras-binding domains (RBDs), a C-terminal Gαi/o-Loco interaction (GoLoco), N-terminal PDZ and PTB (phosphotyrosine-binding) domains. The GoLoco motif/GDI inhibit GDP dissociation- blocks activation A number of events contribute to the termination of signaling by a GPCR. Example: Galpha s signaling 1. Decrease in cAMP levels a. Decrease AC activity b. Hydrolysis of cAMP via cAMP phosphodiesterase c. Reassembly of PKA holoenzyme-C d. Hydrolysis of GTP by Gas enhanced by GAPS/GDIs 2. Decrease transcription a. Dephosphorylation of CREB by protein phosphatase 1 b. Dissociation of CBP from CREB and decreased transcription 2. Desensitization of receptor and receptor down regulation a. Phosphorylation of receptor by kinases such as PKA and GRKs and binding of b-arrestin and GPCRs can be removed from the membrane by endocytosis A number of events contribute to the termination of signaling by a GPCR. Example: Galpha s signaling 1. Decrease in cAMP levels a. Decrease AC activity b. Hydrolysis of cAMP via cAMP phosphodiesterase c. Reassembly of PKA holoenzyme-C d. Hydrolysis of GTP by Gas enhanced by GAPS/GDIs 2. Decrease transcription a. Dephosphorylation of CREB by protein phosphatase 1 b. Dissociation of CBP from CREB and decreased transcription 3. Receptor Desensitization and down regulation a. Phosphorylation of receptor by kinases such as PKA and G-proteincoupled receptor kinase (GRKs) and binding of b-arrestin and subsequently GPCRs can be removed from the membrane by endocytosis Following receptor activation-G-proteins dissociate and receptor is phosphorylated and arrestins bind. This prevents G-proteins from rebinding. Receptor is “desensitized” Receptor internalized into endosome vesicle and either degraded (Down regulated) or recycled to surface G-protein-coupled receptor kinase Recycling (class A) Or degradation (class B) Endosome Phosphorylation of most GPCRs is required for desensitization and internalization Kinases- PKA, PKC and GRKs Experimental evidence: 1. phosphorylation- deficient receptor mutantse.g. serine mutated to alanine Elimination of phosphorylation of GPCRs abolishes arrestin recruitment, receptor desensitization, and internalization 2. Dominant- negative GRK (mutants which are catalytically inactive but retains ability to bind receptor) abolishes arrestin recruitment, receptor desensitization, and internalization. GPCR kinases whever the site willm he phosphorylated GRKs – three subfamilies- phosphorylates sites in C-terminal tail of the receptor but also at other intracellular sites, most notably ICL3 each receptor can be phosphorylated in different ways ****Each receptor has unique phosphorylation sites*** Rhodopsin kinases (GRKs 1 and 7); found in retina and localized to membrane (prenylation on C-terminal). Kinase activity “turned on “ by binding activated receptor Chemical Reviews GPCR kinases β-adrenergic receptor kinases (GRKs 2 and 3); ubiquitously expressed. Located in the cytoplasm and have a C- terminal pleckstrin homology domain that binds Gβγ subunits. Translocate to membrane when receptor is activated and Gβγ released. Kinase activity “turned on “ by binding activated receptor GRK4 subfamily (GRKs 4, 5, and 6). Testes (4) and ubiquitously expressed Located to the membrane by C-terminal by attached palmitic acid) GRK5 is activated by membrane phospholipids and GRK 4,6 appear to be constitutively active and target inactive and active receptors IEMEN:RE:V Rgd Desensitized ne Tppr ax FOBP rhfmTkhmf aTpbncdr- 7“B* hm sgd rhfmZkhmf aZqbncd lncdk* Z qdbdosnq ZbshuZsdc ax khfZmc 7( qdbqthsr jhmZ d Z rhfmZkhmf aZqbncd A( nm sgd B,sdqlhmZk sZhk ne sgd qdbdosnq- Sghr qdrtksr hm sgd qdbqthsldms ne !Zqq Zmc ZbshuZshnm ne deedbs aZqbncd qdrtks hm cheedqdmshZk deedbsnq bntokhmf ax !Zqqr rgnvm Zqd sgd bkZsgqhm ZcZosdq @N,1 Zmc DQJ L@NJ(- 2RGP* rdud qr: Ta* tahpthshm- For most GPCRs, arrestins function as adapters to target receptors bdr hm sgd bnmbYud rtqeYbdr ne sgd !,rgddsr vgdm bnloYqdc vhsg Y qgncnorhm Yqqdrshm,0 ehmfdq to clathrin-coated pits through its scaffolding of clathrin adapter nq ahmchmf Ymc sgd knno adsvddm !,rsqYmcr 0 shcd bnlokdw9 hm sgd qgncnorhm Yqqdrshm,0 rsqtbstqd x) Yqqdrshm,0 Yr Y clathrin sdsqYldq %Yheavy proteinvYr2 bqxrsYkkhydc (AP-2) and chain. rsqtbstqd enq sgd ehmfdq knno vYr qdehmdc %Ehf- 3A) wdk mc vYr mnsdc sn enql chldqr Ymc sdsqYldqr fg cheedqdms eqnl sgnrd nardqudc hm sgd bqxr, lnmnldqhb Yqqdrshm,0 bYm ahmc sn YbshuYsdc vgdqdYr hm sgd qgncnorhm odoshcd bnlokdw) Y qd rsqtbstqd vYr nardqudc %Ehf- 3A) bwXm rshbir( qgncnorhm Yqqdrshm,0 rsqtbstqd gYr sgd YcuYmsYfd ne Arrestins-Four family members: Visual arrestins 1 and 2-located in eye and function with the GPCR-rhodopsin and G protein-transducin and-GRKs1,7. b-arrestin 1 and 2-found other tissues **Bind to phosphorylated GPCR with higher affinity than G-proteins* GPCRs that traffic through the clathrin-dependent endocytic pathway can be divided into two groups, class A and B, based on the characteristics of agonist-dependent barr binding. “Lose or Tight” Class A receptors, such as the b2 adrenergic receptor, bind barr2 with greater affinity than barr1. “Lose” binding In class A interactions, receptors internalized in membrane vesicles and recycling endosomes (tubulovesicular early) that remain at the cellular surface, and barrs dissociate from the receptor at or near the plasma membrane. Class B receptors, such as the V2 vasopressin receptor, bind barr2 and barr1 with approximately equal affinities. Tight binding In class B interactions, barrs form a long-lived complex with the receptor and traffic it into late (multivesicular ) endosomes, which traffic receptors to lysosomes for degradation.. Author Manuscript GRK phosphorylation and b-arrestin binding sites C-terminal domain ICL3 Class B- Tight binder Author Manuscript at least 2 full complete phosphorylation codes at C-terminal end Binds barrestin 1/2 Class A- lose binder A Within C-terminal none, a partial or at most 1 complete phosphorylation codes Partial sites in ICL3 Binds preferential barrestin2 IEMEN:RE:V Rgd !,7ppdrshmr enqlYshnmr lYx qdoqdrdms rsdor hm Y ltksh,rsdo ahmchmf oqnbdrr barrestin: Inactive form: Its own C-Yqd ne !Yqqr sn FOBPr nq lYx qdoqdrdms chrshmbs rsYsdr sgYs terminal has negatively charged YrrnbhYsdc vhsgdomain cheedqdmshYk rhfmYkhmfamino acids that binds to polar domain (positive aminosnacids). RhfmTk SpTmrctbshnm Deedbsnpr Pdbdms rsqtbstqYk rstchdr gYud Ykrn Yccqdrrdc sgd ptdrshnm ne Active form: The phosphorylated peptide of gnv !Yqqr sqYmrlhs rhfmYkr dmbncdc hm sgd qdbdosnq sn deedbsnq C-terminal end of GPCR (this example: lnkdbtkdr- Sgd !Yqqr bYm hmsdqYbs vhsg cnvmrsqdYl deedbsnqr hm vasopressin 2 receptor V2Rpp) displaces the cheedqdms lncdr- Enq dwYlokd) !Yqq0 bYm ahmc adsvddm akYcdr 0 C-terminal barrestin peptide. Ymc 1 ne sgd bkYsgqhm !,oqnodkkdq uhY Ym hmsqhmrhbYkkx chrnqcdqdc Polar region=“Phosphate sensor” bkYsgqhm,ahmchmf anw) ats bYm Ykrn hmsdqYbs vhsg Y ahmchmf onbids enqldc ax akYcdr 3 Ymc 4 ne bkYsgqhm uhY Ym 7,Ylhmn Ybhc This cause change in structure of barrestin rokhbd knno entmc nmkx hm sgd knmf !Yqq0 hrnenql %88(- Etqsgdq and allows binding by “Activation sensor” to hmrhfgsr hmsn sgd Ykknrsdqhb qdftkYshnm ne !Yqq rhfmYkhmf gYud GPCR areas exposed following agonist qdbdmskx addm oqnuhcdc ax Ym MLP rstcx sgYs trdc 08E oqnadr binding hm !Yqq0 sn oqnadsensor: bgYmfdr hm hsr rsqtbstqd hmctbdc ax cheedqdms Activation ognrognodoshcdr sgd U1P B sdqlhmtr %0//(Finger loop cdqhudc bindseqnl to intracellular sites in TM ;ksgntfg Ykk sgd ognrognodoshcdr hmsdqYbsdc vhsg sgd ognr, 7 and TM8 ogYsd rdmrnqfollowing sn hmctbd bgYmfdr hm sgd ehmfdq lhcckd knnor) b-sheet finger loopYmcbinds sgdqd vdqd Ykrn chrshmbs ognrogn,hmsdqYbshnm oYssdqmr sgYsICL3 vdqd Intracellular sites in TM5, TM6 and regar impo This form blocks G protein GPC binding barco tant t micro ciate maco nists, and a FIGURE 4. Structural mechanisms for !arr activation and signaling. A, !arr nove activation occurs through disruption of the polar core (“phosphate sensor”) the c by the phosphorylated C terminus of the receptor, thereby allowing specific motifs in !arr (“activation sensor,” including the finger and lariat loops) to Single particle electron microscopy Identified Core conformation bind to the ligand-activated receptor (inactive structure, Protein Data Bank Ackno identified tail conformation Interactions between the (PDB) 1G4M; active structure, with PDB 4JQI). B, with alternative models for the finger loop interaction from rhodopsin!fingerreceptor loop peptide structure (yellow, Rober interactions between thethe C-terminal transmembrane 4PXF) and the rhodopsin!arrestin-1 structure (cyan, PDB 4ZWJ) with the tail PDB of the receptor with barr domains and C-terminal active receptor (green). C, single particle electron microscopy identifiesend dis(phosphate sensor only) barrs (activation sensor tinct conformations of !2AR!!arr, with a tailwith conformation with interactions Refer between the C-terminal tail of the receptor with arr (phosphate sensor only) and !phosphate sensor). 1. L and a core conformation with interactions between the transmembrane ( domains and !arrs (activation sensor and phosphate sensor). EM images b-arrestins have multiple functions in addition to Desensitization and Internalization MINIREVIEW: The !-Arrestins FIGURE 1. The spectrum of !arr-mediated signaling. !arrs regulate a wide array of pathways downstream of GPCRs (see text). PDEs, phosphodiesterases; EGFR, EGF receptor; PP2A, protein phosphatase 2A; TRP, transient receptor potential. mologo the cell zation desens idues is GRK is tem, G express tract (3 be abs intrace deficien negativ and int !arr b change GRK-m limitin kinetic neity in plexity Activation of MAP Kinase signaling pathway by GPCR Numerous studies have suggested that barrs can adopt multiple conformations that differentially regulate distinct cellular signaling events. Regulation of these unique barr conformations is controlled at a number of levels, through 1. interactions with different ligand-GPCR complexes, 2. Different post-translational modifications of both the receptor and barrs, Barcode” model for receptor-barr signaling. “Signaling Binding of barr to distinct receptor C-terminal/ICL3 phosphorylation patterns (“barcodes”) generated by different kinases results in different conformations of receptor-bound barrs. These different barr conformations are capable of activating distinct downstream signaling events, such as endocytosis, desensitization, or signaling pathways FIGURE 1. The spectrum of !arr-mediated signaling. !arrs regulate a wide array of pathways downstream of GPCRs (see text). PDEs, phosphodiesterases; EGFR, EGF receptor; PP2A, protein phosphatase 2A; TRP, transient receptor potential. signaling. Covalent modification of !arr with ubiquitin (ubiquitination) results in sustained !arr!GPCR complexes and prolonged MAPK activity. Ubiquitination of a GPCR is necessary Distinct Phosphorylation Sites on the β2-Adrenergic Receptor Establish a Barcode That Encodes Differential Functions of βArrestin Mass Spec-based quantitative proteomic approaches used to: 1. Map phosphorylation sites on the β2AR, 2. Determined the GRKs responsible for phosphorylation of the sites 3. Delineated specific β-arrestin associated activities linked to specific phosphorylation. GRK2 sites were primarily responsible for β2AR internalization, GRK6 sites contributed to β-arrestin–mediated ERK activation GRK2/6 sites contributed to desensitization. Model: Different phosphorylation patterns on the β2AR elicited by either GRK2 or GRK6 can induce distinct β-arrestin conformations and activities. Sci Signal. 2011 August 9; 4(185) Distinct G protein-coupled receptor phosphorylation motifs modulate arrestin affinity and activation and global conformation Mayer, D., Damberger, F.F., Samarasimhareddy, M. et al. Nat Commun 10, 1261 (2019) Phosphorylation of most GPCRs is required for desensitization and internalization Kinases- PKA, PKC and GRKs Experimental evidence: 1. phosphorylation- deficient receptor mutantse.g. serine mutated to alanine Elimination of phosphorylation of GPCRs abolishes arrestin recruitment, receptor desensitization, and internalization 2. Dominant- negative GRK (mutants which are catalytically inactive but retains ability to bind receptor) abolishes arrestin recruitment, receptor desensitization, and internalization. GPCR kinases GRKs – three subfamilies- phosphorylates sites in C-terminal tail of the receptor but also at other intracellular sites, most notably ICL3 ****Each receptor has unique phosphorylation sites*** Rhodopsin kinases (GRKs 1 and 7); found in retina and localized to membrane (prenylation on C-terminal). Kinase activity “turned on “ by binding activated receptor Chemical Reviews GPCR kinases β-adrenergic receptor kinases (GRKs 2 and 3); ubiquitously expressed. Located in the cytoplasm and have a C- terminal pleckstrin homology domain that binds Gβγ subunits. Translocate to membrane when receptor is activated and Gβγ released. Kinase activity “turned on “ by binding activated receptor GRK4 subfamily (GRKs 4, 5, and 6). Testes (4) and ubiquitously expressed Located to the membrane by C-terminal by attached palmitic acid) GRK5 is activated by membrane phospholipids and GRK 4,6 appear to be constitutively active and target inactive and active receptors