GPCR Lectures 3 2024 PDF

Lecture 3: GPCRs How do receptors that activated the same transducers/ effectors give different responses in a single cell? The effect of a first messenger depends on: 1. The receptor it binds (location important) 2. The intracellular transducers/effectors activated (location important) 3. The targe...

Lecture 3: GPCRs How do receptors that activated the same transducers/ effectors give different responses in a single cell? The effect of a first messenger depends on: 1. The receptor it binds (location important) 2. The intracellular transducers/effectors activated (location important) 3. The target proteins present in cells (location is important) Table 1. List of GPCRs Activating G Proteins in Intracellular Compartments can be found in different places receptor compartment G protein ref α1A-adrenergic receptor (α1A-AR) α1B-adrenergic receptor (α1B-AR) β1-adrenergic receptor (β1AR) β2-adrenergic receptor (β2AR) calcitonin-gene-related-peptide (CGRP) receptor calcium-sensing receptor (CaSR) cannabinoid type 1 receptor 1 (CB1R) dopamine receptor type 1 (D1R) luteinizing hormone receptor (LHR) melatonin type 1 receptor (MT1R) neurokinin type 1 receptor (NK1R) parathyroid hormone receptor (PTHR) protease-activated receptor-2 (PAR2) sphingosine-1-phosphate 1 receptor (S1P1R) thyroid stimulating hormone receptor (TSHR) vasopressin type 2 receptor (V2R) nucleus nucleus Golgi early endosomes early endosomes early endosomes mitochondria early endosomes early endosomes mitochondria early endosomes early endosomes early endosomes Golgi Golgi early endosomes Gαs Gαs Gαs Gαs Gαs, Gαq Gαq Gαi Gαs Gαs Gαi Gαq Gαs Gαq Gαi Gαs Gαs 30 30 59 25 84 31 32 24 33 34 75 22 193 76 23 69 +,)- 2*3)4)-2 ! A,)B28* (&!'(!)* !$# plasma membrane can be activated on plasma "1$ %&!'(!)* ! +,)-./ endocytosis % &%/. @*3)4)-2 "#$ %&!'(!)* +,)- 2*3)4)-2 23< !)67789:!; ! &%/. "5$ %&!'(!)* +,)- !)6789:!; ! &%/. mhsh]shnm eqnl hmsdqm]k ldlaq]mdr- Dmcnrnld rhfm]khmf l]x ad sdqlhm]sdc ax %,,24!* (&!'(!)* !"# ! % @*3)4)-2 % "#$ %&!'(!)* +,)-./ "00.$ "1$ %&!'(!)* +,)- 2*3)4)-2 ! A,)B28* (&!'(!)* "1$ %&!'(!)* ! +,)-./ Beta arrestin signaling /%.? /%.? !$# 00. % Cell signaling Cell signaling How signaling pathways are activated is dependent on intrinsic characteristics of each receptor and the location of receptor (D) Dimeriza!on ac!va!on (E) Transac!va!on Biphasic ac!va!on (F) Arres n response PM XXXXXXXXXXX Clathrin la ce A pERK ) Cell signaling src P Nuclear targets P Figure 2. Sc Current V Impact of L duced by b pa thw ay cAMP cAMP Cell signaling CRE Sig na lin g Downstream effector pERK Cell signaling ay hw pat ing nal Sig protein-coup mediated ac Time plasma mem duce a rapi access nuc endosomes G-protein β-arres!n vation and s Cytoplasmic Endosome plasm, ther targets nuclear targe vation of Gs Time A and transien cAMP-depen Time cytoplasm. A ) G protein response duces a dela tially accesse PM the cAMP of activation, Figure 1. Classical and Novel Modes of G-Protein-Coupled Receptor (GPCR) Activation. (A) Classical mode, (B) biased activation, (C) intracellular Cytoplasmic shown as an targets (D) dimerization activation,G (E) transactivation, and (F) biphasic activation. are capable o endosome-in Time occur repea 368 Trends in Pharmacological Sciences, April 2018, Vol. 39, No. 4 sence of ago of endocytos curved arrow Endosome Nuclear the b-arrestin targets not require e.g., PCK1 GPCR. Internalization- dependent ERK signaling by barrestin (A) Class A receptors (b2-receptor) interact with barrestin and the complexes are targeted to clathrin-coated pits for transient ERK1/2 activation near the membrane. The binding of barrestin is transient in endosomes and is followed by rapid recycling of the receptors back to the plasma membrane. (B) Class B receptors interact robustly with β-arrestin and the complexes are targeted to endosomes for prolonged ERK1/2 activation and GPCR degradation. (C) β-arrestin is internalized without forming a complex with the β1-adrenergic receptor (β1-AR). β-arrestin briefly ‘kisses’ β1-AR, locates to clathrin-coated structures at the plasma membrane, and then activates ERK1/2. CLASS A/B Internalization dependent ERK activation requires recruiting G proteins cAMP PKA EPAC RAP1 B-RAF MEK. (D) Internalization-dependent activation is mediated by G protein in the cytoplasm. After internalization, GPCRs target the endosome or Golgi. The receptors then recruit the Gs protein, resulting in cAMP accumulation that induces ERK1/2 activation in endosome or Golgi. ERK GEF Small G protein MAP Kinase pathway EPAC-(exchange protein directly activated by cAMP) Mediates Sustained G Protein Signal Class B: GPCR-G Protein-b-Arrestin Super-Complex Mediates Graphical Sustained G Abstract Protein Signaling and Barrestin/ERK signaling Authors Alex R.B Thomas Michel B Corresp michel.b C-terminal tail binding lefko001 In Brief Megaple simultan and b-ar following GPCRS can reside on membrane of organelles. Two patterns of internalization-independent intracellular activation are proposed: GPCRs can reside on either the Golgi, mitochondria or ER (E) or nuclear membrane (F) and then initiate internal signaling which is independent of receptor internalization from the plasma membrane. How is GPCR localization to sites in PM or Subcellular sites? Receptor compartmentalization occurs via Scaffold proteins containing PDZ domain Localized to specific site in the plasma membrane Localized to specific Intracellular site Also localized by non-PDZ scaffolds (b-arrestin and AKAPs) dmg]mbdc cdrdmrhshy]shnm ne 4,GS1BQ“ldch]sdc B]1 ]bbt, ltk]shnm %F]u]qhmh ds ]k-+ 1//5(- Hm ORC,84 mtkk lhbd+ rdqnsnmhm 1B qdbdosnq %4,GS1BQ(“ldch]sdc benr hmctbshnm hr hlo]hqdc %@aa]r ds ]k-+ 1//8(- Cdrohsd rhfmhehb]ms rdptdmbd gnlnknfx+ ORC,84 ]ood]qr sn g]ud noonrhmf qnkdr hm qdftk]s, hmf hsr sq]eehbjhmf ]mc rhfm]khmf o]sgv]xr ne 4,GS1@Q ]mc Large number of PDZ containing proteins PDZ domains 80-90 amino acids- folded cup-like structure that recognizes short peptides. Many GPCR has PDZ binding domains at C-terminal end S@AKD 0 Deedbs ne OCY oqnsdhmr nm FOBQ sq]eehbjhmf OCY Oqnsdhm Sq]eehbjhmf Etmbshnm ORC,84 FOBQ Rxmsdmhm,0 ↓ Ldlaq]md knb]khy]shnm RMW16 ↓ Qdbxbkhmf a0@Q+ 4,GS1@Q C0Q FOQ2/ 4,GS1BQ+ C0Q BQEQ0+ 4,GS1@Q a0@Q @1@ qdbdosnq 4,GS1BQ UO@B0 a0@Q lFktQ0] a1@Q+ SOa a1@Q+ gtl]m g nohnhc qdbdosnq RRSQ4+ OSG0Q Rdqnsnmhm 3@ qdbdosnq Eqhyykdc 3 BBQ4+ ok]sdkds,]bshu]shmf e]bsnq qdbdosnq+ O1X01Q 4,GS1@Q gHOQ gHOQ lFktQ4+ BK0 C1@Q kNQ 4,GS1@Q RRSQ2 lFktQ0 lFktQ4 C0C@Q Oqnk]bshm,qdkd]rhmf odoshcd qdbdosnq Fqnvsg gnqlnmd“qdkd]rhmf gnqlnmd qdbdosnq F oqnsdhm“bntokdc qdbdosnq 26 %dmcnsgdkhm qdbdosnq sxod A,khjd( a1@Q+ a0@Q+ RRSQ4 FHOB ↓ Dmcnrnld.Fnkfh knb]khy]shnm ↓ Sq]eehbjhmf sn d]qkx dmcnrnld ↓ Ldlaq]md rs]ahkhsx Ldlaq]md knb]khy]shnm C1Q+ cno]lhmd 2 qdbdosnq KO@0Q Gtl]m ktsdhmhyhmf gnqlnmd qdbdosnq a0@Q+ RRSQ4 Qdbxbkhmf ↓ Fnkfh knb]khy]shnm a0@Q RRSQ4 R@O86 R@O0/1 LOO2 L@FH,1 MGDQE0 ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ OCYJ0 OCYJ1 Rg]mj0 Rohmnoghkhm LTOO0 S]l]khm Rxmsqnoghmr OHBJ0 B@K Dmcnbxsnrhr Qdbxbkhmf Ldlaq]md knb]khy]shnm Dmcnbxsnrhr Dmcnbxsnrhr Qdbxbkhmf Lnahkhsx Ldlaq]md knb]khy]shnm Dmcnbxsnrhr Dmcnbxsnrhr Ldlaq]md knb]khy]shnm Dmcnbxsnrhr Qdbxbkhmf Ldlaq]md knb]khy]shnm Lhbqnuhkkh knb]khy]shnm Bxsnrjdkds]k knb]khy]shnm Dmcnbxsnrhr Dmcnbxsnrhr ↓ Ldlaq]md knb]khy]shnm ↓ Ldlaq]md knb]khy]shnm ↓ Bktrsdqhmf Dmcnbxsnrhr ↓ Dmcnbxsnrhr ↓ Ldlaq]md knb]khy]shnm ↓ Shfgs itmbshnm knb]khy]shnm ↓ Ldlaq]md knb]khy]shnm ↓ Mdtqhsd knb]khy]shnm ↓ Ldlaq]md knb]khy]shnm ↓ Hmsq]bdkktk]q bktrsdqhmf Qdbxbkhmf Qdedqdmbd Gt ds ]k-+ 1///: Wh] ds ]k-+ 1//2 Rtm ds ]k-+ 1//8 @j]l] ds ]k-+ 1/02: Aqnrdkhc ds ]k-+ 1/03 F]u]qhmh ds ]k-+ 1//5: Yg]mf ds ]k-+ 1//6a Ctmm ds ]k-+ 1/02+ 1/03 F]qcmdq ds ]k-+ 1//6 Sgtqmdq ds ]k-+ 1/03 F]u]qhmh ds ]k-+ 1//5 Fdd ds ]k-+ 1//8 Wt ds ]k-+ 1//0 Rtfh ds ]k-+ 1//6 Qnbgch ]mc O]qdms+ 1//2: V]mf ds ]k-+ 1//6 B]n ds ]k-+ 0888: Kh ds ]k-+ 1//1 Vgddkdq ds ]k-+ 1//7: A]tbg ds ]k-+ 1/03 Intadqs ds ]k-+ 1//3 Vgddkdq ds ]k-+ 1/00 G]ll]c ds ]k-+ 1/0/: Ctoqä ds ]k-+ 1/01: Mhr]q ds ]k-+ 1/01 V]ksgdq ds ]k-+ 1/04 Stqmdq ds ]k-+ 1/00 Qdhc ds ]k-+ 1/01 St ds ]k-+ 0888: Sna]adm ds ]k-+ 1/// Aq]cx ds ]k-+ 1//2 Bg]qksnm ds ]k-+ 1//7 Inmdr ds ]k-+ 1//8 Khdv ds ]k-+ 1//8 Jhs]mn ds ]k-+ 1//1: Rtfh ds ]k-+ 1//6 Jhs]mn ds ]k-+ 1//1 Bgdm ds ]k-+ 1//5: Kxrr]mc ds ]k-+ 1//7+ 1/00 Khm ds ]k-+ 1//0: L]crdm ds ]k-+ 1/01 J]srtrghl] ds ]k-+ 1/02 Ctmg]l ds ]k-+ 1//8 K]teedq ds ]k-+ 1/0/: Sdljhm ds ]k-+ 1/00: M]j]f]v] ]mc @r]gh+ 1/02: A]tbg ds ]k-+ 1/03 Id]mmdsd]t ds ]k-+ 1//3 U]qr]mn ds ]k-+ 1/01 Ghq]j]v] ds ]k-+ 1//2 Gd ds ]k-+ 1//3: A]tbg ds ]k-+ 1/03: Jnkhvdq ds ]k-+ 1/04 Jnkhvdq ds ]k-+ 1/04 Vdmsd ds ]k-+ 1//4: A]tbg ds ]k-+ 1/03 Downloaded from molpharm.aspetjournals.org at ASPET Journals on December 22, 2020 shnm+ sgdqdax rs]ahkhyhmf sgd qdbdosnq ]s sgd bdkk rtqe]bd %Gt ds ]k-+ 1///( %S]akd 0(- Cdrohsd sgd onsdmsh]shnm ne a0@Q ldlaq]md dwoqdrrhnm+ sghr hmsdq]bshnm ]ood]qr sn g]ud mn etmbshnm]k bnmrdptdmbd nm FCr,bntokdc rhfm]khmf+ ]r ld], rtqdc ax b@LO ]bbtltk]shnm %Gt ds ]k-+ 1///(- Hm bnmsq]rs+ ORC,84 hmsdq]bshnmr vhsg sgd rdqnsnmhm 1@ qdbdosnq %4,GS1@Q( Localization of the signaling machinery by non-PDZ proteins AKAP (A kinase anchoring proteins) epienephrine/norepinefrine AKAP localizes b AR, PKA, PDEs and Adenylate cyclase near L type calcium channels in cardiac muscle T-tubules In normal heart cells β1AR/ β2AR bind to Norepinephrine and are coupled to Gs/AC/cAMP b1 AR plays dominant role in chronotropy and inotropy b2 AR-modest role in chronotropy but little role in inotropy Inappropriate increase in sympathetic activity leads to heart failure. In the case of heart failure-β1AR promotes cardiomyocyte hypertrophy and apoptosis, whereas β2AR prevents these events. Hypothesis: They localize to different subcellular sites, and this may mean they activate different target proteins kiss and run B-arrestin signaling GC at membrane B-arrestin abd Gs signaling in endosome Kiss and run b-arrestin signaliing GS at membrane b-arrestin and Gs signaling in endosome Kiss and run b-arrestin signaliing GS signaling not in endosome GS cAMP PKA PDE prevents PKA activation in cytosol b-arrestin and Gs signaling in endosome Gs cAMP EPAC were fixed with 4% formaldehyde and imaged on a LEICA DMi8 microscope in confocal mode with a 20 x air lens. NE β1AR Oct3 Plasma membrane cAMP EPAC? PDE3 Oct3 Golgi Gs cAMP mAKAP ! PI4P IP2+DAG PLCε EPAC β1AR Golgi PKD Cytosol Daisioglycerol from m Nucleus Figure 9. Signal transduction by cell surface and Golgi b1ARs. b1ARs are located on both the plasma membrane and the Golgi apparatus in cardiac myocytes. Stimulation of cell surface b1ARs leads to production of cytosolic cAMP but this cAMP cannot access the Epac/PLCe/mAKAPb due to PDE3 dependent hydrolysis of cAMP. To PKD=protein kinase D Activate transcription factors Can knowledge about subcellular localization allow us to understand why some drugs work or help us to design better drugs? Metoprolol (hydrophobic) and Atenolol (hydrophilic) are drugs (antagonist) that selectively bind β1AR and block signaling. Kiss and run b-arrestin signaliing GS not in endosome GS cAMP PKA CREB PDE b-arrestin and Gs signaling in endosome Gs cAMP EPAC GCPR Biased signaling Fig. 1. GPCRs signaling toward multiple pathways. A: GPCRs relay signals through cognate/non-cognate G proteins, β-arrestins, and G pr toward each of these pathways can activate different effector molecules, resulting in different cellular responses. B: Different ligand receptor conformations favoring one among all downstream pathways. Signaling is not a matter of YES/NO bimodal system; rather, d terms of the magnitude or rate of activation downstream repertoire of a single receptor. Thus, presuming a balanced ligand which potency, biased ligands (a-x) induce a different pattern of activation of signaling pathways (1-n) compared to that of the balanced l cyclase; AP2, activating protein 2, GIRK, G protein inward rectifying K channel; GRK, GPCR kinase; MAPK, mitogen-activated phospholipase C; PKD, protein kinase D; PLC, phospholipase C; PP2A, protein phosphatase 2A; GPCRs exist in a dynamic equilibrium between the inactive state and multiple active states These states then uniquely couple to downstream G proteins, barrestins, and GPCR kinases, resulting in distinct cellular responses. If a receptor can activate many signaling pathways How is one specific signaling pathway selected? 1. Biased receptors-are modified to change ability to activate specific transducers Post translation modification or splice variants, binding of cofactors, membrane phospholipids Receptor dimerization )«TÒ)«TÑS6‡æ$Tí Ò$æç $Ò 6çÒÒ ‡¾6ç æT Òæ$ìš6‡æç ÍÒs ¾šæ å‡í Òæ$ìš6‡æç Í$ ìTÑç d7 Vdifbß\ I«ç ŸT66TÕ$í{ ìTŒç6 $Ò Ò«TÕí $í ž${ YÁ š)Tí ‡{Tí$Òæ ¾$íŒ$í{s æ«ç Ñçåç)æTÑtå‡æ‡6SÒçŒ ‡åæ$ô‡æ$Tí TŸ ÍÒ Òæ$ìš6‡æçÒ Cognate Receptor expression Method M. Seyedabadi et al. / Pharmacology ‡ŒçíS6S6 åSå6‡Òç ‡åæ$ô$æS˜Non-cognate æ«ç ÑçÒš6æ$í{ Ñ$Òç $í $íæчåç66š6‡Ñ åöa¶ Gα Gα (endogenous or heterologous) ‡åæ$ô‡æçÒ ¶+ö ‡íŒ æ«ç ‡{Tí$ÒætTååš)$çŒ Ñçåç)æTÑ $Ò )«TÒ)«TÑS6‡æçŒ s3Cjffi S DqT )bh )b n–[*Sb7 br *)fi !wTF Sh fi1:S fi3 rb Shb fi7[–S7fiwS73:8fi3 of the coupling of themS*)beta2öŸæçÑ )«TÒ)«TÑS6‡æ$Tís æ«ç Ñçåç)æTÑ ÒÕ$æå«çÒ $æÒ åTš)6$í{ Ò)çå$må$æS Switching )bh )b n–[*Sb7 br yTDqM gfi––h mfi fi * [7hSfi7*–n * [7hrfi8*fi3 )[fix[pp–:*Sw Gi Gs, Gz, Gq endogenous/heterologous GTPγS l J æT Í$ ÍI¶t¾TšíŒ Í$D Œ$ÒÒTå$‡æçÒ ŸÑTì æ«ç «çæçÑTŒ$ìçÑ$å Í!"s ‡íŒ adrenergic 2 fiS*)fi [–b7fito b mS*) mS–3w*n fi !EwTFv b mS*)analysis !EwTF 7S7w*[ppfi3 f J( a ]]6Twx[ receptor different Meth Cognate Non-cognate Receptor expression ŸÑçç Í!" Òš¾ší$æÒ ìçŒ$‡æç ‡åæ$ô‡æ$Tí TŸ æ«ç aö¶ Ì$í‡Òç Ò${í‡66$í{ 7Sp)* –[8JS7p )bh )b n–[*Sb7 hS*fih rb DqT a!EwTFx:*2M gfi––h mfi fi h*[ 5fi3 b5fioligonu Gi Gs heterologous second G proteins by protein Gα Gα (endogenous or kinase A )‡æ«Õ‡S $í æ«ç Ò‡ìç Õ‡S ‡Ò Í$tåTš)6çŒ Ñçåç)æTÑÒ Xfirb fi [hh[nS7p rb Shb fi7[–S7fiwS73:8fi3 yTDq )bh )b n–[*Sb7M k[–:fih and h)bm7 CTX 1 heterologous) Gq/11 Gi/o, G12/13 heterologous Yfi Daaka R JJ[73Lefkowitz ‹ç)çíŒ$í{ Tí åç66 æS)çs ‡í $íåÑç‡Òç $í $íæчåç66š6‡Ñ åöa¶ ì‡S [ fi fiV fihhfi3 second [h rb–3 fihfi7* xfi[7h # h, fiLbr M )bh Luttrell, )b n–[*fi3 ]]6Twx[ treatme ß]s÷− constru Gi/o Gs, co-p sGq Œ$ŸŸçÑçíæ$‡æ$Tí÷ßs÷÷ TÑ {ÑTÕæ«endogenous/heterologous ÑçÒš6æ $í åç66š6‡Ñ )ÑT6$Ÿçчæ$Tí S78 fi[hfi b5fi :7h*Sx:–[*fi3 8fi––hM +)fi fiV fihhSb7 br fi8fi *b m[h [h rb––bmh4 Affiliations Gq/11 G13 endogenous/heterologous second ÷ù $í«$¾$æ$Tí TŸ /‡Ÿß ‡íæ‡{Tí$ÆçÒ aö¶ ‡ÑÑçÒæ åöa¶tŒç)çíŒçíæGi v l99R mess fi73bpfi7b:h !EwTFv l9 rxb– xp ( b*fiS7i b5fi fiV fihhfi3 !EwTF b !EwTFx:*GTPγS Gq/11 , Go, G13 endogenous/heterologous l 9363896 Ì$í‡Òç ‡åæ$ô‡æ$Tí $í m¾ÑT¾6‡ÒæÒ÷Xs÷Y ‡æ ‡ Òæç) $í æ«ç Ò${í‡66$í{ å‡Ò凌ç PMID: [8fi––:w ]99 rxb– xp ( b*fiS7M k[–:fih rb *)fi Shb fi7[–S7fiwS73:8fi3 S78 fi[hfi br S7* capture oligonu Gq/11 Gi/o ‡ŸæçÑ /‡Ò ‡åæ$ô‡æ$Tí (í åTíæчÒæs ‡í $íåÑç‡Òç $í $íæчåç66š6‡Ñ åöa¶ $íheterologous –[ 8TyD mfi fi [h rb––bmh4 fi73bpfi7b:h !EwTFv a(A H # 9 L2wrb–3i b5fi fiV messen fihhfi3 secon Gs Gi, Gq/11 heterologous Gs heterologous GTPγS éTÒt² åç66Ò ¾S ‡ ÑTšæç íTæ G13 $íôT6ô$í{ ‡ Ñçåç)æTÑs $íå6šŒ$í{ çþ)ÑçÒÒ$Tí !EwTFv aEY A # ( E2wrb–3i b5fi fiV fihhfi3 !EwTFx:*v aEC L # ( l2wrb–3 fi–[*S5fi *b :7h*Sw co-pl transcri mess TŸ ‡ åTíÒæ$æšæ$ôç6S ‡åæ$ôç ìšæ‡íæ ÍÒ s TÑ çþ)TÒšÑç æT \t¾ÑTìTt x:–[*fi3 8fi––hM p115Rh Gs Gq/11 endogenous secon \s÷~ Gs Gi/o heterologous GTPγS l åöa¶ TÑ ŸTÑÒÌT6$ís ÑçÒš6æÒ $í aö¶ Ì$í‡Òç ‡åæ$ô‡æ$Tí (í f%+÷]ù analysis Gα s Gs Gi,TÑGq/11, GTPγS l åç66Òs æÑç‡æìçíæ Õ$æ« ŸTÑÒÌT6$í $ÒT)Ñçí‡6$íç $íåÑç‡ÒçÒ åöa¶heterologous ¾S Gi Gq, G13 heterologous GTPγ analysis XY # ÷ ÷ ‡íŒ ß² # ß Y G12 æ$ìçÒs ÑçÒ)çåæ$ôç6S žTÑÒÌT6$í æÑç‡æìçíæ SRE and Gi Gi, Gs, Gq heterologous GTPγS l $팚åçÒ ‡í Î\ ÷ # − ~JtŸT6Œ $íåÑç‡Òç $í aö¶ Ì$í‡Òç )«TÒ)«TÑS6‡t endogenous/heterologousanalysis GTPγ Gq/11 G13, Gi2 æ$Tís Òš{{çÒæ$í{ 櫇æ åöa¶ ì‡S ìçŒ$‡æç aö¶ Ì$í‡Òç ‡åæ$ô‡æ$Tí heterologous ¾S ‡ Gi Gq/11 co-prec secon Gi Gs heterologous second Ñçåç)æTÑt$íŒç)çíŒçíæ )‡æ«Õ‡S fTÕçôçÑs ŸTÑÒÌT6$ítÒæ$ìš6‡æçŒ Gs Gi/o, Gq/11, endogenous/heterologousco-prec GTPaö¶ Ì$í‡Òç )«TÒ)«TÑS6‡æ$Tí $í f%+÷]ù åç66Ò $Ò $íÒçíÒ$æ$ôç æT Gs Gi heterologous PTX trea G12, G13 mess analysis ¾Tæ« ¶IÚ ‡íŒ !tö/+åæGi/o, çþ)ÑçÒÒ$TísGq, $íŒ$å‡æ$í{ æ«‡æ ŸTÑÒÌT6$í Gs/olf G15 endogenous/heterologous GTPγS o ÕTÑÌÒ ¾S ‡ Œ$ŸŸçÑçíæ ìç嫇í$Òì messen Gq/11 Gi3ŸÑTì æ«‡æ šÒçŒ ¾S æ«ç !tö/ heterologous GTPfusion, N ÉS åTš)6$í{ æT Œ$ŸŸçÑçíæ å6‡ÒÒçÒ TŸ Í )ÑTæç$ís Ñçåç)æTÑÒ heterologous å‡í mess Gq Gi second Table 1 Non-exhaustive list of GPCRs coupling to non-cognate G proteins. Receptor Class A Table 1 (continued) 5-HT1A Receptor 5-HT1E 5-HT2A Opioid μ 5-HT2B 5-HT2C 3 Oxytocin OT Prostanoid IP 5-HT4A Prostanoid EP2 5-HT4B 5-HT7 Lysophospholipid S1P3 and S1P5 Adenosine A1 Proteinase-activated PAR Adenosine A3 Adrenergic α2 Thyrotropin Adrenergic β1 Adrenergic β2 Vasopressin V1a Angiotensin AT1A Bradykinin B2 Class B Calcitonin Cannabinoid CB1 3 Gq/11, Gi/o, G14 Gs Gi/o Gs Gq/11, Gs, Gq/11, G16 G14/15/16 heterologous endogenous/heterologous Corticotrophin-releasing Cholecystokinin CCK1 hormone Dopamine D1A Glucagon Gs Gq/11 Gs Gs and Gz Gs, Gi, Gq heterologous endogenous Dopamine D3 Glucagon like peptide Galanin GAL2 GLP-1 Gonadotropin releasing Parathyroid hormone Gi Gs Gs, Gz Gi/o, Gq/11 Gi/o, G12 heterologous heterologous endogenous/heterologous Gq/11 Gs Gi, Gs, Gq/11, G14/15 G14/15/16 Gi2 endogenous/heterologous hormone Luteinizing hormone Histamine H2 Vasoactive intestinal Melanin peptide concentrating hormone MCH1 Melanocortin MC4 Class C Calcium sensing Melatonin MT1 CaSR Muscarinic M1 Metabotropic glutamate Muscarinic M2 1a Muscarinic M Metabotropic glutamate 3 5 Muscarinic M4 Gs Gq/11 Gs Gs Gs Gi/o Gq/11, Gi, Go endogenous/heterologous Gi Gi1 Gi, Gq/11, Gi G14/15 Gq, Gs endogenous endogenous heterologous endogenous/heterologous endogenous/heterologous endogenous heterologous s3Cjffi d frrfi8* br *)fi 8TyDw3fi fi73fi7* b*fiS7 JS7[hfi T S7)SXS*b 6wYC b7 yTD GTPase messen capture secon second CRE-luc domina GTPco-prec second GTPγS o GTPco-prec mess messen second GTPγ GTP-AA mess messen second GTPand CTX mess GTP-AA messen GTP-AA secon messen GTPγS l antib analysis JS7[hfi [8*S5[*Sb7M 6fqECl 8fi––h mfi fi S78:X[*fi3 b5fi 7Sp)* S7 hfi :xwr fifi xfi3S[ s3Cjffi A yb3fi– rb !EwTFwxfi3S[*fi3 ;w b*fiS7 hmS*8)S7p *b [8*S5[*fi yTD JS7[hfiM G15 Xfirb fi l9 xS7 fi* fi[*xfi7* mS*) (9 $y 6wYCM uhb fi7[–S7fi a( $y2 b -DT a(9 $y2 D)bh )b n–[*Sb7 br yTD JS7[hfi Xn *)fi !wTF b8fifi3h *) b:p) fi8fi *b 8b: –S7p analysis Gs Gq/11, Gi/o endogenous/heterologous GTPγS l Gα kno mfi fi [33fi3 *b h[x –fih rb A xS7 S7 *)fi fihfi78fi br 6wYC [73 [8*S5fi yTDq –fi5fi–h *b GTPγ *b ;h [73 [8*S5[*Sb7 br DqTM T8*S5[*fi3 DqT )bh )b n–[*fih *)fi !wTF –fi[3S7p Gi/o, G12/13, Gs endogenous/heterologous Gi2, Gi3 Gq/11 endogenous/heterologous co-prec 3fi*fi xS7fi3M k[–:fih h)bm7 fi fihfi7* xfi[7h # h fi r bx l fiV fi Sxfi7*h [73 [ fi !wTF mess fi8fi *b 8b: –S7p *bv [73 [8*S5[*Sb7 brv ;SM ;!" fi–fi[hfi3 r bx *)fi ;Sw8b: –fi3 messen Gq/11 Gq/11 Gi, Gs, endogenous/heterologous GTPγS o fiV fihhfi3 [h rb–3 S78 fi[hfi b5fi :7h*Sx:–[*fi3 8fi––hM [8*S5[*fih yTD JS7[hfi S7 [ t 8w [73 F[hw3fi fi73fi7* [*)m[nM p115 messen Gq/11 Gi/o g9 Gq/11 Gq/11 Gi/o Gi/o, Gs heterologous Gi, Gs, Gq/11 heterologous Gi, Gs G12, Gs heterologous heterologous Gi, Gs heterologous GTPγ Second ñöI1/% ® 2dï ù]− ® ~ ñd2%aÉ%/ ß]]² analy siRNA, P GTPγS o secon messen NanoBR second Dimerization of receptors can be required for signaling, alter transducer used and change choice of effectors used If a receptor can activate many signaling pathways How is one specific signaling pathway selected? 2. Biased Ligands which favor specific alteration of intracellular domains of receptor that selectively activate specific transducers 3. Systems bias-differential high expression of transducers proximal to receptor such as GRKs or b-arrestins or types of G proteins its improtant because if we can sort th edrug effect in benificail and side effect an dif we can control the side-effect to turn into benificial n b-arrestin and theb-arrestin-mediated class but increases cardiomyocyte contractility and promotes cardiac output in vivo. TRV120027 has activated the potential totrigger treat acutetwo heart failure. By contrast,pathways in some Then, theThus, receptor rapidlywhen Many GPCR or more signal GPCRs, the G protein pathway mediates beneficial effects, while the b-arrestin pathway rast to class Aligands receptors, can selectively activate these pathways Biased mediates undesirable adverse effects. Therefore, G protein-biased ligands have been develdosomes fortreat subsequent opedµ-opioid to relevant diseases. receptor andFor example, the m-opioid receptor (MOR) is the target of the prototypical m-opioid most opioid narcotics, morphine activates 1,52]. Third, b1-AR was receptor agonist, morphine. Similar to TRV130-predominately Morphine activates both pathways has adverse effects, such as pain tolerance, constipation, and respiratory suppression, thereby (A) G protein pathway Ligand (B) (C) β-arres n β-arres n Endosome G protein Kis s ru and n Clathrincoated pit β-arres n β-arres n G protein β-arres n β-arres n ERK1/2 ERK1/2 P Downstream effector P Downstream effector Good: Pain Good: Pain relief (F) relief Bad: Constipation, decrease Gi track Figure 2. Biased Activation. Two major patterns of based activation: (A) b-arrestin-mediated biased signaling and (B) G Respiratory suppression Transloca on