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Fine-tuning of GPCR activity by receptor-interacting proteins
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"G protein-coupled receptors (GPCRs) are the largest family of transmembrane proteins in vertebrates and they are the molecular targets for nearly half of the therapeutic drugs that are prescribed worldwide 1 . The approximately 1,000 members of the GPCR family exhibit a conserved 7-transmembrane domain topology and can be divided into 3 main subfamilies, termed A, B and C, based on sequence similarity. The canonical view of how GPCRs regulate cellular physiology is that the binding of ligands (such as hormones, neurotransmitters or sensory stimuli) induces conformational changes in the transmembrane and intracellular domains of the receptor, thereby allow- ing interactions with heterotrimeric G proteins. Activated GPCRs act as guanine nucleotide exchange factors (GEFs) for the ? subunits of heterotrimeric G proteins, catalysing the release of GDP and the binding of GTP for G protein activation. The activated G protein subunits (??GTP and ??) can then associate with downstream effectors to modulate various aspects of cellular physiology. In addition to interacting with G proteins, agonist- bound GPCRs associate with GPCR kinases (GRKs), leading to receptor phosphorylation. GRKs are a family of seven related kinases (GRK1?GRK7) that have differ- ential patterns of distribution across tissues and distinct preferences for binding to certain receptors 2?4 . However, a common outcome of GPCR phosphorylation by GRKs is a decrease in GPCR interactions with G proteins and an increase in GPCR interactions with arrestins (mem- bers of a family of four closely related scaffold proteins). The interaction of GPCRs with arrestins further inhibits GPCR signalling through G proteins and simultaneously turns on other signalling pathways that are initiated by the arrestin-mediated recruitment of signalling proteins to activated GPCRs 2 . Furthermore, arrestins can directly link active receptors to clathrin-coated pits to facilitate receptor endocytosis, which is an important process controlling the desensitization and resensitization of GPCR activity 5 . Interactions with G proteins, GRKs and arrestins have been intensively studied for numerous GPCRs and have been exhaustively reviewed elsewhere 2?5 . For this reason, these broadly important interactions, which now represent a canonical model of GPCR regulation (BOX 1), are not further reviewed here. Similarly, the importance of homomeric and heteromeric interactions between GPCRs has also been thoroughly reviewed elsewhere 6?8 and is not addressed here. The focus of this Review is on recent advances in the characterization of receptor- selective GPCR associations with various proteins out- side the four previously mentioned families of general GPCR-interacting proteins (G proteins, GRKs, arrestins and other receptors). These GPCR-selective partners can mediate GPCR signalling, organize GPCR signal- ling through G proteins, direct GPCR trafficking, anchor GPCRs in particular subcellular areas and/or influence GPCR pharmacology. As many of these partners exhibit limited patterns of tissue expression, these interactions can also help to explain cell type-specific fine-tuning of GPCR functional activity. Mediation of GPCR signalling For a GPCR-interacting protein to be considered a mediator of GPCR signalling, it would seem that the protein?s interaction with the receptor needs to be regulated by agonist stimulation, as agonist-induced Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia, 30322, USA. Correspondence to R.A.H. e-mail: rhall@pharm.emory.edu doi:10.1038/nrm2803 Agonist A molecule that binds to and stimulates a receptor to trigger a cellular response. Clathrin-coated pit An invaginated membrane structure involved in receptor endocytosis. It consists of a cluster of transmembrane receptors that are attached by adaptor proteins to the protein clathrin, on the cytosolic side of the membrane. Fine-tuning of GPCR activity by receptor-interacting proteins Stefanie L. Ritter and Randy A. Hall Abstract | G protein-coupled receptors (GPCRs) mediate physiological responses to various ligands, such as hormones, neurotransmitters and sensory stimuli. The signalling and trafficking properties of GPCRs are often highly malleable depending on the cellular context. Such fine-tuning of GPCR function can be attributed in many cases to receptor-interacting proteins that are differentially expressed in distinct cell types. In some cases these GPCR-interacting partners directly mediate receptor signalling, whereas in other cases they act mainly as scaffolds to modulate G protein-mediated signalling. Furthermore, GPCR-interacting proteins can have a big impact on the regulation of GPCR trafficking, localization and/or pharmacological properties. REVIEWS NATURE REVIEWS | Molecular cell Biology VOLUME 10 | DECEMBER 2009 | 819 � 2009 Macmillan Publishers Limited. All rights reserved Plasma membrane Nature Reviews | Molecular Cell Biology ? ? ? 12/13 RhoGEFAdenylyl cyclase PLC? ?cAMP PKA RhoA PKC ?Ca 2+ DAG Ins(1,4,5)P 3 P P ? ? ? GRK ? ? ? GTP P P P P ? ? ? GTP G protein-mediated signalling P P Endosome Recycling endosome Resensitization Desensitization Lysosome ERK Other pathways AP2 Clathrin GPCR Ion channel Agonist a G protein-mediated signalling by GPCRs b Arrestin-mediated signalling by GPCRs GPCR Agonist Plasma membrane Arrestin Arrestin Arrestin G protein ? s ? q ? i G protein Ion changes are the essence of receptor-initiated signalling. For example, GPCR interactions with G proteins, GRKs and arrestins are strongly enhanced by agonist stimu- lation 1?5 . In addition, other GPCR-interacting proteins interact with speci fic GPCRs in an agonist-promoted manner to mediate particular aspects of receptor signal- ling, and these examples are considered in this section. By contrast, some GPCR-interacting proteins associate with receptors in an agonist-independent manner. Such proteins can potentially modulate G protein-mediated signal ling, as described in the next section, but they should not be considered mediators of GPCR signalling as their interactions with GPCRs are not influenced by agonist stimulation. If a GPCR can be considered analo- gous in some ways to a gun, then ligand-dependent inter- actors that mediate signalling are analogous to bullets and ligand-independent interactors that modulate signalling are analogous to silencers and scopes, which influence gun function but do not directly mediate the effects of guns on targets. Agonist-promoted interactors that mediate signalling. Several GPCRs have been shown to initiate cellular signalling through agonist-promoted interactions with members of the janus kinase (Jak) family of non- receptor protein tyrosine kinases. For example, the Box 1 | Canonical mechanisms of GPCR signalling In the classical view of G protein-coupled receptor (GPCR) signalling, an agonist binds to extracellular and/or transmembrane regions of the receptor, leading to its interaction with heterotrimeric G proteins. The GPCR acts as a guanine nucleotide exchange factor, catalysing the exchange of GDP for GTP on the G? subunit and inducing dissociation of the G? and G?? subunits from each other and from the GPCR (see the figure, part a). Activated ??GTP subunits, of which there are multiple subtypes, including G? s , G? i , G? 12/13 and G? q , subsequently bind to and regulate the activity of effectors such as adenylyl cyclase, RhoGEF and phospholipase C? (PLC?). These modulate downstream effectors directly or by generating second messengers (such as cyclic AMP, diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (Ins(1,4,5)P 3 ) that modulate further downstream effectors, such as protein kinase A (PKA) and protein kinase C (PKC). Following their liberation from the heterotrimeric G protein complex, the ?? subunits can also bind to and regulate certain downstream effectors, such as ion channels and PLC?. G protein-mediated signalling by agonist-activated GPCRs can be terminated through GPCR phosphorylation by GPCR kinases (GRKs) and concomitant GPCR association with arrestins, which interact with clathrin and the clathrin adaptor AP2 to drive GPCR internalization into endosomes (see the figure, part b) 2null . GPCR internalization regulates the functional process of receptor desensitization. Recruitment of arrestins to activated GPCRs can also lead to the initiation of distinct arrestin-mediated signalling pathways, including activation of the mitogen-activated protein kinase extracellular signal-regulated kinase (ERK) pathway. Following internalization after association with arrestins, GPCRs can be trafficked to lysosomes, where they are ultimately degraded, or to recycling endosomes for recycling back to the cell surface in the functional process of resensitization nullwhereby the cell is resensitized for another round of signalling. Interestingly, nulliasednullagonists have been recently characterized that specifically activate G protein-mediated signalling pathways over arrestin-mediated GPCR signalling pathways, or vice versa 151,152 . This new concept illustrates the importance of characterizing all GPCR downstream signalling pathways in order to fully exploit the therapeutic potential of clinically important receptors. REVIEWS 820 | DECEMBER 2009 | VOLUME 10 www.nature.com/reviews/molcellbio � 2009 Macmillan Publishers Limited. All rights reserved Nature Reviews | Molecular Cell Biology AGTR1 Angiotensin II a Agonist-promoted signalling Plasma membrane JAK2SHP2 P SSTR2 Somatostatin b Agonist-inhibited signalling STAT P P STAT STAT P P STAT STAT Altered transcription to regulate cell growth Nucleus P13K p85 p110 Stronger signalling Weaker signalling p85 p110 Akt signalling pathway Cell survival and proliferation agonist-dependent activation of type 1 angiotensin II receptor (AGTR1) can recruit a complex of JAK2 and tyrosine phosphatase non-receptor type 11 (PTPN11; also known as SHP2) to associate with the AGTR1 car- boxyl terminus, which facilitates JAK2 phosphorylation and activation 9,10 . Activated JAK2 can then recruit and phosphorylate members of the signal transducers and activators of transcription (STAT) family of trans- cription factors. Phosphorylated STAT dissociates from JAK2 and translocates to the nucleus to regulate the transcription of target genes (FIG. 1a). The agonist- promoted interaction between JAK2 and AGTR1 shows an additional signalling avenue for AGTR1, beyond the receptor?s well-established coupling to the G protein subunit G? q . It can also help to explain certain effects of AGTR1 stimulation on cellular physiology that are not explained by G protein-mediated signalling 11 . Interestingly, AGTR1 coupling to G? q can induce a rise in intracellular Ca 2+ that further potentiates Jak?STAT signalling by this GPCR 12,13 , providing an example of how G protein-dependent and G protein-independent signalling mechanisms can in some cases work syner- gistically. Another GPCR that can interact with JAK2 is platelet-activating factor receptor (PTAFR), which asso- ciates with a tyrosine kinase 2 (TYK2)?JAK2 complex in an agonist-regulated manner 14,15 . A mutant version of PTAFR that does not couple to G proteins but still inter- acts with the TYK2?JAK2 complex is fully capable of activating downstream STATs, showing the physiological importance of PTAFR?s recruitment of JAK2 (REF. 15). GPCR interactions with proteins that possess PDZ domains can also, in some cases, mediate agonist- promoted GPCR signalling. PDZ domains, named after the first three proteins in which they were discovered (Postsynaptic density protein 95, Discs large and Zonula occludens protein 1), can mediate high-affinity inter- actions with specific motifs at the distal C termini of target proteins 16 . For example, the PDZ protein Na + ?H + exchange regulatory factor 1 (NHERF1; also known as EBP50 and SLC9A3R1) has been shown to associate in an agonist-promoted manner with the C terminus of the ? 2 -adrenergic receptor (? 2 AR) 17,18 . The recruitment of NHERF1 to ? 2 AR disrupts the ability of NHERF1 to inhibit Na + ?H + exchanger type 3 (NHE3; also known as SLC9A3), providing a G protein-independent mecha- nism by which ? 2 AR can activate Na + ?H + exchange in kidney cells 17 . The ?-type opioid receptor (?OPR) is another GPCR that can regulate Na + ?H + exchange through agonist-induced interactions with NHERF1 (REFS 19,20). Interestingly, the studies on ?OPR provide an example of how interactions between a GPCR and a protein such as NHERF1 can confer cell type-specific signalling to a given GPCR: ?OPR stimulation robustly activates NHE3 activity in cell lines expressing high levels of NHERF1 but not in other cell lines that lack significant NHERF1 expression 20 . Agonist-disrupted interactors that mediate signalling. In addition to the above examples of how agonist-promoted associations of GPCR-interacting proteins with GPCRs mediate aspects of GPCR signalling, the disruption of interactions between a GPCR and a cytoplasmic binding partner can also initiate cellular signalling. For example, it has been shown that agonist stimulation of somatostatin receptor type 2 (SSTR2) disrupts its constitutive associa- tion with the phosphoinositide 3-kinase (PI3K) subunit p85 to negatively regulate PI3K signalling 21 (FIG. 1b). In the absence of agonist, association of p85 with the first intracellular loop of SSTR2 constitutively enhances PI3K activity to promote cell survival through the Akt pathway. However, following agonist stimulation of SSTR2, asso- ciation of the receptor with p85 is disrupted, leading to decreased PI3K activity and sensitization of cells to stimu li that induce apoptosis 21 . Thus, even though the class- ical examples of proteins that mediate GPCR signalling (G proteins and arrestins) exhibit enhanced associations with receptors following agonist stimulation, it is evident that as long as a GPCR-interacting partner exhibits some type of change in its location and/or activity in response to agonist stimulation, this can be sufficient to initiate cellular signalling. Modulation of GPCR signalling Some GPCR-interacting partners increase the speed and efficiency of GPCR signalling by acting as scaffolds to tether downstream effectors in close proximity to the receptor. Other GPCR-interacting partners can decrease the intensity and/or time course of GPCR signalling by disrupting the association of GPCRs with G proteins or, in some cases, by recruiting negative regulators of GPCR signalling. By finely tuning the spatial and Figure 1 | gPcr signalling can be mediated by receptor-interacting proteins. a | Certain G protein-coupled receptor (GPCR)-interacting proteins can act as mediators of agonist-induced GPCR signalling, independently of G protein- or arrestin-mediated signalling pathways. An example of this phenomenon is the interaction of the non-receptor tyrosine kinase janus kinase 2 (JAK2) with type 1 angiotensin II receptor (AGTR1). Association of JAK2 with tyrosine protein phosphatase non-receptor type 11 (PTPN11; also known as SHP2) and stimulation of AGTR1 with angiotensin II together promote JAK2 association with AGTR1 and the initiation of JAK2-dependent signalling. Activated JAK2 can phosphorylate members of the signal transducers and activators of transcription (STAT) family of transcription factors, which leads to STAT dimerization, STAT translocation into the nucleus and the regulation of genes controlling cell growth. b | In other cases, the agonist-dependent dissociation of an interacting protein from a GPCR can alter the activity of an intracellular signalling pathway. For example, the interaction between the p85 regulatory subunit of phosphoinositide 3-kinase (PI3K) and somatostatin receptor type 2 (SSTR2) is disrupted by agonist stimulation, leading to reduced PI3K-mediated Akt signalling and the suppression of cell survival pathways. REVIEWS NATURE REVIEWS | Molecular cell Biology VOLUME 10 | DECEMBER 2009 | 821 � 2009 Macmillan Publishers Limited. All rights reserved Nature Reviews | Molecular Cell Biology ? ?GTP GTP PTH1R Parathyroid hormone a Plasma membrane PTH1R PKCPLC? NHERF TRPC PKCPLC? NHERF TRPC ? ? ? ? ? Adenylyl cyclase Preferential G? s signalling Preferential G? q signalling ? ?GTP �OPR �OPR Enkephalinb Stronger G protein signalling Weaker G protein signalling ? Periplakin Periplakin ? ? ? G protein G protein G protein G protein ? s ? q ? q Second messenger An intracellular signal, such as cAMP, diacylglycerol or inositol triphosphate, that is rapidly and transiently synthesized following receptor activation in order to further amplify the signal transduction cascade. temporal resolution of signalling, certain GPCR inter- actors can dramatically affect the ability of GPCRs to transduce extracellular stimuli into changes in cellular physiology. Interactors that enhance G protein-mediated signalling. The visual system of Drosophila melanogaster has long been an important model system for studying fast and efficient G protein-mediated signalling. Visual signal- ling in D. melanogaster is mediated by light stimu lation of the GPCR rhodopsin, which couples to G? q to activ- ate phospholipase C? (PLC?). Active PLC? leads to the generation of the second messengers inositol-1,4,5- trisphosphate (Ins(1,4,5)P 3 ) and diacylglycerol and to the opening of the Ca 2+ transient receptor potential channels (TRPCs). The influx of Ca 2+ and the production of dia- cylglycerol lead to activation of protein kinase C (PKC), which plays a key part in the termination of visual sig- nalling. Interestingly, almost all of the components of the D. melanogaster visual signalling pathway are tethered together by a large PDZ domain-containing scaffold protein known as Inactivation-no-after-potential D pro- tein (INAD), which associates with rhodopsin, TRPC, PLC? and PKC 22?28 . By tethering these downstream effec- tors in close proximity to rhodopsin, INAD creates an efficient signalling complex that dramatically increases the speed and amplitude of physiological responses to light stimulation 29 . Analogous to D. melanogaster INAD, some mamma- lian PDZ scaffold proteins have been found to interact with GPCRs to enhance the efficiency of GPCR-stimulated G protein signalling. For example, the association of NHERF1 and/or the closely related protein NHERF2 (also known as SLC9A3R2) with various GPCRs, including parathyroid hormone 1 receptor (PTH1R) 30?34 , lysophos- phatidic acid receptor 2 (LPAR2) 35 , purinergic recep- tor (P2RY1) 36 and metabotropic glutamate receptor 5 (mGluR5) 37 , can enhance their PLC?-mediated signal- ling. Unlike the aforementioned interactions of NHERF1 with ? 2 AR and ?OPR, which are regulated by agonists, the associations of the NHERF proteins with PTH1R, LPAR2, P2RY1 and mGluR5 are not altered by agonist stimulation and they mainly seem to enhance G protein-mediated signalling. Interestingly, many known NHERF-binding partners (in addition to GPCRs and NHE3) are com- ponents of G? q ?PLC? signalling pathways; for example, G? q 38 , several TRPCs 39,40 , various isoforms of PLC? 36,41 , PKC 42 and protein kinase D (PKD) 43 . The interaction of NHERF2 with PTH1R pro- vides a particularly compelling example as to how receptor-interacting scaffolds can help to explain cell type-specific fine-tuning of GPCR signalling. In osteoblast-like ROS 17/2.8 cells (which do not express detectable levels of NHERF proteins) PTH1R signals mainly by regulating adenylyl cyclase, but in human umbilical vein endo thelial ECV304 cells (which contain high levels of both NHERF1 and NHERF2) PTH1R signals mainly through PLC? regulation 30 (FIG. 2a). Thus, the enigmatic ability of PTH1R to signal by regulating adeny- lyl cyclase in some cell types but PLC? in other cell types might be accounted for in many cases by differential cellular expression of the NHERF proteins 44 . In addition to NHERF proteins and INAD, various other PDZ scaffold proteins have been shown to asso- ciate with specific GPCRs to enhance certain signal- ling pathways (TABLE 1). For example, association of multiple PDZ domain protein (MUPP1; also known as MPDZ) with ?-aminobutyric acid B (GABA B ) receptors 45 and melatonin type 1 receptors 46 results in markedly enhanced G? i -mediated signalling following receptor stimulation. Similarly, interactions of the PDZ scaffol d membrane-associated guanylate kinase, WW and PDZ domain-containing protein 3 (MAGI3) with the GPCRs Frizzled 4 (REF. 47) and LPAR2 (REF. 48) enhance receptor-mediate d activation of the mitogen-activated protein kinase (MAPK) extra cellular signal-regulated kinase (ERK) pathway. Conversely, associations of LPAR2 with two related PDZ scaffold proteins, PDZ- RhoGEF and leukaemia-associated RhoGEF (LARG; also known as ARHGEF12), do not result in enhanced downstream MAPK activation but rather potentiate LPAR2 -induced stimulation of Rho signalling to modify cytoskeleton dynamics 49 . Thus, LPAR2 can preferen- tially couple to downstream PLC? activation 35 , MAPK activation 48 or Rho activation 49 , in a cell type-specific manner, depending on which LPAR2-interacting PDZ scaffold protein is expressed. Other prominent examples of GPCR-associated proteins that enhance the efficiency of G protein-mediated signalling are the Homer proteins, Figure 2 | gPcr-interacting proteins can modulate g protein-mediated signalling. a | Interaction between the Na + null + exchange regulatory factor (NHERF) scaffold proteins and parathyroid hormone 1 receptor (PTH1R) leads to a preferential enhancement of downstream G? q -mediated signalling by PTH1R. Through its various proteinnullrotein interaction domains, NHERF not only binds to PTH1R but also tethers multiple downstream signalling effectors, such as phospholipase C? (PLC?), protein kinase C (PKC) and transient receptor potential channel (TRPC), in close proximity to the PTH1R. This creates an efficient complex for preferential G? q -mediated signalling. However, when PTH1R is in a cell type or cellular compartment in which NHERF proteins are absent, PTH1R preferentially signals through G? s to activate adenylyl cyclase. b | By contrast, periplakin can associate with uni03BC-type opioid receptor (uni03BCOPR) to impair G protein-mediated signalling by an unknown mechanism. However, in cell types or cellular compartments where periplakin is not found, uni03BCOPR ligands can more robustly activate the receptor to stimulate signalling by G proteins. GPCR, G protein-coupled receptor. REVIEWS 822 | DECEMBER 2009 | VOLUME 10 www.nature.com/reviews/molcellbio � 2009 Macmillan Publishers Limited. All rights reserved which associate with mGluR1 and mGluR5 (REFS 50?53), and members of the A-kinase anchoring protein (AKAP) family, which interact with ?-adrenergic receptors 54?58 . In addition, Homer proteins also interact with intra- cellular Ins(1,4,5)P 3 receptors, thereby linking Ins(1,4,5)P 3 receptors, mGluRs and other components to increase the efficiency of mGluR-stimulate d Ca 2+ signalling 52,59,60 . The AKAPs were originally named because of their asso- ciations with protein kinase A (PKA). Indeed, the inter- actions of A-kinase anchor protein 79 kDa (AKAP79; also known as AKAP5) 55 and AKAP250 (also known as AKAP12 and gravin) 54,56,57 with ? 2 AR tether PKA in the vicinity of the receptor and increase the efficiency of PKA-mediated phosphorylation of various substrates that Table 1 | GPCR interactors that mediate or modulate GPCR signalling interactor* associated gPcr Site of interaction impact on gPcr refs AKAP79 (AKAP5) and AKAP250 (AKAP12, gravin) ? 2 AR and ? 1 AR CT Tethers PKA near to the receptor 54null8 Calmodulin 5-HT1A i3L Competes with PKC for GPCR phosphorylation 78 5-HT2A i2L and CT Impairs G protein coupling 79 5-HT2C CT Promotes arrestin-dependent ERK activation 80 D2R i3L Modulates G protein signalling 81,82 mGluR7 CT Regulates GPCR phosphorylation 76,77 PTH1R CT Inhibits GPCR activity 85 V2R CT Enhances GPCR-induced Ca 2+ signalling 84 uni03BCOPR i3L Inhibits G protein coupling 83 Homer mGluR1 and mGluR5 CT Regulates GPCR signalling and localization 51,52, 126null28, 153,154 INAD Rhodopsin CT Enhances speed and efficiency of GPCR signalling 22null9 JAK2 AGTR1 and PTAFR CT Promotes JaknullTAT signalling 9,10,12null5 LARG (ARHGEF12) LPAR2 CT Facilitates GPCR-mediated activation of Rho 49 MAGI3 Frizzled 4 and LPAR2 CT Potentiates GPCR-mediated activation of the MAPK ERK 47,48 MUPP1 (MPDZ) GABA B and MT 1 CT Enhances GPCR-mediated G? i signalling 45,46 Neurochondrin MCHR1 CT Disrupts G protein-mediated signalling 88 NHERF1 (EBP50, SLC9A3R1) PTH1R CT Enhances G? q -mediated receptor signalling 31null4 ? 2 AR and ?OPR CT Mediates activation of Na + null + exchange 17null0,122 NHERF2 (SLC9A3R2) LPAR2 CT Enhances G? q -mediated receptor signalling 35 mGluR5 and P2RY1 CT Prolongs GPCR-mediated Ca 2+ signalling 36,37 PTH1R CT Enhances G? q -mediated receptor signalling 30 p85 SSTR2 i1L and CT Mediates survival signalling by receptor 21 PDZ-RhoGEF (ARHGEF11) LPAR2 CT Facilitates GPCR-mediated activation of Rho 49 Periplakin MCHR1 and uni03BCOPR CT Impairs G protein-mediated signalling 86,87 Spinophilin D2R i3L Reduces G protein and arrestin-mediated signalling 61 ? 2 AR and mAChR i3L Reduces GPCR-mediated Ca 2+ signalling 62null5 *Not included in this list are GPCR interactions with G proteins, GPCR kinases, arrestins, regulators of G protein signalling proteins or other receptors. Alternative protein names are provided in brackets. AGTR1, type 1 angiotensin II receptor; AKAP, A-kinase anchor protein; AR, adrenergic receptor; CT, carboxyl terminus, D2R, D2 dopamine receptor; GABA, ?-aminobutyric acid; GEF, guanine nucleotide exchange factor; GPCR, G protein-coupled receptor; i1L, first intracellular loop; i2L, second intracellular loop; i3L, third intracellular loop; INAD, Inactivation-no-after-potential D protein; JAK2, janus kinase 2; LARG, leukaemia-associated RhoGEF; LPAR2, lysophosphatidic acid receptor 2; mAChR, muscarinic acetylcholine receptor; MAGI3, membrane-associated guanylate kinase, WW and PDZ domain-containing protein 3; MAPK, mitogen-activated protein kinase; MCHR1, melanin- concentrating hormone receptor 1; mGluR, metabotropic glutamate receptor; MT1, melatonin type 1 receptor; MUPP1, multiple PDZ domain protein; NHERF, Na + null + exchange regulatory factor; OPR, opioid receptor; P2RY1, purinergic receptor; PKA, protein kinase A; PKC, protein kinase C; PTAFR, platelet-activating factor receptor ; PTH1R, parathyroid hormone 1 receptor; SSTR2, somatostatin receptor type 2; STAT, signal transducers and activators of transcription; V2R, vasopressin V2 receptor. REVIEWS NATURE REVIEWS | Molecular cell Biology VOLUME 10 | DECEMBER 2009 | 823 � 2009 Macmillan Publishers Limited. All rights reserved Heterologous cell A cell that lacks endogenous expression of a gene of interest but is manipulated, for example by transfection or viral infection, to express the gene. are downstream of receptor activation, including ? 2 AR itself. The consequences of the increased functional rela- tionship between PKA and ? 2 AR include the enhanced efficiency of receptor resensitization 54,57 and more robust ? 2 AR-mediated ERK signalling 55 . Interactors that reduce G protein-mediated signalling. In contrast to the above examples of GPCR-interacting proteins, which increase the efficiency of certain GPCR- stimulated signalling pathways, some GPCR-interacting proteins associate with GPCRs to decrease the effi- ciency of G protein-mediated signalling. The arrestins are perhaps the most general example of this phenom- enon. A more receptor-specific example is spinophilin, which interacts with the third intracellular loop of a few GPCRs, including members of the dopamine, adren- ergic and muscarinic acetylcholine receptor families 61?65 . Spinophilin also binds to several members of the regu- lators of G protein signalling (RGS) family of proteins. RGS proteins tightly regulate the intensity and time course of GPCR signalling by accelerating the inherent GTPase activity of activated G? subunits. Thus, spinophilin tethers RGS proteins in close proximity to GPCRs to attenuate receptor-stimulate d G protein signalling 63,64 . Interestingly, RGS proteins can also associate directly with the intra- cellular regions of some GPCRs to inhibit their signalling and exert cell type-specific regulation of their activity 66?71 . RGS proteins have been reviewed in detail elsewhere 72,73 and are therefore not discussed further here. Calmodulin is another protein that can interact with a variety of GPCRs to modulate their functional properties. This widely expressed Ca 2+ -binding protein can associate in a Ca 2+ -sensitive manner with mGluRs 74?77 and sero- tonin (also known as 5-hydroxytryptamine (5-HT)) 78?80 , dopamine 81,82 and other 83?85 receptors. The functional effects of calmodulin interaction vary depending on the GPCR, but perhaps the most commonly reported effect of calmodulin?GPCR interaction is an attenuation of G pro- tein coupling 79,81,83,85 . As stimulation of many GPCRs can result in a downstream increase in cellular Ca 2+ levels, the Ca 2+ -dependent interaction of calmodulin with GPCRs can, in some cases, represent a form of feedback inhibition that restrains GPCR-initiated G protein signalling. However, G protein-independent signalling pathways can actually be potentiated by GPCR inter actions with calmodulin in some situations, as calmodulin association with the 5-HT2C receptor strongly promotes arrestin-mediated signalling (but not G protein-mediated signalling) by the receptor in both transfected cells and cultured neurons 80 . Two other proteins that can associate with a few GPCRs to tone down G protein-mediated signalling are periplakin and neurochondrin. Periplakin was first reported to associate with the C-terminus of uni03BC-type opioid receptor (uni03BCOPR) 86 and melanin-concentrating hormone receptor 1 (MCHR1) 87 , and neurochondrin was found to interact with the same region of the MCHR1 C terminus as periplakin 88 . Both periplakin and neuro- chondrin have recently been shown to also interact with a few other GPCRs 89 . For all of the GPCRs that associate with periplakin and neurochondrin, the primary func- tional consequence is an attenuation of G protein-mediated signalling 86?89 (FIG. 2b). As periplakin and neurochondrin exhibit discrete patterns of distribution in the brain and other tissues 89 , it seems likely that they contribute to the cell-context-dependent sculpting of receptor signalling for the various GPCRs with which they interact. Considered together with the other GPCR partners discussed in this section, the emerging theme is that GPCR-interacting proteins can exert bidirectional effects on the efficiency of G protein-mediated signalling to impart cell type-specific fine-tuning of GPCR activity. Regulation of GPCR trafficking GPCRs are typically trafficked to the plasma membrane to achieve functional activity. Following agonist stimulation, most receptors are internalized into endosomes and then either targeted for lysosomal degradation or recycled back to the plasma membrane. A number of GPCR-interacting proteins have been shown to exert dramatic effects on both the biosynthetic trafficking and the post-endocytic sorting of particular GPCRs. Interactors that regulate biosynthetic trafficking. GPCRs must be properly folded after translation and, in most cases, transported to the plasma membrane to achieve functional activity. A number of GPCR-interacting pro- teins can regulate the folding, biosynthetic trafficking and surface expression of receptors in a cell type- and receptor- specific manner. A classic example is the D. melano- gaster neither inactivation nor afterpotential protein A (NINAA), which associates with the GPCR rhodopsin to enhance the receptor?s folding and forward trafficking 90,91 . Similarly, Ran-binding protein 2 is a vertebrate homo- logue of NINAA that associates with vertebrate opsins to enhance the biosynthetic trafficking of these GPCRs 92 . Another protein that regulates rhodopsin trafficking is the dynein light chain component T-complex testis-specific protein 1 homologue (TCTEX1; also known as DYNLT1), which directly associates with vertebrate rhodopsin to promote its trafficking to the cell surface 93,94 . Other verte- brate proteins that have been found to act as chaperones to enhance GPCR-specific surface expression include glandular epithelial cell protein 1 (GEC1; also known as GABARAPL1) 95,96 , receptor of activated protein kinase C1 (RACK1; also known as GNB2L1) 97 , dopamine receptor- interacting protein of 78 kDa (DRIP78; also known as DNAJC14) 98,99 , AT2 receptor binding protein of 50 kDa (ATBP50) 100 and ubiquitin-specific-processing protease 4 (USP4) 101 , which are listed in TABLE 2. For each of these GPCR-interacting proteins, their expression level in a given cell type can strongly influence the level of func- tional receptor expression for the particular GPCRs with which they interact. Olfactory receptors, the largest subfamily of GPCRs, have proved notoriously difficult to study in heterologous cells (that is, in cells other than olfactory sensory neu- rons, which naturally express olfactory GPCRs) because of their poor trafficking to the plasma membrane 102 . This trafficking deficiency suggests a key role for chaperone proteins in the cell type-specific control of anterograde olfactory receptor trafficking in olfactory sensory neu- rons. Indeed, Caenorhabditis elegans odorant response REVIEWS 824 | DECEMBER 2009 | VOLUME 10 www.nature.com/reviews/molcellbio � 2009 Macmillan Publishers Limited. All rights reserved Table 2 | GPCR interactors that regulate GPCR trafficking, targeting and/or ligand binding interactor* associated gPcr Site of interaction impact on gPcr refs ATBP50 AGTR2 CT Enhances GPCR surface expression 100 DRIP78 (DNAJC14) AGTR1 and D1R CT Promotes GPCR surface expression 98,99 GASP1 CNR1, D2R and ?OPR CT Targets GPCR to lysosomes for degradation 114null17 GEC1 (GABARAPL1) EP3R and ?OPR CT Enhances GPCR surface expression 95,96 Homer mGluR1 and mGluR5 CT Regulates GPCR signalling and localization 51,52,126null 128,153,154 M10 MHC V2R Unknown Enhances GPCR surface expression 108 MAGI2 ? 1 AR CT Promotes agonist-induced ? 1 -AR internalization 138 MPP3 5-HT2C CT Inhibits agonist-induced 5-HT2C internalization 139 MRAP and MRAP2 MC2R NT and TM Promotes forward trafficking of GPCR 109null13 MUPP1 5-HT2C CT Induces GPCR clustering 155 GABA B CT Increases GPCR stability 45 SSTR3 CT Targets GPCR to epithelial tight junctions 144 NHERF1 (EBP50, SLC9A3R1) ? 2 AR and ?OPR CT Promotes GPCR recycling 17null0,122 NINAA Rhodopsin Unknown Promotes GPCR biogenesis and trafficking 90,91 ODR4 ODR10 Unknown Facilitates GPCR folding and surface expression 103 PICK1 mGluR7a CT Facilitates GPCR clustering in presynaptic active zones 140,141,156 PSD95 5-HT2A CT Impairs GPCR internalization and directs localization 134,135 ? 1 AR CT Attenuates agonist-promoted GPCR internalization 136,138 RACK1 (GNB2L1) TXA2R i1L and CT Promotes forward trafficking of GPCR 97 RAMP1 CRLR NT and TM Forms functional CGRP receptors 146 CALCR NT and TM Forms functional amylin receptors 149,150 RAMP2 CRLR NT and TM Forms functional adrenomedullin receptors 146 RAMP3 CRLR NT and TM Forms functional adrenomedullin receptors 147,148 CALCR NT and TM Forms functional amylin receptors 149,150 RanBP2 Opsin Unknown Promotes forward trafficking of GPCR 92 REEPs OR Unknown Promotes forward trafficking of GPCR 104 RTPs OR Unknown Promotes forward trafficking of GPCR 104,105 RTPs and REEPs T2R Unknown Promotes GPCR surface expression 106 RTP4 uni03BCOPR and ?OPR CT Promotes heterodimer surface expression 107 Shank LPHN1 (CL1) CT Promotes GPCR clustering 132 mGluR1 and mGluR5 CT Anchors GPCR in mature dendritic spines 129 SNX1 PAR1 (F2R) CT Facilitates agonist-promoted GPCR degradation 119,120 Syntrophin ? 1D AR CT Enhances GPCR stability 142,143 TCTEX1 (DYNLT1) Rhodopsin CT Promotes apical delivery of GPCR in polarized cells 93,94 USP4 ? 2A AR CT Enhances GPCR surface expression 101 *Not included in this list are GPCR interactions with G proteins, GPCR kinases, arrestins, regulators of G protein signalling proteins or other receptors. Alternative protein names are provided in brackets. AR, adrenergic receptor; AGTR1, type-1 angiotensin II receptor; AGTR2, type-2 angiotensin II receptor; ATBP50, AT2 receptor binding protein of 50 kDa; CALCR, calcitonin receptor; CGRP, calcitonin gene-related peptide; CNR1, cannabinoid receptor 1; CRLR, calcitonin receptor-like receptor; CT, carboxyl terminus; D1R, D1 dopamine receptor; D2R, D2 dopamine receptor; DRIP78, dopamine receptor- interacting protein of 78 kDa; EP3R, prostaglandin E receptor 3; GABA B , ?-aminobutyric acid B; GASP1, GPCR-associated sorting protein 1; GEC1, glandular epithelial cell protein 1; GPCR, G protein-coupled receptor; i1L, first intracellular loop; LPHN1, latrophilin 1; MAGI2, membrane-associated guanylate kinase, WW and PDZ domain-containing protein 2; mGluR, metabotropic glutamate receptor; MHC, major histocompatibility complex; MPP3, MAGUK p55 subfamily member 3; MC2R, melanocortin receptor 2; MRAP, MC2R-accessory protein; MUPP1, multiple PDZ domain protein; NHERF1, Na + null H + exchange regulatory factor 1; NINAA, neither inactivation nor afterpotential protein A; NT, amino terminus; ODR, odorant response abnormal protein; OPR, opioid receptor; OR, olfactory receptor; PAR1, proteinase-activated receptor 1; PICK1, protein interacting with C kinase; PSD95, postsynaptic density protein 95; RACK1, receptor of activated protein kinase C1; RanBP2, Ran-binding protein 2; RAMP, receptor activity-modifying protein; REEP, receptor expression-enhancing protein; RTP, receptor transporting protein; SNX1, sorting nexin 1; SSTR3, somatostatin receptor type 3; TCTEXI, T-complex testis-specific protein 1; T2R, taste receptor type 2; TM, transmembrane; TXA2R, thromboxane A2 receptor; USP4, ubiquitin-specific-processing protease 4; V2R, vasopressin V2 receptor. REVIEWS NATURE REVIEWS | Molecular cell Biology VOLUME 10 | DECEMBER 2009 | 825 � 2009 Macmillan Publishers Limited. All rights reserved NHERF1 NHERF1 NHERF1 NHERF1 GASP1 GASP1 GASP1 Nature Reviews | Molecular Cell Biology ? ?GTP ?OPR ?OPR ?OPR downregulation Enkephalina G protein-mediated signalling ? ? ?GTP ? G protein-mediated signalling Arrestin P P Arrestin GASP1 P P P P Endosome Arrestin Recycling endosome P P EndosomeLysosome Arrestin ? ?GTP ? 2 AR ? 2 AR ? 2 AR downregulation Adrenalineb G protein-mediated signalling ? ? ?GTP ? G protein- and NHERF-mediated signalling Arrestin P P Arrestin P P P P Endosome Arrestin Recycling endosome P P EndosomeLysosome Arrestin G protein G protein G protein G protein Familial glucocorticoid deficiency type 2 A rare, autosomal recessive disorder in which affected individuals are unresponsive to adrenocorticotropin owing to mutations in the gene encoding MRAP. abnormal protein 4 (ODR-4) is expressed exclusively in chemosensory neurons, where it regulates the forward trafficking of chemosensory receptors such as the GPCR ODR-10 (REF. 103). In vertebrates, two unrelated families of transmembrane proteins have been shown to associate with olfactory receptors to enhance receptor trafficking to the plasma membrane: the receptor transporting proteins (RTPs) and the receptor expression-enhancing pro- teins (REEPs) 104,105 . Certain RTP and REEP isoforms are expressed exclusively in the olfactory epithelium, where they exert a cell type-specific enhancement of olfactory receptor trafficking 104 . However, other RTP and REEP isoforms are more widely expressed and can interact with other GPCRs, including bitter taste receptor type 2 (REF. 106) and opioid receptors 107 , to enhance the trafficking of these GPCRs to the plasma membrane. Analogous to the RTPs and REEPs, two other types of single-transmembrane proteins have been shown to control the biosynthetic trafficking of particular GPCRs. Vomeronasal type 2 receptors (V2Rs) had proved difficult to study in heterologous cells until the observation that they interact with M10 major histocompatibility complex (MHC) molecules, which associate with the MHC com- ponent ?2-microglobulin to promote the surface expres- sion of V2Rs in heterologous cells 108 . In a similar manner, melanocortin receptor 2 (MC2R)-accessory protein (MRAP) and MRAP2 can directly interact with MC2R and dramatically enhance the receptor?s surface expres- sion 109?113 . The physiological importance of MC2R?MRAP interactions in vivo has been well established by studies revealing that naturally occurring MRAP mutations cause defects in MC2R trafficking and function, resulting in an inherited disease known as familial glucocorticoid deficiency type 2 (REF. 109). Interactors that influence post-endocytic trafficking. Most GPCRs undergo significant endocytosis from the plasma membrane in response to agonist stimulation. In some cases the receptors are recycled back to the plasma mem- brane, but in other cases they are targeted to lysosomes for degradation 5 . GPCR internalization is heavily influenced by two of the canonical families of GPCR-interacting proteins, the GRKs and the arrestins 2?5 . However, cer- tain other GPCR-interacting proteins can also regulate Figure 3 | gPcr-interacting proteins can regulate the post-endocytic trafficking of gPcrs. Following agonist-induced receptor endocytosis, some G protein-coupled receptors (GPCRs) are targeted for proteolytic and/or lysosomal degradation, whereas other GPCRs rapidly recycle back to the plasma membrane. a | The interaction between GPCR-associated sorting protein 1 (GASP1) and ?-type opioid receptor (?OPR) promotes the endocytic targeting of agonist-internalized ?OPRs to lysosomes, where the receptors are degraded. However, in a distinct cellular compartment (or distinct cell type) that lacks GASP1, as shown on the left, ?OPRs are rapidly recycled back to the plasma membrane. b | By contrast, the interaction between the GPCR-interacting protein Na + null + exchange regulatory factor 1 (NHERF1; also known as EBP50 and SLC9A3R1) and the ? 2 -adrenergic receptor (? 2 AR) promotes the rapid recycling of receptors following agonist-promoted internalization. However, in a distinct cellular compartment (or distinct cell type) that lacks NHERF1, as shown on the left, ? 2 ARs are preferentially targeted to lysosomes for degradation. REVIEWS 826 | DECEMBER 2009 | VOLUME 10 www.nature.com/reviews/molcellbio � 2009 Macmillan Publishers Limited. All rights reserved Postsynaptic density A specialized postsynaptic compartment, highly enriched in various scaffold proteins and receptors, that is found at all asymmetric (usually glutamatergic) synapses in vertebrate central nervous systems. Cortical pyramidal neuron The predominant type of neuron in the neocortex. It is named after its triangular cell body. Palmitoylation Post-translational modification of a protein by the covalent attachment of a palmitate (a 16-carbon saturated fatty acid) to a cysteine residue through a thioester bond. Dystrophin-associated glycoprotein complex A series of protein subcomplexes, including complexes of the adaptor protein dystrophin, cytoskeletal proteins, the sarcoglycan complex and the ?,?-dystroglycan complex, which together are required to link the cytoskeleton to the extracelluar matrix in muscle cells. the endocytic trafficking of GPCRs in a more receptor- selective manner. For example, the GPCR-associated sorting proteins (GASPs) comprise a family of ten pro- teins, and the founding member, GASP1, was originally identified in a yeast two-hybrid screen for proteins that interact with the C terminus of the ?-type opioid receptor (?OPR) 114 . Association of ?OPR with GASP1 promotes receptor trafficking to lysosomes following agonist- stimulated endocytosis 114 (FIG. 3a), and GASP1 has a simi- lar effect on a few other GPCRs, including D2 dopamine receptor 115 and cannabinoid receptor 1 (REFS 116,117). Most of the GASP family members are preferentially expressed in the central nervous system (CNS) 118 , suggesting that they might act in a cell type-specific manner to control the post-endocytic fate (recycling versus degradation) of certain CNS-enriched GPCRs. In addition to the members of the GASP family, a few other GPCR-interacting proteins influence the post- endocytic sorting of particular receptors. For example, sorting nexin 1 (SNX1) directly associates with proteinase- activated receptor 1 (PAR1; also known as F2R) to promote PAR1 post-endocytic trafficking to lysosomes 119,120 . SNX1 can also bind to the C termini of several other GPCRs 121 , but how this influences the trafficking of these receptors is still unknown. In contrast to the effects of GASPs and SNX1, which associate with GPCRs to decrease receptor recycling to the plasma membrane, GPCR interactions with NHERF1 have been found to promote receptor recy- cling following endocytosis 19,122?124 . As mentioned earlier, NHERF1 interactions with ? 2 AR and ?OPR mediate certain aspects of GPCR signalling, and NHERF inter- actions with other GPCRs can modulate signalling by G proteins. Interestingly, mutant versions of ? 2 AR that cannot associate with NHERF1 are targeted much more robustly than wild-type receptors to lysosomes after ago- nist stimulation 122 , revealing that their interaction with NHERF1 promotes receptor recycling (FIG. 3b). The asso- ciation of ?OPR with NHERF1 also favours GPCR recy- cling over GPCR targeting to lysosomes 19 , and interaction of PTH1R with NHERF1 increases the plasma membrane retention of this receptor 125 . Moreover, transplantation of the NHERF1 binding motif onto the C termini of GPCRs that do not normally interact with NHERF1, such as ?OPR, dramatically enhances the efficiency of receptor recycling back to the plasma membrane following endo- cytosis 123,124 . These findings provide an example of how certain GPCR-interacting proteins, such as NHERF1, can act as signalling intermediates, regulators of G protein sig- nalling and regulators of receptor trafficking, analogous to the multiple roles of arrestins in the signalling and trafficking of many GPCRs 2 . Control of GPCR targeting In addition to the effects of many of the aforementioned GPCR-interacting proteins on GPCR signalling and trafficking, some of these interactions control GPCR anchoring to discrete regions of the plasma membrane. For example, Homer proteins not only enhance the effi- ciency of mGluR1- and mGluR5-mediated Ca 2+ signal- ling (as described above), they also facilitate the clustering and anchoring of mGluR1- and mGluR5 in postsynaptic dendritic spines 126?128 . Consequently, mGluR1 and mGluR5 can be selectively localized near the postsynaptic density, positioning them to respond to the glutamate that is released into the synaptic cleft. Members of the Shank family of PDZ scaffold proteins interact with both Homer proteins and mGluRs to further strengthen the anchoring of mGluRs to postsynaptic regions 129?131 . The Shank family of scaf- folding proteins also induce clustering of the latrotoxin- binding GPCR latrophilin 1 (LPHN1; also known as CL1 and CIRL1) in heterologous cells and colocalize with latrophilin 1 at synapses in native brain tissue 132,133 . A variety of PDZ scaffold proteins, in addition to the Shank family, have been shown to regulate GPCR-specific clustering and anchoring. For example, interaction of PSD95 (also known as DLG4) with 5-HT2A receptors induces the clustering of 5-HT2A receptors in heter- ologous cells 134 and facilitates the targeting of 5-HT2A receptors to postsynaptic dendritic compartments in cul- tured cortical pyramidal neurons 135 . PSD95 also associates with the C terminus of ? 1 AR to facilitate the clustering of this receptor with other components of the postsynaptic density, such as NMDA (N-methyl-d.sc-aspartate) recep- tors 136 . The anchoring of both 5-HT2A receptor and ? 1 AR to the plasma membrane as a result of their asso- ciation with PSD95, which is known to be palmitoylated and therefore tightly membrane associated 137 , greatly reduces their agonist-induced internalization 134,136,138 . By contrast, PSD95 association with 5-HT2C recep- tors facilitates agonist-dependent internalization, and another PDZ scaffold protein, MAGUK p55 subfamily member 3 (MPP3), associates with 5-HT2C receptors to prevent agonist-dependent internalization and to stabi- lize the receptors at the cell surface in primary cortical neurons 139 . Furthermore, interaction of the mGluR7a C terminus with the PDZ scaffold protein PICK1 (protein interacting with C kinase) results in the specific clustering of mGluR7a at presynaptic sites 140,141 , interaction of the ? 1D -adrenergic receptor with syntrophins enables linkage to the dystrophin-associated glycoprotein complex in smooth muscle cells 142,143 , and the interaction of SSTR3 with MUPP1 enables SSTR3 to be targeted to tight junctions in epithelial cells 144 . These examples show how certain GPCR-interacting partners can selectively target GPCRs to specialized cellular compartments to promote the receptors? physiological activities. Regulation of ligand binding The vast majority of GPCR-interacting proteins described above associate with intracellular regions of the GPCRs. Therefore, they have no direct effects on the pharma- cological properties of the GPCRs, which are typically determined by their extracellular and/or transmembrane domains. However, a few GPCR-interacting proteins have been shown to exert striking effects on the agonist selec- tivity of the GPCRs with which they interact. The most intensively studied examples of GPCR-interacting partners that regulate receptor pharmacology are receptor activity- modifying protein 1 (RAMP1), RAMP2 and RAMP3 (REF. 145). The RAMP proteins were initially identified in experiments designed to search for the receptor that was activated by calcitonin gene-related peptide (CGRP). REVIEWS NATURE REVIEWS | Molecular cell Biology VOLUME 10 | DECEMBER 2009 | 827 � 2009 Macmillan Publishers Limited. All rights reserved Surprisingly, it was found that expression of a functional CGRP receptor required co-expression of an orphan GPCR, known as the calcitonin receptor-like recep- tor (CALCRL; also known as CRLR), with a receptor- interacting partner ? the single-transmembrane protein RAMP1 (REF. 146). When CALCRL was co-expressed with RAMP2, this was shown to result in the forma- tion of receptors activated not by CGRP, but rather by a related peptide known as adrenomedullin 146 . Subsequent work has revealed that CALCRL can also interact with RAMP3 to form a distinct subtype of adrenomedullin receptor 147,148 . Moreover, expression of a distinct receptor, calcitonin receptor (CALCR), with any of the RAMPs results in the formation of receptors with unique pharma- cological properties, including the preferential activation of some RAMP?CALCR combinations by a distinct peptide known as amylin 149,150 . It is clear from work in this area that the pharmacological properties of CALCR and CALCRL are heavily regulated in a cell type-specific manner depending on which of the RAMP proteins is expressed, and furthermore the RAMP proteins can also dramatically affect the surface expression levels of CALCR and CALCRL 145 . Thus, a comprehensive under- standing of RAMP?GPCR interactions is essential for developing therapeutics that might target the various RAMP-interacting receptors 145 . Conclusions and perspectives The signalling and trafficking of most GPCRs involves receptor interactions with G proteins, GRKs, arrestins and other receptors. In addition to these widespread canonical GPCR associations, there are many other types of GPCR-interacting proteins that can interact with par- ticular receptors to fine-tune receptor activity. GPCRs are important drug targets and, because it is often desir- able to achieve cell type-specific drug action in order to minimize side effects, it can be clinically useful to consider the ways in which GPCRs can be differentially modulated by therapeutics depending on the cellular context. 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Knock-in mice lacking the PDZ-ligand motif of mGluR7a show impaired PKC-dependent autoinhibition of glutamate release, spatial working memory deficits, and increased susceptibility to pentylenetetrazol. J. Neurosci. 28, 8604?8614 (2008). Acknowledgements The authors? research is funded by the National Institutes of Health, USA. DATABASES UniProtKB: http://www.uniprot.org AGTR1 | AKAP79 | AKAP250 | ? 1 AR | ? 2 AR | CALCR | CALCRL | CGRP | ?OPR | DRIP78 | GASP1 | GEC1 | 5-HT2A | 5-HT2C | INAD | JAK2 | ?OPR | LARG | LPHN1 | LPAR2 | MAGI3 | MCHR1 | mGluR5 | uni03BCOPR | MPP3 | MRAP | MRAP2 | MUPP1 | NHE3 | NHERF1 | NHERF2 | ODR-4 | ODR-10 | PAR1 | PICK1 | P2RY1 | PSD95 | PTAFR | PTH1R | PTPN11 | RACK1 | RAMP1 | RAMP2 | RAMP3 | SNX1 | SSTR2 | SSTR3 | TCTEX1 | USP4 FURTHER INFORMATION Randy A. Hallnulls homepage: http://www.pharm.emory.edu/rhall/ all linkS are active in the online Pdf REVIEWS 830 | DECEMBER 2009 | VOLUME 10 www.nature.com/reviews/molcellbio � 2009 Macmillan Publishers Limited. All rights reserved "
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