SUMMARY Class B G protein-coupled receptors are major targets for treatment of chronic diseases, including osteoporosis, diabetes and obesity. Here we report the structure of a full-length class B receptor, the calcitonin receptor, in complex with peptide ligand and heterotrimeric Gαβγs protein determined by Volta phase plate single-particle cryo-electron microscopy. The peptide agonist engages the receptor through binding to an extended hydrophobic pocket facilitated by the large outward movement of the extracellular ends of transmembrane helices 6 and 7. This conformation is accompanied by a 60° kink in helix 6 and large outward movement of the intracellular end of this helix, opening the bundle to accommodate interactions with the α5-helix of Gαs. Also observed is an extended intracellular helix 8 that contributes to both receptor stability and functional G protein coupling via interaction with the Gβ subunit. This structure provides a new framework for understanding G protein-coupled receptor function.

Receptor activity-modifying proteins (RAMPs) are single-transmembrane proteins that transport the calcitonin receptor-like receptor (CRLR) to the cell surface. RAMP 1-transported CRLR is a calcitonin gene-related peptide (CGRP) receptor. RAMP 2- or RAMP 3-transported CRLR is an adrenomedullin receptor. The role of RAMPs beyond their interaction with CRLR, a class II G protein-coupled receptor, is unclear. In this study, we have examined the role of RAMPs in generating amylin receptor phenotypes from the calcitonin (CT) receptor gene product. Cotransfection of RAMP 1 or RAMP 3 with the human CT receptor lacking the 16-amino acid insert in intracellular domain 1 (hCTRI1-) into COS-7 cells induced specific 125I-labeled rat amylin binding. RAMP 2 or vector cotransfection did not cause significant increases in specific amylin binding. Competition-binding characterization of the RAMP-induced amylin receptors revealed two distinct phenotypes. The RAMP 1-derived amylin receptor demonstrated the highest affinity for salmon CT (IC50, 3.01 +/- 1.44 x 10(-10) M), a high to moderate affinity for rat amylin (IC50, 7.86 +/- 4.49 x 10(-9) M) and human CGRPalpha (IC50, 2.09 +/- 1.63 x 10(-8) M), and a low affinity for human CT (IC50, 4.47 +/- 0.78 x 10(-7) M). In contrast, whereas affinities for amylin and the CTs were similar for the RAMP 3-derived receptor, the efficacy of human CGRPalpha was markedly reduced (IC50, 1.12 +/- 0.45 x 10(-7) M; P <.05 versus RAMP 1). Functional cyclic AMP responses in COS-7 cells cotransfected with individual RAMPs and hCTRI1- were reflective of the phenotypes seen in competition for amylin binding. Confocal microscopic localization of c-myc-tagged RAMP 1 indicated that, when transfected alone, RAMP 1 almost exclusively was located intracellularly. Cotransfection with calcitonin receptor (CTR)I1- induced cell surface expression of RAMP 1. The results of experiments cross-linking 125I-labeled amylin to RAMP 1/hCTR-transfected cells with bis succidimidyl suberate were suggestive of a cell-surface association of RAMP 1 and the receptors. Our data suggest that in the CT family of receptors, and potentially in other class II G protein-coupled receptors, the cellular phenotype is likely to be dynamic in regard to the level and combination of both the receptor and the RAMP proteins.

The receptor activity-modifying proteins (RAMPs) comprise a family of three accessory proteins that heterodimerize with the calcitonin receptor-like receptor (CL receptor) or with the calcitonin receptor (CTR) to generate different receptor phenotypes. However, RAMPs are more widely distributed across cell and tissue types than the CTR and CL receptor, suggesting additional roles for RAMPs in cellular processes. We have investigated the potential for RAMP interaction with a number of Class II G protein-coupled receptors (GPCRs) in addition to the CL receptor and the CTR. Using immunofluorescence confocal microscopy, we demonstrate, for the first time, that RAMPs interact with at least four additional receptors, the VPAC1 vasoactive intestinal polypeptide/pituitary adenylate cyclaseactivating peptide receptor with all three RAMPs; the glucagon and PTH1 parathyroid hormone receptors with RAMP2; and the PTH2 receptor with RAMP3. Unlike the interaction of RAMPs with the CL receptor or the CTR, VPAC1R-RAMP complexes do not show altered phenotypic behavior compared with the VPAC1R alone, as determined using radioligand binding in COS-7 cells. However, the VPAC1R-RAMP2 heterodimer displays a significant enhancement of agonist-mediated phosphoinositide hydrolysis with no change in cAMP stimulation compared with the VPAC1R alone. Our findings identify a new functional consequence of RAMPreceptor interaction, suggesting that RAMPs play a more general role in modulating cell signaling through other GPCRs than is currently appreciated.The discovery of receptor activity-modifying proteins (RAMPs) 1 has led to a re-evaluation of what defines G proteincoupled receptor (GPCR) phenotypic behavior toward agonists and/or G proteins (1). The RAMP family comprises three accessory proteins (designated RAMP1, RAMP2, and RAMP3) that were originally identified during attempts to clone the receptor for calcitonin gene-related peptide (CGRP). Phenotypic receptor behavior corresponding to that of the native CGRP receptor could be demonstrated only in recombinant expression systems when another seven-transmembrane receptor, the calcitonin receptor-like receptor (CL receptor) was co-expressed with RAMP1 (1). Additional studies extended these observations to identify a general role of RAMPs in modifying the expression and the pharmacology of receptors related to the calcitonin family of peptides (1-5).To date, studies of RAMP-GPCR interactions have focused predominantly on the receptors for calcitonin and its related peptides (i.e. CGRP, adrenomedullin, and amylin). However, these receptors belong to the Class II family of GPCRs, members of which share a number of structural features and are all activated by peptide ligands (6). Given these similarities, it is possible that other Class II GPCRs may also interact with RAMP proteins to yield novel receptor phenotypes or receptors with altered pharmacological profiles. Importantly, RAMPs display a ubiquitous tissue distribution (1,7,8), and cellular background can have a significant impact on ...

Calcitonin (CT) receptors dimerize with receptor activity-modifying proteins (RAMPs) to create high-affinity amylin (AMY) receptors, but there is no reliable means of pharmacologically distinguishing these receptors. We used agonists and antagonists to define their pharmacology, expressing the CT (a) receptor alone or with RAMPs in COS-7 cells and measuring cAMP accumulation. Intermedin short, otherwise known as adrenomedullin 2, mirrored the action of ␣CGRP, being a weak agonist at CT (a) , AMY 2(a) , and AMY 3(a) receptors but considerably more potent at AMY 1(a) receptors. Likewise, the linear calcitonin gene-related peptide (CGRP) analogs (Cys(ACM) 2,7 )h␣CGRP and (Cys(Et) 2,7 )h␣CGRP were only effective at AMY 1(a) receptors, but they were partial agonists. As previously observed in COS-7 cells, there was little induction of the AMY 2(a) receptor phenotype; thus, AMY 2(a) was not examined further in this study. The antagonist peptide salmon calcitonin 8-32 (sCT 8-32 ) did not discriminate strongly between CT and AMY receptors; however, AC187 was a more effective antagonist of AMY responses at AMY receptors, and AC413 additionally showed modest selectivity for AMY 1(a) over AMY 3(a) receptors. CGRP 8-37 also demonstrated receptor-dependent effects. CGRP 8-37 more effectively antagonized AMY at AMY 1(a) than AMY 3(a) receptors, although it was only a weak antagonist of both, but it did not inhibit responses at the CT (a) receptor. Low CGRP 8-37 affinity and agonism by linear CGRP analogs at AMY 1(a) are the classic signature of a CGRP 2 receptor. Our data indicate that careful use of combinations of agonists and antagonists may allow pharmacological discrimination of CT (a) , AMY 1(a) , and AMY 3(a) receptors, providing a means to delineate the physiological significance of these receptors.

Highlights d Cryo-EM structure reveals how CRF1R interacts with CRF and the Gs signaling protein d Cryo-EM structure reveals interactions of Pac1nR with PACAP-38 and Gs d Evolutionary related GPCRs have greater conservation in peptide and G protein binding

Receptor activity-modifying proteins (RAMPs) 1, 2, and 3 are prototypic G protein-coupled receptor accessory proteins that can alter not only receptor trafficking but also receptor phenotype. Specific RAMP interaction with the calcitonin receptor (CTR) generates novel and distinct receptors for the peptide amylin; however, the role of RAMPs in receptor signaling is not understood. The current study demonstrates that RAMP interaction with the CTRa in COS-7 or HEK-293 cells leads to selective modulation of signaling pathways activated by the receptor complex. There was a 20- to 30-fold induction in amylin potency at CTR/RAMP1 (AMY1) and CTR/RAMP3 (AMY3) receptors, compared with CTR alone, for formation of the second-messenger cAMP that parallels an increase in amylin binding affinity. In contrast, only 2- to 5-fold induction of amylin potency was seen for mobilization of intracellular Ca++ or activation of ERK1/2. In addition, in COS-7 cells, the increase in amylin potency for Ca++ mobilization was 2-fold greater for AMY3 receptors, compared with AMY1 receptors and this paralleled the relative capacity of overexpression of Galphaq proteins to augment induction of high affinity 125I-amylin binding. These data demonstrate that RAMP-complexed receptors have a different signaling profile to CTRs expressed in the absence of RAMPs, and this is likely due to direct effects of the RAMP on G protein-coupling efficiency.

Calcitonin-family receptors comprise calcitonin receptor-like receptor (CL) or calcitonin receptor and receptor activity-modifying protein (RAMP) pairings. Calcitonin gene-related peptide (CGRP) receptors are CL/RAMP1, whereas adrenomedullin (AM) receptors are CL/RAMP2 (AM 1 receptor) or CL/RAMP3 (AM 2 receptor). Amylin (Amy) receptors are RAMP hetero-oligomers with the calcitonin receptor (AMY 1 , AMY 2 , and AMY 3 , respectively). How RAMPs change G protein-coupled receptor pharmacology is not fully understood. We exploited sequence differences between RAMP1 and RAMP3 to identify individual residues capable of altering receptor pharmacology. Alignment of human RAMPs revealed eight residues that are conserved in RAMP2 and RAMP3 but are different in RAMP1. We hypothesized that residues in RAMP2 and RAMP3, but not RAMP1, are responsible for making CL/RAMP2 and CL/RAMP3 AM receptors. Using site-directed mutagenesis, we introduced individual RAMP3 residues into RAMP1 and vice versa in these eight positions. Mutant or wild-type RAMPs were transfected into Cos7 cells with CL or the insert-negative form of the calcitonin receptor [CT (a) ]. Agonist-stimulated cAMP production and cellsurface expression of constructs were measured. Position 74 in RAMP1 and RAMP3 was critical for determining AM potency and affinity, and Phe93 in RAMP1 was an important contributor to ␣CGRP potency at CGRP receptors. Mutant RAMP/CT (a) receptor complexes displayed different phenotypes. It is noteworthy that RAMP1 S103N and W74E mutations led to enhanced rAmy potency, probably related to increased cellsurface expression of these complexes. This differs from the effect on CL-based receptors where expression was unchanged. Targeted substitution has emphasized the importance of position 74 in RAMP1/RAMP3 as a key determinant of AM pharmacology.The calcitonin peptide family, in humans, comprises calcitonin, amylin (Amy), two forms of calcitonin gene-related peptide (CGRP; ␣ and ␤) and adrenomedullin (AM). A second version of AM, a 47-amino acid peptide named intermedin (IMD) or AM2, has been identified more recently (Fig. 1A) (Roh et al., 2004;Takei et al., 2004). Calcitonin-peptide family receptors are composed of G protein-coupled receptors and receptor activity-modifying proteins (RAMPs) (Poyner et al., 2002). For example, AM receptors are heteromers of the calcitonin receptor-like receptor (CL) with RAMP2 (AM 1 receptors) or RAMP3 (AM 2 receptors). CGRP 1 receptors are formed from CL/RAMP1 complexes. In contrast, Amy receptors comprise the calcitonin receptor with individual RAMPs to generate AMY 1 , AMY 2 , and AMY 3 receptor subtypes.Pharmacological profiles have been determined for most of these receptor complexes in heterologous expression systems (Christopoulos et al., 1999;Nagoshi et al., 2002;Hay et al., 2003Hay et al., , 2005Kuwasako et al., 2003a;Bailey and Hay, 2006 ABBREVIATIONS: AMY, amylin receptor phenotype; AM, adrenomedullin; BIBN4096BS, 1-piperidinecarboxamide, N-[2-[[5amino-l-[[4-(4- Hay et al., 2005). Such obs...

The most well-characterized subgroup of family B G protein-coupledreceptors (GPCRs) comprises receptors for peptide hormones, such as secretin, calcitonin (CT), glucagon, and vasoactive intestinal peptide (VIP). Recent data suggest that many of these receptors can interact with a novel family of GPCR accessory proteins termed receptor activity modifying proteins (RAMPs). RAMP interaction with receptors can lead to a variety of actions that include chaperoning of the receptor protein to the cell surface as is the case for the calcitonin receptor-like receptor (CLR) and the generation of novel receptor phenotypes. RAMP heterodimerization with the CLR and related CT receptor is required for the formation of specific CT gene-related peptide, adrenomedullin (AM) or amylin receptors. More recent work has revealed that the specific RAMP present in a heterodimer may modulate other functions such as receptor internalization and recycling and also the strength of activation of downstream signaling pathways. In this article we review our current state of knowledge of the consequence of RAMP interaction with family B GPCRs.