Altered cell surface expression of human MC1R variant receptor alleles associated with red hair and skin cancer risk

The human melanocortin-1 receptor gene (MC1R) encodes a G-protein coupled receptor that is primarily expressed on melanocytes, where it plays a key role in pigmentation regulation. Variant alleles are associated with red hair colour and fair skin, known as the RHC phenotype, as well as skin cancer risk. The R151C, R160W and D294H alleles, designated 'R', are strongly associated with the RHC phenotype and have been proposed to result in loss of function receptors due to impaired G-protein coupling. We recently provided evidence that the R151C and R160W variants can efficiently couple to G-proteins in response to alpha-melanocyte stimulating hormone. The possibility that altered cellular localization of the R151C and R160W variant receptors could underlie their association with RHC was therefore considered. Using immunofluorescence and ligand binding studies, we found that melanocytic cells exogenously or endogenously expressing MC1R show strong surface localization of the wild-type and D294H alleles but markedly reduced cell surface expression of the R151C and R160W receptors. In additional exogenous expression studies, the R variant D84E and the rare I155T variant, also demonstrated a significant reduction in plasma membrane receptor numbers. The V60L, V92M and R163Q weakly associated RHC alleles, designated 'r', were expressed with normal or intermediate cell surface receptor levels. These results indicate that reduced receptor coupling activity may not be the only contributing factor to the genetic association between the MC1R variants and the RHC phenotype, with MC1R polymorphisms now linked to a change in receptor localization.

Solution structure and characterization of the LGR8 receptor binding surface of insulin-like peptide 3

Insulin-like peptide 3 (INSL3), a member of the relaxin peptide family, is produced in testicular Leydig cells and ovarian thecal cells. Gene knock-out experiments have identified a key biological role in initiating testes descent during fetal development. Additionally, INSL3 has an important function in mediating male and female germ cell function. These actions are elicited via its recently identified receptor, LGR8, a member of the leucine-rich repeat-containing G-protein- coupled receptor family. To identify the structural features that are responsible for the interaction of INSL3 with its receptor, its solution structure was determined by NMR spectroscopy together with in vitro assays of a series of B-chain alanine-substituted analogs. Synthetic human INSL3 was found to adopt a characteristic relaxin/ insulin-like fold in solution but is a highly dynamic molecule. The four termini of this two-chain peptide are disordered, and additional conformational exchange is evident in the molecular core. Alanine-substituted analogs were used to identify the key residues of INSL3 that are responsible for the interaction with the ectodomain of LGR8. These include Arg(B16) and Val(B19), with His(B12) and Arg(B20) playing a secondary role, as evident from the synergistic effect on the activity in double and triple mutants involving these residues. Together, these amino acids combine with the previously identified critical residue, Trp(B27), to form the receptor binding surface. The current results provide clear direction for the design of novel specific agonists and antagonists of this receptor.

Endothelin A receptor localization in the internal mammary artery

Introduction: The vasoconstricting peptide endothelin-1 (ET-1) binds two G-protein-coupled receptor subtypes, the Endothelin A (ETA) and Endothelin B (ETB) receptors. The ETB receptor subtype has been predominantly localised to the arterial and venous endothelial cells both in-vivo and in culture. Stimulation of ET-1 through this receptor subtype can modulate the expression of endothelial nitric oxide and accelerate endothelial cell wound healing. In comparison the ETA receptor is abundantly expressed in medial vascular smooth muscle cells and mediates the vasoconstrictor action of ET-1 and is thought to play a key role in angiogenesis. Aims: To determine the levels of ETA receptor expression and localisation in the internal mammary artery (IMA). Methods: Twenty-four IMA sections were examined from patients undergoing coronary artery bypass (CABG) surgery (5F; 19M; mean age 67 years). And 14 organ donor IMA specimens were used as controls (7M; 7F; mean age 45 years. The tissue was fixed in formalin and processed for histology. Immunohistochemistry was performed on cross-sections of the left distal IMA to assess the areas of ETA receptor staining. The percentage are of ETA receptor staining in the media was calculated using image analysis software connected to an optical microscope and semiquantitative assessment was used to grade staining intensity, that is, mild (+), moderate (++) and strong (+++). Results: ETA receptor staining was significantly elevated in the media of the CABG specimens compared with the donor controls (46.88+/11.52% Vs 18.58+/7.65%, P = .0001). Interestingly, the endothelium (++) of the IMA, as well as the small microvessels in the adventitia (+++) stained positive for ETA receptor expression. Without using a haematoxylin counterstain, the nuclei of the cell stained more intensely (+++) with respect to the cytoplasm in both the medial smooth muscle (++) and endothelial cells (++). Fibroblasts in the medial adventitia junction were also positive for ETA receptor expression (+++). Further, this receptor subtype was also strongly expressed by inflammatory cells (monocytes and macrophages). Conclusions: These results demonstrate that the ETA receptor expression is increased in the medial SMC layer of the CABG IMA specimens and also present in the endothelium, vasa vasorum, fibroblasts and inflammatory cell types. Thus it is possible that in addition to affecting vascular tone, ET-1 may play an important role in IMA remodelling.

The second intracellular loop of the calcitonin gene-related peptide receptor provides molecular determinants for signal transduction and cell surface expression

The calcitonin gene-related peptide (CGRP) receptor is a heterodimer of a family B G-protein-coupled receptor, calcitonin receptor-like receptor (CLR), and the accessory protein receptor activity modifying protein 1. It couples to Gs, but it is not known which intracellular loops mediate this. We have identified the boundaries of this loop based on the relative position and length of the juxtamembrane transmembrane regions 3 and 4. The loop has been analyzed by systematic mutagenesis of all residues to alanine, measuring cAMP accumulation, CGRP affinity, and receptor expression. Unlike rhodopsin, ICL2 of the CGRP receptor plays a part in the conformational switch after agonist interaction. His-216 and Lys-227 were essential for a functional CGRP-induced cAMP response. The effect of (H216A)CLR is due to a disruption to the cell surface transport or surface stability of the mutant receptor. In contrast, (K227A)CLR had wild-type expression and agonist affinity, suggesting a direct disruption to the downstream signal transduction mechanism of the CGRP receptor. Modeling suggests that the loop undergoes a significant shift in position during receptor activation, exposing a potential G-protein binding pocket. Lys-227 changes position to point into the pocket, potentially allowing it to interact with bound G-proteins. His-216 occupies a position similar to that of Tyr-136 in bovine rhodopsin, part of the DRY motif of the latter receptor. This is the first comprehensive analysis of an entire intracellular loop within the calcitonin family of G-protein-coupled receptor. These data help to define the structural and functional characteristics of the CGRP-receptor and of family B G-protein-coupled receptors in general. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

Adrenomedullin: receptor and signal transduction

Adrenomedullin is a vascular tissue peptide and a member of the calcitonin family of peptides, which includes calcitonin calcitonin-gene-related peptide (CGRP) and amylin. Its many biological actions are mediated via CGRP type 1 (CGRP(1)) receptors and by specific adrenomedullin receptors. Although the pharmacology of these receptors is distinct, they are both represented in molecular terms by the type II family G-protein-coupled receptor, calcitonin-receptor-like receptor (CRLR). The specificity here is defined by co-expression of receptor-activity-modifying proteins (RAMPs). CGRP(1) receptors are represented by CRLR and RAMP1, and specific adrenomedullin receptors by CRLR and RAMP2 or 3. Here we discuss how CRLR/RAMP2 relates to adrenomedullin binding, pharmacology and pathophysiology, and how chemical cross-linking of receptor-ligand complexes in tissue relates to that in CRLR/RAMP2-expressing cells. CRLR, like other type II family G-protein-coupled receptors, signals via G(s) and adenylate cyclase activation. We demonstrated that adrenomedullin signalling in cell lines expressing specific adrenomedullin receptors followed this expected pattern.

Ligand binding and activation of the CGRP receptor

The receptor for CGRP (calcitonin gene-related peptide) is a heterodimer between a GPCR (G-protein-coupled receptor), CLR (calcitonin receptor-like receptor) and an accessory protein, RAMP1 (receptor activity-modifying protein 1). Models have been produced of RAMP1 and CLR. It is likely that the C-terminus of CGRP interacts with the extracellular N-termini of CLR and RAMP1; the extreme N-terminus of CLR is particularly important and may interact directly with CGRP and also with RAMP1. The N-terminus of CGRP interacts with the TM (transmembrane) portion of the receptor; the second ECL (extracellular loop) is especially important. Receptor activation is likely to involve the relative movements of TMs 3 and 6 to create a G-protein-binding pocket, as in Family A GPCRs. Pro321 in TM6 appears to act as a pivot. At the base of TMs 2 and 3, Arg151, His155 and Glu211 may form a loose equivalent of the Family A DRY (Asp-Arg-Tyr) motif. Although the details of this proposed activation mechanism clearly do not apply to all Family B GPCRs, the broad outlines may be conserved. ©The Authors.

Diverse functional motifs within the three intracellular loops of the CGRP1 receptor

The CGRP1 receptor exists as a heterodimeric complex between a single-pass transmembrane accessory protein (RAMP1) and a family B G-protein-coupled receptor (GPCR) called the calcitonin receptor-like receptor (CLR). This study investigated the structural motifs found in the intracellular loops (ICLs) of this receptor. Molecular modeling was used to predict active and inactive conformations of each ICL. Conserved residues were altered to alanine by site-directed mutagenesis. cAMP accumulation, cell-surface expression, agonist affinity, and CGRP-stimulated receptor internalization were characterized. Within ICL1, L147 and particularly R151 were important for coupling to Gs. R151 may interact directly with the G-protein, accessing it following conformational changes involving ICL2 and ICL3. At the proximal end of ICL3, I290 and L294, probably lying on the same face of an α helix, formed a G-protein coupling motif. The largest effects on coupling were observed with I290A; additionally, it reduced CGRP affinity and impaired internalization. 1290 may interact with TM6 to stabilize the conformation of ICL3, but it could also interact directly with Gs. R314, at the distal end of ICL3, impaired G-protein coupling and to a lesser extent reduced CGRP affinity; it may stabilize the TM6-ICL3 junction by interacting with the polar headgroups of membrane phospholipids. Y215 and L214 in ICL2 are required for cell-surface expression; they form a microdomain with H216 which has the same function. This study reveals similarities between the activation of CLR and other GPCRs in the role of TM6 and ICL3 but shows that other conserved motifs differ in their function. © 2006 American Chemical Society.

Functional characterization of two human receptor activity-modifying protein 3 variants

Adrenomedullin (AM) and amylin are involved in angiogenesis/lymphangiogenesis and glucose homeostasis/food intake, respectively. They activate receptor activity-modifying protein (RAMP)/G protein-coupled receptor (GPCR) complexes. RAMP3 with the calcitonin receptor-like receptor (CLR) forms the AM(2) receptor, whereas when paired with the calcitonin receptor AMY(3) receptors are formed. RAMP3 interacts with other GPCRs although the consequences of these interactions are poorly understood. Therefore, variations in the RAMP3 sequence, such as single nucleotide polymorphisms or mutations could be relevant to human health. Variants of RAMP3 have been identified. In particular, analysis of AK222469 (Homo sapiens mRNA for receptor (calcitonin) activity-modifying protein 3 precursor variant) revealed several nucleotide differences, three of which encoded amino acid changes (Cys40Trp, Phe100Ser, Leu147Pro). Trp56Arg RAMP3 is a polymorphic variant of human RAMP3 at a conserved amino acid position. To determine their function we used wild-type (WT) human RAMP3 as a template for introducing amino acid mutations. Mutant or WT RAMP3 function was determined in Cos-7 cells with CLR or the calcitonin receptor (CT((a))). Cys40Trp/Phe100Ser/Leu147Pro RAMP3 was functionally compromised, with reduced AM and amylin potency at the respective AM(2) and AMY(3(a)) receptor complexes. Cys40Trp and Phe100Ser mutations contributed to this phenotype, unlike Leu147Pro. Reduced cell-surface expression of mutant receptor complexes probably explains the functional data. In contrast, Trp56Arg RAMP3 was WT in phenotype. This study provides insight into the role of these residues in RAMP3. The existence of AK222469 in the human population has implications for the function of RAMP3/GPCR complexes, particularly AM and amylin receptors.

The role of the extracellular loops of the CGRP receptor, a family B GPCR

The CGRP (calcitonin gene-related peptide) receptor is a family B GPCR (G-protein-coupled receptor). It consists of a GPCR, CLR (calcitonin receptor-like receptor) and an accessory protein, RAMP1 (receptor activity-modifying protein 1). RAMP1 is needed for CGRP binding and also cell-surface expression of CLR. There have been few systematic studies of the ECLs (extracellular loops) of family B GPCRs. However, they are likely to be especially important for the interaction of the N-termini of the peptide agonists that are the natural agonists for these receptors. We have carried out alanine scans on all three ECLs of CLR, as well as their associated juxtamembrane regions. Residues within all three loops influence CGRP binding and receptor activation. Mutation of Ala203 and Ala206 on ECL1 to leucine increased the affinity of CGRP. Residues at the top of TM (transmembrane) helices 2 and 3 influenced CGRP binding and receptor activation. L351A and E357A in TM6/ECL3 reduced receptor expression and may be needed for CLR association with RAMP1. ECL2 seems especially important for CLR function; of the 16 residues so far examined in this loop, eight residues reduce the potency of CGRP at stimulating cAMP production when mutated to alanine.

The activation of the CGRP receptor

The CGRP (calcitonin gene-related peptide) receptor is a family B GPCR (G-protein-coupled receptor). It consists of a GPCR, CLR (calcitonin receptor-like receptor) and an accessory protein, RAMP1 (receptor activity modifying protein 1). RAMP1 is needed for CGRP binding and also cell-surface expression of CLR. CLR is an example of a family B GPCR. Unlike family A GPCRs, little is known about how these receptors are activated by their endogenous ligands. This review considers what is known about the activation of family B GPCRs and then considers how this might be applied to CLR, particularly in light of new knowledge of the crystal structures of family A GPCRs.

G-protein-coupled receptors:from structural insights to functional mechanisms

The papers resulting from the recent Biochemical Society Focused Meeting 'G-Protein-Coupled Receptors: from Structural Insights to Functional Mechanisms' held in Prato in September 2012 are introduced in the present overview. A number of future goals for GPCR (G-protein-coupled receptor) research are considered, including the need to develop biophysical and computational methods to explore the full range of GPCR conformations and their dynamics, the need to develop methods to take this into account for drug discovery and the importance of relating observations on isolated receptors or receptors expressed in model systems to receptor function in vivo. © 2013 Biochemical Society.

The role of ECL2 in CGRP receptor activation:a combined modelling and experimental approach

The calcitonin gene-related peptide (CGRP) receptor is a complex of a cal-citonin receptor-like receptor (CLR), which is a family B G-protein-coupled receptor (GPCR) and receptor activity modifying protein 1. The role of the second extracellular loop (ECL2) of CLR in binding CGRP and coupling to Gs was investigated using a combination of mutagenesis and modelling. An alanine scan of residues 271-294 of CLR showed that the ability of CGRP to produce cAMP was impaired by point mutations at 13 residues; most of these also impaired the response to adrenomedullin (AM). These data were used to select probable ECL2-modelled conformations that are involved in agonist binding, allowing the identification of the likely contacts between the peptide and receptor. The implications of the most likely structures for receptor activation are discussed. © 2013 The Authors.

Investigation of the activation mechanism and structural characterization of the CGRP receptor

The calcitonin gene-related peptide (CGRP) receptor is an unusual G protein-coupled receptor (GPCR) in that it comprises the calcitonin receptor-like receptor (CLR), receptor activity modifying protein 1 (RAMP1) and the receptor component protein (RCP). The RAMP1 has two other homologues – RAMP2 and RAMP3. The endogenous ligand for this receptor is CGRP, a 37 amino acid neuropeptide that act as a vasodilator. This peptide has been implicated in the aetiology of health conditions such as inflammation, Reynaud’s disease and migraine. A clear understanding of the mode of activation of this receptor could be key in developing therapeutic agents for associated health conditions. Although the crystal structure of the N-terminal extracellular domain (ECD) of this receptor (in complex with an antagonist) has been published, the details of receptor-agonist interactions at this domain, and so ultimately the mechanism of receptor activation, are still unclear. Also, the C-terminus of the CLR (in the CGRP receptor), especially around the presumed helix 8 (H8) region, has not been well studied for its role in receptor signalling. This research project investigated these questions. In this study, certain residues making up the putative N-terminal ligand-binding core of the CLR (in the CGRP receptor) were mapped out and found to be crucial for receptor signalling. They included W69 and D70 of the WDG motif in family B GPCRs, as well as Y91, F92, D94 and F95 in loop 2 of CLR N-terminus. Also, F163 at the cytoplasmic end of TM1 and certain residues spanning H8 and associated C-terminal region of CLR were found to be required for CGRP receptor signalling. These residues were investigated by site-directed mutagenesis where they were mutated to alanine (or other residues in specific cases) and the effect of the mutations on receptor pharmacology assessed by evaluating cAMP production, cell surface expression, total cell expression and aCGRP-mediated receptor internalization. Moreover, the N-terminal ECDs of the CLR and RAMPs (RAMP1, RAMP2 and RAMP3) were produced in a yeast host strain (Pichia pastoris) for the purpose of structural interaction study by surface plasmon resonance (SPR). Following expression and purification, these receptor proteins were found to individually retain their secondary structures when analysed by circular dichroism (CD). Results were analysed and interpreted with the knowledge of the secretin family receptor paradigm. The research described in this thesis has produced novel data that contributes to a clearer understanding of CGRP receptor pharmacology. The study on CLR and RAMPs ECDs could be a useful tool in determining novel interacting GPCR partners of RAMPs.

Differential impact of amino acid substitutions on critical residues of the human glucagon-like peptide-1 receptor involved in peptide activity and small-molecule allosterys

The glucagon-like peptide-1 receptor (GLP-1R) is a class B G protein-coupled receptor that has a critical role in the regulation of glucose homeostasis, principally through the regulation of insulin secretion. The receptor systemis highly complex, able to be activated by both endogenous [GLP-1(1-36)NH2, GLP-1(1-37), GLP-1(7-36)NH2, GLP-1(7-37), oxyntomodulin], and exogenous (exendin-4) peptides in addition to small-molecule allosteric agonists (compound 2 [6,7-dichloro-2-methylsulfonyl-3-tertbutylaminoquinoxaline], BETP [4-(3-benzyloxy)phenyl)-2-ethylsulfinyl-6-(trifluoromethyl)pyrimidine]). Furthermore, the GLP-1R is subject to single-nucleotide polymorphic variance, resulting in amino acid changes in the receptor protein. In this study, we investigated two polymorphic variants previously reported to impact peptidemediated receptor activity (M149) and small-molecule allostery (C333). These residues were mutated to a series of alternate amino acids, and their functionality was monitored across physiologically significant signaling pathways, including cAMP, extracellular signal-regulated kinase 1 and 2 phosphorylation, and intracellular Ca2+ mobilization, in addition to peptide binding and cell-surface expression. We observed that residue 149 is highly sensitive to mutation, with almost all peptide responses significantly attenuated at mutated receptors. However, most reductions in activity were able to be restored by the small-molecule allosteric agonist compound 2. Conversely, mutation of residue 333 had little impact on peptide-mediated receptor activation, but this activity could not be modulated by compound 2 to the same extent as that observed at the wild-type receptor. These results provide insight into the importance of residues 149 and 333 in peptide function and highlight the complexities of allosteric modulation within this receptor system.

Identifying key residues important for CGRP binding to its receptor

The calcitonin gene related peptide (CGRP) is a 37 amino acid neuropeptide. Its receptor is a heterodimeric complex of calcitonin receptor-like receptor (CLR) – a family B G-protein coupled receptor – and a single-pass transmembrane protein, receptoractivity modifying protein 1 (RAMP1). Here, we identify residues, within the N-terminal extracellular domain (ECD) of CLR, potentially involved in ligand binding.Certain residues presumed to be possible sites of contact for the CGRP were picked from the CLR/RAMP1 ECD crystal structure (PDB 3N7S). Residues were mutated to alanine (A) bysite-directed mutagenesis (QuikChangeTM, Stratagene). Mutants were analysed for their ability to stimulate cAMP and cell surface expression as previously described [1]. All mutants showed reduced potency, though to varying degrees as indicated by their pEC50 values. W69A and D70Ashowed significant reduction in cell surface expression.These findings suggest that these residues are important for the interaction of CGRP with its receptor. W69A and D70A, part of the WDG motif of family B GPCRs, are thought to rather play a role in receptor stability [2]. The data is consistent with CGRP binding in agroove between CLR and RAMP1. This project was supported byAston School of Life and Health Sciences.References1. Barwell J, Conner A & Poyner D (2011) Extracellular loops 1and 3 and their associated transmembrane regions of the calcitonin receptor-like receptor are needed for CGRP receptor function. Biochim Biophys Acta 1813, 1906–1916.2. Kumar S, Pioszak A, Zhang C et al. (2011) Crystal Structure of the PAC1R Extracellular Domain Unifies a Consensus Fold for Hormone Recognition by Class B G-Protein Cou-pled Receptors. PLoS One 6, e19682