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.

RAMPs and CGRP receptors

Receptor activity modifying protein 1 (RAMP1) forms a complex with calcitonin receptor-like receptor (CLR) to produce the receptor for calcitonin gene-related peptide (CGRP). RAMP1 has two main roles. It facilitates the cell-surface expression of CLR. It is also essential for the binding of CGRP to the receptor. It seems likely that Y66, F93, H97 and F101, amongst other residues, form a binding site for CLR. These cluster together on the same face of the extracellular portion of RAMP1, probably close to where it enters the plasma membrane. Residues at the other end of RAMP1 are most likely to be involved in CGRP recognition, although it is currently unclear how they do this. Within this area, W74 is important for the binding of the nonpeptide antagonist, BIBN4096BS, although it does not seem to be involved in the binding of CGRP itself. It has been shown that there is an epitope within residues 23-60 of CLR that are essential for RAMP recognition. Under some circumstances, changes in the expression of RAMP1 can alter the sensitivity of cells to CGRP, demonstrating that regulation of its levels may be of physiological or pathophysiological importance.

RAMPs as drug targets

The receptor activity-modifying protein (RAMP) family of membrane proteins regulates G protein-coupled receptor (GPCR) function in several ways. RAMPs can alter their pharmacology and signalling as well as the trafficking of these receptors to and from the cell surface. Accordingly, RAMPs may be exploited as drug targets, offering new opportunities for regulating the function of therapeutically relevant RAMP-interacting GPCRs. For example, several small molecule antagonists of RAMP1/ calcitonin receptor-like receptor complexes, which block the actions of the neuropeptide calcitonin gene-related peptide are in development for the treatment of migraine headache.

Secretin family (Class B) G protein-coupled receptors - from molecular to clinical perspectives

Family B G protein-coupled receptors represent an important but under-researched group of receptors. This edition of the British Journal of Pharmacology considers the roles and pharmacology of a number of these receptors. Whilst common themes emerge, it is clear that more work is needed to understand the details of each receptor in order to properly exploit them therapeutically.

The pharmacology of adrenomedullin 2/intermedin

Adrenomedullin 2 (AM2) or intermedin is a member of the calcitonin gene-related peptide (CGRP)/calcitonin family of peptides and was discovered in 2004. Unlike other members of this family, no unique receptor has yet been identified for it. It is extensively distributed throughout the body. It causes hypotension when given peripherally, but when given into the CNS, it increases blood pressure and causes sympathetic activation. It also increases prolactin release, is anti-diuretic and natriuretic and reduces food intake. Whilst its effects resemble those of AM, it is frequently more potent. Some characterization of AM2 has been done on molecularly defined receptors; the existing data suggest that it preferentially activates the AM receptor formed from calcitonin receptor-like receptor and receptor activity modifying protein 3. On this complex, its potency is generally equivalent to that of AM. There is no known receptor-activity where it is more potent than AM. In tissues and in animals it is frequently antagonised by CGRP and AM antagonists; however, situations exist in which an AM2 response is maintained even in the presence of supramaximal concentrations of these antagonists. Thus, there is a partial mismatch between the pharmacology seen in tissues and that on cloned receptors. The only AM2 antagonists are peptide fragments, and these have limited selectivity. It remains unclear as to whether novel AM2 receptors exist or whether the mismatch in pharmacology can be explained by factors such as metabolism. © 2011 The British Pharmacological Society.

Calcitonin and calcitonin receptor-like receptors:common themes with family B GPCRs?

The calcitonin receptor (CTR) and calcitonin receptor-like receptor (CLR) are two of the 15 human family B (or Secretin-like) GPCRs. CTR and CLR are of considerable biological interest as their pharmacology is moulded by interactions with receptor activity-modifying proteins. They also have therapeutic relevance for many conditions, such as osteoporosis, diabetes, obesity, lymphatic insufficiency, migraine and cardiovascular disease. In light of recent advances in understanding ligand docking and receptor activation in both the family as a whole and in CLR and CTR specifically, this review reflects how applicable general family B GPCR themes are to these two idiosyncratic receptors. We review the main functional domains of the receptors; the N-terminal extracellular domain, the juxtamembrane domain and ligand interface, the transmembrane domain and the intracellular C-terminal domain. Structural and functional findings from the CLR and CTR along with other family B GPCRs are critically appraised to gain insight into how these domains may function. The ability for CTR and CLR to interact with receptor activity-modifying proteins adds another level of sophistication to these receptor systems but means careful consideration is needed when trying to apply generic GPCR principles. This review encapsulates current thinking in the realm of family B GPCR research by highlighting both conflicting and recurring themes and how such findings relate to two unusual but important receptors, CTR and CLR.

Structure-function analysis of amino acid 74 of human RAMP1 and RAMP3 and its role in peptide interactions with adrenomedullin and calcitonin gene-related peptide receptors

The receptors for calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) are complexes of the calcitonin receptor-like receptor (CLR) and receptor activity-modifying proteins (RAMP). The CGRP receptor is a CLR/RAMP1 pairing whereas CLR/RAMP2 and CLR/RAMP3 constitute two subtypes of AM receptor: AM(1) and AM(2), respectively. Previous studies identified Glu74 in RAMP3 to be important for AM binding and potency. To further understand the importance of this residue and its equivalent in RAMP1 (Trp74) we substituted the native amino acids with several others. In RAMP3, these were Trp, Phe, Tyr, Ala, Ser, Thr, Arg and Asn; in RAMP1, Glu, Phe, Tyr, Ala and Asn substitutions were made. The mutant RAMPs were co-expressed with CLR in Cos7 cells; receptor function in response to AM, AM(2)/intermedin and CGRP was measured in a cAMP assay and cell surface expression was determined by ELISA. Phe reduced AM potency in RAMP3 but had no effect in RAMP1. In contrast, Tyr had no effect in RAMP3 but enhanced AM potency in RAMP1. Most other substitutions had a small effect on AM potency in both receptors whereas there was little impact on CGRP or AM(2) potency. Overall, these data suggest that the geometry and charge of the residue at position 74 contribute to how AM interacts with the AM(2) and CGRP receptors and confirms the role of this position in dictating differential AM pharmacology at the AM(2) and CGRP receptors.

Adrenomedullin and calcitonin gene-related peptide receptors in endocrine-related cancers:opportunities and challenges

Adrenomedullin (AM), adrenomedullin 2 (AM2/intermedin) and calcitonin gene-related peptide (CGRP) are members of the calcitonin family of peptides. They can act as growth or survival factors for a number of tumours, including those that are endocrine-related. One mechanism through which this occurs is stimulating angiogenesis and lymphangiogenesis. AM is expressed by numerous tumour types and for some cancers, plasma AM levels can be correlated with the severity of the disease. In cancer models, lowering AM content or blocking AM receptors can reduce tumour mass. AM receptors are complexes formed between a seven transmembrane protein, calcitonin receptor-like receptor and one of the two accessory proteins, receptor activity-modifying proteins (RAMPs) 2 or 3 to give the AM1 and AM2 receptors respectively. AM also has affinity at the CGRP receptor, which uses RAMP1. Unfortunately, due to a lack of selective pharmacological tools or antibodies to distinguish AM and CGRP receptors, the precise receptors and signal transduction pathways used by the peptides are often uncertain. Two other membrane proteins, RDC1 and L1/G10D (the 'ADMR'), are not currently considered to be genuine CGRP or AM receptors. In order to properly evaluate whether AM or CGRP receptor inhibition has a role in cancer therapy, it is important to identify which receptors mediate the effects of these peptides. To effectively distinguish AM1 and AM2 receptors, selective receptor antagonists need to be developed. The development of specific CGRP receptor antagonists suggests that this is now feasible.

Regulation of signal transduction by calcitonin gene-related peptide receptors

Calcitonin gene-related peptide (CGRP) plays a pivotal role in migraine, activating its cognate receptor to initiate intracellular signalling. This atypical receptor comprises a distinct assembly, made up of a G protein-coupled receptor (GPCR), a single transmembrane protein, and an additional protein that is required for Ga(s) coupling. By altering the expression of individual receptor components, it might be possible to adjust cellular sensitivity to CGRP. In recognition of the increasing clinical significance of CGRP receptors, it is timely to review the signalling pathways that might be controlled by this receptor, how the activity of the receptor itself is regulated, and our current understanding of the molecular mechanisms involved in these processes. Like many GPCRs, the CGRP receptor appears to be promiscuous, potentially coupling to several G proteins and intracellular pathways. Their precise composition is likely to be cell type-dependent, and much work is needed to ascertain their physiological significance.

Calcitonin gene-related peptide, adrenomedullin and flushing

Administration of calcitonin gene-related peptide (CGRP) or adrenomedullin (AM) can cause facial flushing, suggesting that the peptides may be important in hot flushes experienced particularly by post-menopausal women. Five studies have measured plasma CGRP concentrations in post-menopausal women who suffer from flushes; all demonstrated elevations of between 170% and 320% over control. Three of the studies showed a temporal relationship between flushes and CGRP elevation. A further study has shown that CGRP is elevated in the urine of women who suffer from flushes. Only a single study has investigated flushes in pre-menopausal women; no elevation of CGRP was observed. Flushes are also experienced by men undergoing androgen deprivation therapy. Whilst one study failed to find any increase in CGRP in the urine of these individuals, a small study has identified an increase in plasma CGRP. No studies have investigated plasma AM or the related peptide, intermedin/AM2. Overall, there is good evidence to show that flushes in post-menopausal women are accompanied by an increase in CGRP. CGRP could act centrally on the thermoregulatory centre of the hypothalamus as well as peripherally to cause vasodilation and sweating. However, it remains to be demonstrated that the elevated CGRP causes flushes. Recently developed CGRP antagonists provide an opportunity to test this hypothesis. If they are successful, they may represent a useful alternative to oestrogen replacement therapy.

Novel peptide antagonists of adrenomedullin and calcitonin gene-related peptide receptors:identification, pharmacological characterization, and interactions with position 74 in receptor activity-modifying protein 1/3

Human adrenomedullin (AM) is a 52-amino acid peptide belonging to the calcitonin peptide family, which also includes calcitonin gene-related peptide (CGRP) and AM2. The two AM receptors, AM(1) and AM(2), are calcitonin receptor-like receptor (CL)/receptor activity-modifying protein (RAMP) (RAMP2 and RAMP3, respectively) heterodimers. CGRP receptors comprise CL/RAMP1. The only human AM receptor antagonist (AM(22-52)) is a truncated form of AM; it has low affinity and is only weakly selective for AM(1) over AM(2) receptors. To develop novel AM receptor antagonists, we explored the importance of different regions of AM in interactions with AM(1), AM(2), and CGRP receptors. AM(22-52) was the framework for generating further AM fragments (AM(26-52) and AM(30-52)), novel AM/alphaCGRP chimeras (C1-C5 and C9), and AM/AM(2) chimeras (C6-C8). cAMP assays were used to screen the antagonists at all receptors to determine their affinity and selectivity. Circular dichroism spectroscopy was used to investigate the secondary structures of AM and its related peptides. The data indicate that the structures of AM, AM2, and alphaCGRP differ from one another. Our chimeric approach enabled the identification of two nonselective high-affinity antagonists of AM(1), AM(2), and CGRP receptors (C2 and C6), one high-affinity antagonist of AM(2) receptors (C7), and a weak antagonist selective for the CGRP receptor (C5). By use of receptor mutagenesis, we also determined that the C-terminal nine amino acids of AM seem to be responsible for its interaction with Glu74 of RAMP3. We provide new information on the structure-activity relationship of AM, alphaCGRP, and AM2 and how AM interacts with CGRP and AM(2) receptors.

Structure-activity relationships for α-calcitonin gene-related peptide

Calcitonin gene-related peptide (CGRP) is a member of the calcitonin (CT) family of peptides. It is a widely distributed neuropeptide implicated in conditions such as neurogenic inflammation. With other members of the CT family, it shares an N-terminal disulphide-bonded ring which is essential for biological activity, an area of potential α-helix, and a C-terminal amide. CGRP binds to the calcitonin receptor-like receptor (CLR) in complex with receptor activity-modifying protein 1 (RAMP1), a member of the family B (or secretin-like) GPCRs. It can also activate other CLR or calcitonin-receptor/RAMP complexes. This 37 amino acid peptide comprises the N-terminal ring that is required for receptor activation (residues 1-7); an α-helix (residues 8-18), a region incorporating a β-bend (residues 19-26) and the C-terminal portion (residues 27-37), that is characterized by bends between residues 28-30 and 33-34. A few residues have been identified that seem to make major contributions to receptor binding and activation, with a larger number contributing either to minor interactions (which collectively may be significant), or to maintaining the conformation of the bound peptide. It is not clear if CGRP follows the pattern of other family B GPCRs in binding largely as an α-helix. Linked Articles This article is part of a themed section on Neuropeptides. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.170.issue-7 © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

Similarity between class A and class B G-protein-coupled receptors exemplified through calcitonin gene-related peptide receptor modelling and mutagenesis studies

Modelling class B G-protein-coupled receptors (GPCRs) using class A GPCR structural templates is difficult due to lack of homology. The plant GPCR, GCR1, has homology to both class A and class B GPCRs. We have used this to generate a class A-class B alignment, and by incorporating maximum lagged correlation of entropy and hydrophobicity into a consensus score, we have been able to align receptor transmembrane regions. We have applied this analysis to generate active and inactive homology models of the class B calcitonin gene-related peptide (CGRP) receptor, and have supported it with site-directed mutagenesis data using 122 CGRP receptor residues and 144 published mutagenesis results on other class B GPCRs. The variation of sequence variability with structure, the analysis of polarity violations, the alignment of group-conserved residues and the mutagenesis results at 27 key positions were particularly informative in distinguishing between the proposed and plausible alternative alignments. Furthermore, we have been able to associate the key molecular features of the class B GPCR signalling machinery with their class A counterparts for the first time. These include the [K/R]KLH motif in intracellular loop 1, [I/L]xxxL and KxxK at the intracellular end of TM5 and TM6, the NPXXY/VAVLY motif on TM7 and small group-conserved residues in TM1, TM2, TM3 and TM7. The equivalent of the class A DRY motif is proposed to involve Arg(2.39), His(2.43) and Glu(3.46), which makes a polar lock with T(6.37). These alignments and models provide useful tools for understanding class B GPCR function.

Placements and degree performance:do placements lead to better marks, or do better students choose placements?

There has been a strong move recently to make degrees more applicable to employment; including work placements as part of the programme is one way of achieving this. Such placements are advocated to increase employability, but also for improving academic performance. This paper examines the relationship between undertaking a work placement and the class of degree achieved. It challenges earlier findings that undertaking a placement increases degree results. Studying seven cohorts of students, a well tested approach was employed that allows for sample selection – i.e. whether better students do placements rather than whether placements produce better students. The paper concludes that the sample selection is much stronger, i.e. placement students do better because they are better students. The results highlight that it is not merely doing a placement that matters, but a successful placement adds significantly to subsequent performance. The paper concludes with advice to students and policy makers.

Lymphoid aggregates that resemble tertiary lymphoid organs define a specific pathological subset in metal-on-metal hip replacements

Aseptic lymphocyte-dominated vasculitis-associated lesion (ALVAL) has been used to describe the histological lesion associated with metal-on-metal (M-M) bearings. We tested the hypothesis that the lymphoid aggregates, associated with ALVAL lesions resemble tertiary lymphoid organs (TLOs). Histopathological changes were examined in the periprosthetic tissue of 62 M-M hip replacements requiring revision surgery, with particular emphasis on the characteristics and pattern of the lymphocytic infiltrate. Immunofluorescence and immunohistochemistry were used to study the classical features of TLOs in cases where large organized lymphoid follicles were present. Synchrotron X-ray fluorescence (XRF) measurements were undertaken to detect localisation of implant derived ions/particles within the samples. Based on type of lymphocytic infiltrates, three different categories were recognised; diffuse aggregates (51%), T cell aggregates (20%), and organised lymphoid aggregates (29%). Further investigation of tissues with organised lymphoid aggregates showed that these tissues recapitulate many of the features of TLOs with T cells and B cells organised into discrete areas, the presence of follicular dendritic cells, acquisition of high endothelial venule like phenotype by blood vessels, expression of lymphoid chemokines and the presence of plasma cells. Co-localisation of implant-derived metals with lymphoid aggregates was observed. These findings suggest that in addition to the well described general foreign body reaction mediated by macrophages and a T cell mediated type IV hypersensitivity response, an under-recognized immunological reaction to metal wear debris involving B cells and the formation of tertiary lymphoid organs occurs in a distinct subset of patients with M-M implants. © 2013 Mittal et al.