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.

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.

Structure-activity relationships of the N-terminus of calcitonin gene-related peptide:key roles of alanine-5 and threonine-6 in receptor activation

Background and purpose - The N-terminus of calcitonin gene-related peptide (CGRP) is important for receptor activation, especially the disulphide-bonded ring (residues 1-7). However, the roles of individual amino acids within this region have not been examined and so the molecular determinants of agonism are unknown. This study has examined the role of residues 1, 3-6 and 8-9, excluding Cys-2 and Cys-7. Experimental approach - CGRP derivatives were substituted with either cysteine or alanine; further residues were introduced at position 6. Their affinity was measured by radioligand binding and their efficacy by measuring cAMP production in SK-N-MC cells and ß-arrestin 2 translocation in CHO-K1 cells at the CGRP receptor. Key results - Substitution of Ala-5 by cysteine reduced affinity 270-fold and reduced efficacy for production of cAMP in SK-N-MCs. Potency at ß-arrestin translocation was reduced by 9-fold. Substitution of Thr-6 by cysteine destroyed all measurable efficacy of both cAMP and ß-arrestin responses; substitution with either alanine or serine impaired potency. Substitutions at positions 1, 4, 8 and 9 resulted in approximately 10-fold reductions in potency at both responses. Similar observations were made at a second CGRP-activated receptor, the AMY1(a) receptor. Conclusions and implications - Ala-5 and Thr-6 are key determinants of agonist activity for CGRP. Ala-5 is also very important for receptor binding. Residues outside of the 1-7 ring also contribute to agonist activity.

The preclinical pharmacology of BIBN4096BS, a CGRP antagonist

CGRP is an important neuropeptide found throughout the cardiovascular system. However, until recently it has been difficult to define its pharmacology or physiological role because of the lack of suitable antagonists. BIBN4096BS is a high-affinity, nonpeptide antagonist that shows much greater selectivity for human CGRP1 receptors compared to any other drug. Its pharmacology has been defined with studies on transfected cells or cell lines endogenously expressing receptors of known composition. These have allowed confirmation that in many human blood vessels, CGRP is working via CGRP1 receptors. However, it also interacts with other CGRP-activated receptors, of unknown composition. In vivo, clinical studies have shown that BIBN4096BS is likely to be useful in the treatment of migraine. It has also been used to define the role of CGRP in phenomena such as plasma extravasation and cardioprotection following ischemia.

Characterization of CGRP receptor binding

CGRP receptor binding may be measured using homogenates of cell membranes or intact cells. Here a microcentrifugation-based assay is described that utilizes radioiodinated CGRP in displacement studies to determine the binding parameters for any ligand that interacts with CGRP receptors. A similar assay is described for binding to cultured cells. The protocols may be adapted for other radioligands or for filtration-based assays. The main problems with CGRP binding assays can usually be traced to degradation of the radioligand or displacing ligands. Also, some cell lines fail to express CGRP receptors after extensive passage. CGRP binding assays provide a rapid and easy approach for distinguishing between receptors for CGRP and related peptides such as adrenomedullin and amylin.

Sequence and chemistry requirements for a novel aptameric oligonucleotide inhibitor of EGF receptor tyrosine kinase activity

We have previously identified a phosphorothioate oligonucleotide (PS-ODN) that inhibited epidermal growth factor receptor tyrosine kinase (TK) activity both in cell fractions and in intact A431 cells. Since ODN-based TK inhibitors may have anti-cancer applications and may also help understand the non-antisense mediated effects of PS-ODNs, we have further studied the sequence and chemistry requirements of the parent PS-ODN (sequence: 5′-GGA GGG TCG CAT CGC-3′) as a sequence-dependent TK inhibitor. Sequence deletion and substitution studies revealed that the 5′-terminal GGA GGG hexamer sequence in the parent compound was essential for anti-TK activity in A431 cells. Site-specific substitution of any G with a T in this 5′-terminal motif within the parent compound caused a significant loss in anti-TK activity. The fully PS-modified hexameric motif alone exhibited equipotent activity as the parent 15-mer whereas phosphodiester (PO) or 2′-O-methyl-modified versions of this motif had significantly reduced anti-TK activity. Further, T substitutions within the two 5′-terminal G residues of the hexameric PS-ODN to produce a sequence, TTA GGG, representing the telomeric repeats in human chromosomes, also did not exhibit a significant anti-TK activity. Multiple repeats of the active hexameric motif in PS-ODNs resulted in more potent inhibitors of TK activity than the parent ODN. These results suggested that PS-ODNs, but not PO or 2′-O-methyl modified ODNs, containing the GGA GGG motif can exert potent anti-TK activity which may be desirable in some anti-tumor applications. Additionally, the presence of this previously unidentified motif in antisense PS-ODN constructs may contribute to their biological effects in vitro and in vivo and should be accounted for in the design of the PS-modified antisense ODNs. © 2002 Published by Elsevier Science Inc.

Serious games for energy social science research

This paper proposes a set of criteria for evaluation of serious games (SGs) which are intended as effective methods of engaging energy users and lowering consumption. We discuss opportunities for using SGs in energy research which go beyond existing feedback mechanisms, including use of immersive virtual worlds for learning and testing behaviours, and sparking conversations within households. From a review of existing SG evaluation criteria, we define a tailored set of criteria for energy SG development and evaluation. The criteria emphasise the need for the game to increase energy literacy through applicability to real-life energy use/management; clear, actionable goals and feedback; ways of comparing usage socially and personal relevance. Three existing energy games are evaluated according to this framework. The paper concludes by outlining directions for future development of SGs as an effective tool in social science research, including games which inspire reflection on trade-offs and usage at different scales.

Structural basis for receptor activity-modifying protein-dependent selective peptide recognition by a G protein-coupled receptor

Association of receptor activity-modifying proteins (RAMP1-3) with the G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) enables selective recognition of the peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) that have diverse functions in the cardiovascular and lymphatic systems. How peptides selectively bind GPCR:RAMP complexes is unknown. We report crystal structures of CGRP analog-bound CLR:RAMP1 and AM-bound CLR:RAMP2 extracellular domain heterodimers at 2.5 and 1.8 Å resolutions, respectively. The peptides similarly occupy a shared binding site on CLR with conformations characterized by a β-turn structure near their C termini rather than the α-helical structure common to peptides that bind related GPCRs. The RAMPs augment the binding site with distinct contacts to the variable C-terminal peptide residues and elicit subtly different CLR conformations. The structures and accompanying pharmacology data reveal how a class of accessory membrane proteins modulate ligand binding of a GPCR and may inform drug development targeting CLR:RAMP complexes.