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 calcitonin 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.</jats:p

Effects of diabetes family history and exercise training on the expression of adiponectin and leptin and their receptors

The daughters of patients with diabetes have reduced insulin sensitivity index (ISI) scores compared with women with no family history of diabetes, but their ISI increase more in response to exercise training(1). The present study aimed to determine whether differences between these groups in exercise-induced changes in circulating adiponectin and leptin concentrations and expression of their genes and receptors in subcutaneous adipose tissue (SAT), could explain differences in the exercise-induced changes in ISI between women with and without a family history of diabetes

Receptor activity-modifying protein-dependent effects of mutations in the calcitonin receptor-like receptor:implications for adrenomedullin and calcitonin gene-related peptide pharmacology

Background and Purpose Receptor activity-modifying proteins (RAMPs) define the pharmacology of the calcitonin receptor-like receptor (CLR). The interactions of the different RAMPs with this class B GPCR yield high-affinity calcitonin gene-related peptide (CGRP) or adrenomedullin (AM) receptors. However, the mechanism for this is unclear. Experimental Approach Guided by receptor models, we mutated residues in the N-terminal helix of CLR, RAMP2 and RAMP3 hypothesized to be involved in peptide interactions. These were assayed for cAMP production with AM, AM2 and CGRP together with their cell surface expression. Binding studies were also conducted for selected mutants. Key Results An important domain for peptide interactions on CLR from I32 to I52 was defined. Although I41 was universally important for binding and receptor function, the role of other residues depended on both ligand and RAMP. Peptide binding to CLR/RAMP3 involved a more restricted range of residues than that to CLR/RAMP1 or CLR/RAMP2. E101 of RAMP2 had a major role in AM interactions, and F111/W84 of RAMP2/3 was important with each peptide. Conclusions and Implications RAMP-dependent effects of CLR mutations suggest that the different RAMPs control accessibility of peptides to binding residues situated on the CLR N-terminus. RAMP3 appears to alter the role of specific residues at the CLR-RAMP interface compared with RAMP1 and RAMP2

Non-peptidic antagonists of the CGRP receptor, BIBN4096BS and MK-0974, interact with the calcitonin receptor-like receptor via methionine-42 and RAMP1 via tryptophan-74

The receptor for calcitonin gene-related peptide (CGRP) has been the target for the development of novel small molecule antagonists for the treatment of migraine. Two such antagonists, BIBN4096BS and MK-0974, have shown great promise in clinical trials and hence a deeper understanding of the mechanism of their interaction with the receptor is now required. The structure of the CGRP receptor is unusual since it is comprised of a hetero-oligomeric complex between the calcitonin receptor-like receptor (CRL) and an accessory protein (RAMP1). Both the CLR and RAMP1 components have extracellular domains which interact with each other and together form part of the peptide-binding site. It seems likely that the antagonist binding site will also be located on the extracellular domains and indeed Trp-74 of RAMP1 has been shown to form part of the binding site for BIBN4096BS. However, despite a chimeric study demonstrating the role of the N-terminal domain of CLR in antagonist binding, no specific residues have been identified. Here we carry out a mutagenic screen of the extreme N-terminal domain of CLR (residues 23-63) and identify a mutant, Met-42-Ala, which displays 48-fold lower affinity for BIBN4096BS and almost 900-fold lower affinity for MK-0974. In addition, we confirm that the Trp-74-Lys mutation at human RAMP1 reduces BIBN4096BS affinity by over 300-fold and show for the first time a similar effect for MK-0974 affinity. The data suggest that the non-peptide antagonists occupy a binding site close to the interface of the N-terminal domains of CLR and RAMP1

Morphological and geographical traits of the British Odonata

Trait data are fundamental for many aspects of ecological research, particularly for modeling species response to environmental change. We synthesised information from the literature (mainly field guides) and direct measurements from museum specimens, providing a comprehensive dataset of 26 attributes, covering the 43 resident species of Odonata in Britain. Traits included in this database range from morphological traits (e.g. body length) to attributes based on the distribution of the species (e.g. climatic restriction). We measured 11 morphometric traits from five adult males and five adult females per species. Using digital callipers, these measurements were taken from dry museum specimens, all of which were wild caught individuals. Repeated measures were also taken to estimate measurement error. The trait data are stored in an online repository (https://github.com/BiologicalRecordsCentre/Odonata_traits), alongside R code designed to give an overview of the morphometric data, and to combine the morphometric data to the single value per trait per species data

Can cutaneous telangiectasiae as late normal-tissue injury predict cardiovascular disease in women receiving radiotherapy for breast cancer?

Background: Overall, ~5% of patients show late normal-tissue damage after radiotherapy with a smaller number having a risk of radiation-induced heart disease. Although the data are conflicting, large studies have shown increased risks of cardiovascular disease (CVD) for irradiated patients compared with non-irradiated ones, or for those treated to the left breast or chest wall compared with those treated to the right. Cutaneous telangiectasiae as late normal-tissue injury have so far only been regarded as a cosmetic burden. Methods: The relationship between late normal-tissue radiation injury phenotypes in 149 irradiated breast cancer patients and the presence of cardiovascular disease were examined. Results: A statistically significant association between the presence of skin telangiectasiae and the long-term risk of CVD was shown in these patients (P=0.017; Fisher's exact test). Interpretation: This association may represent initial evidence that telangiectasiae can be used as a marker of future radiation-induced cardiac complications. It could also suggest a common biological pathway for the development of both telangiectasiae and CVD on the basis of a genetically predisposed endothelium. To our knowledge this is the first reported study looking at this association

Family Resemblances? Ligand Binding and Activation of Family A and B G-Protein-Coupled Receptors Ligand binding and activation of the CGRP receptor

Abstract The receptor for CGRP (calcitonin gene-related peptide) is a heterodimer between a GPCR (G-proteincoupled receptor), CLR (calcitonin receptor-like receptor) and an accessory protein, RAMP1 (receptor activitymodifying 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. Pro 321 in TM6 appears to act as a pivot. At the base of TMs 2 and 3, Arg 151 , His 155 and Glu 211 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

Is telomere length in peripheral blood lymphocytes correlated with cancer susceptibility or radiosensitivity?

Mean terminal restriction fragment (TRF) lengths in white blood cells (WBCs) have been previously found to be associated with breast cancer. To assess whether this marker could be used as a test for breast cancer susceptibility in women, TRF length was measured in 72 treated female breast cancer patients and 1696 unaffected female controls between the ages of 45 and 77 from the Twin Research Unit at St Thomas' Hospital, as well as 140 newly diagnosed breast cancer cases and 108 mammographically screened unaffected controls from Guy's Hospital. Mean TRF was also tested for correlation with chromosome radiosensitivity and apoptotic response in the Guy's Hospital patients. After adjusting for age, smoking and body mass index, there was no significant difference in TRF lengths between the treated breast cancer patients and unaffected controls (P=0.71). A positive correlation between age-adjusted apoptotic response and mean TRF in newly diagnosed untreated breast cancer patients (P=0.008) was identified but no significant difference in TRF lengths between breast cancer patients and unaffected controls was detected (P=0.53). This suggests that TRF lengths in WBC, is not a marker of breast cancer susceptibility and does not vary significantly between affected women before and after treatment