Institute of Membrane and Systems Biology

Leeds, United Kingdom

Institute of Membrane and Systems Biology

Leeds, United Kingdom
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Miller P.S.,Institute of Membrane and Systems Biology | Barwell J.,Aston University | Poyner D.R.,Aston University | Wigglesworth M.J.,Glaxosmithkline | And 2 more authors.
Biochemical and Biophysical Research Communications | Year: 2010

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. © 2009 Elsevier Inc. All rights reserved.

Barwell J.,Aston University | Miller P.S.,Institute of Membrane and Systems Biology | Donnelly D.,Institute of Membrane and Systems Biology | Poyner D.R.,Aston University
Peptides | Year: 2010

The calcitonin receptor-like receptor (CLR) acts as a receptor for the calcitonin gene-related peptide (CGRP) but in order to recognize CGRP, it must form a complex with an accessory protein, receptor activity modifying protein 1 (RAMP1). Identifying the protein/protein and protein/ligand interfaces in this unusual complex would aid drug design. The role of the extreme N-terminus of CLR (Glu23-Ala60) was examined by an alanine scan and the results were interpreted with the help of a molecular model. The potency of CGRP at stimulating cAMP production was reduced at Leu41Ala, Gln45Ala, Cys48Ala and Tyr49Ala; furthermore, CGRP-induced receptor internalization at all of these receptors was also impaired. Ile32Ala, Gly35Ala and Thr37Ala all increased CGRP potency. CGRP specific binding was abolished at Leu41Ala, Ala44Leu, Cys48Ala and Tyr49Ala. There was significant impairment of cell surface expression of Gln45Ala, Cys48Ala and Tyr49Ala. Cys48 takes part in a highly conserved disulfide bond and is probably needed for correct folding of CLR. The model suggests that Gln45 and Tyr49 mediate their effects by interacting with RAMP1 whereas Leu41 and Ala44 are likely to be involved in binding CGRP. Ile32, Gly35 and Thr37 form a separate cluster of residues which modulate CGRP binding. The results from this study may be applicable to other family B GPCRs which can associate with RAMPs. © 2009 Elsevier Inc. All rights reserved.

Hatano N.,Aichi Gakuin University | Itoh Y.,Aichi Gakuin University | Itoh Y.,Nagoya City University | Suzuki H.,Aichi Gakuin University | And 6 more authors.
Journal of Biological Chemistry | Year: 2012

Transient receptor potential ankyrin repeat 1 (TRPA1) forms calcium (Ca2+)- and zinc (Zn2+)-permeable ion channels that sense noxious substances. Despite the biological and clinical importance of TRPA1, there is little knowledge of the mechanisms that lead to transcriptional regulation of TRPA1 and of the functional role of transcriptionally induced TRPA1. Here we show induction of TRPA1 by inflammatory mediators and delineate the underlying molecular mechanisms and functional relevance. In human fibroblast-like synoviocytes, key inflammatory mediators (tumor necrosis factor-α and interleukin-1α) induced TRPA1 gene expression via nuclear factor-κB signaling and downstream activation of the transcription factor hypoxiainducible factor-1α (HIF1α). HIF1α unexpectedly acted by binding to a specific hypoxia response element-like motif and its flanking regions in the TRPA1 gene. The induced TRPA1 channels, which were intrinsically activated by endogenous hydrogen peroxide and Zn2+, suppressed secretion of interleukin-6 and interleukin-8. The data suggest a previously unrecognized HIF1α mechanism that links inflammatory mediators to ion channel expression. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

Mak S.,Sinai University | Witte K.K.,Institute of Membrane and Systems Biology | Al-Hesayen A.,St Michaels Hospital | Granton J.J.,Sinai University | Parker J.D.,Sinai University
American Journal of Physiology - Regulatory Integrative and Comparative Physiology | Year: 2012

Patients with congestive heart failure (CHF) due to left ventricular (LV) dysfunction have sympathetic activation specifically directed to the myocardium. Although pulmonary arterial hypertension (PAH) is associated with increased systemic sympathetic activity, its impact on sympathetic drive to ventricular myocardium is unknown. Fifteen patients with PAH (9 women; 54 ± 12 years) were studied: 10 with idiopathic PAH and 5 with a connective tissue disorder. We measured hemodynamics, as well as radiolabeled and endogenous concentrations of arterial and coronary sinus norepinephrine (NE). These measures were repeated after inhaled nitric oxide (NO). Measurement of transcardiac NE concentrations and the cardiac extraction of radiolabeled NE allowed calculation of the corrected transcardiac gradient of NE (CTCG of NE). Comparative data were collected from 15 patients (9 women: 55 ± 12 yr) with normal LV function and 15 patients with CHF (10 women; 53 ± 12 yr). PAH patients had elevated arterial NE concentrations compared with those with normal LV function but were similar to those with CHF. The CTCG of NE was higher in those with PAH than in the normal LV group (3.6 ± 2.2 vs. 1.5 ± 0.9 pmol/ml; P < 0.01) but similar to that seen in those with CHF (3.3 ± 1.4; P = NS). Inhaled NO, which reduced pulmonary artery pressure and increased cardiac output, had no effect on cardiac sympathetic activity. Therefore, cardiac sympathetic activation occurs in PAH. The mechanism of this activation remains uncertain but does not involve elevations in left heart filling pressure. © 2012 the American Physiological Society.

Gilbert S.H.,Institute of Membrane and Systems Biology | McConnell F.J.,University of Liverpool | Holden A.V.,Institute of Membrane and Systems Biology | Sivananthan M.U.,Cardiac MRI Unit | Dukes-McEwan J.,University of Liverpool
Veterinary Journal | Year: 2010

Over the last decade, magnetic resonance imaging (MRI) has become established as a useful referral diagnostic method in veterinary medicine that is widely used in small animal brain and spinal diseases, aural, nasal and orbital disorders, planning soft tissue surgery, oncology and small animal and equine orthopaedics. The use of MRI in these disciplines has grown due to its unparalleled capability to image soft tissue structures. This has been exploited in human cardiology where, despite the inherent difficulties in imaging a moving, contractile structure, cardiac MRI (CMRI) has become the optimal technique for the morphological assessment and quantification of ventricular function. Both CMRI hardware and software systems have developed rapidly in the last 10 years but although several preliminary veterinary CMRI studies have been reported, the technique's growth has been limited and is currently used primarily in clinical research. A review of published studies is presented with a description of CMRI technology and the potential of CMRI is discussed along with some of the reasons for its limited usage. © 2008 Elsevier Ltd. All rights reserved.

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