Addressing the architecture, dynamics and activation of the CGRP receptor

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The CGRP receptor is a particularly interesting family B G-protein coupled receptor (GPCR) having an absolute requirement for an auxiliary protein known as Receptor activity modifying protein 1 (RAMP1). Class B GPCRs consist of a large extracellular domain (ECD) and a transmembrane domain (TMD). They frequently associate with accessory proteins belonging to the family of RAMPs. They act as receptors for a number of peptide hormones and neurotransmitters. They are attractive therapeutic targets but it has proved very difficult to obtain drugs that target them. Several crystal structures exist for the ECDs and there are crystal structures for two class B GPCRs (glucagon and CRF), but neither have bound peptides and the orientation between the TMD and ECD for any receptor remains speculative, as does the mechanism whereby agonists activate the receptors.

We have recently used a combination of site-directed mutagenesis and molecular modelling to propose a structure for CGRP bound to the TMD of CLR. This shows excellent agreement with the crystal structures, which were published after our modelled structures were deposited. Thus we propose that our methodology is robust. Furthermore, the presence of the RAMP provides additional constraints on the orientation of the ECD relative to the TMD, making the CGRP receptor especially amenable to modelling by greatly reducing the number of ways in which it could be modelled incorrectly.

We propose a strategy of photoaffinity cross-linking, disulphide trapping and point mutagenesis to provide experimental information on the architecture of the receptor when bound to CGRP and as a test for the modelling. This information will then be used to produce a model of the complex. We will use molecular dynamics and other modelling techniques to plan the experiments, to interpret the results and hence to determine the conformational changes caused by CGRP binding and so establish how the receptor is activated by its native agonist.

The most immediate beneficiaries would be those companies with research programmes directed towards the development of CGRP antagonists for migraine, where there is clinical evidence of the effectiveness of these agents. Migraine alone is estimated to cost the UK economy £2.25 billion per annum (Steiner TJ., Lecture to the All Party Parliamentary Group on Primary Headache Disorders., 19 November 2008) and CGRP antagonists have been shown to be effective against migraine in clinical trials. There have been 66 new patent applications filed worldwide for CGRP antagonists since January 2010. Thus the development of new agents to target the CGRP receptor would be of considerable benefit both to the UK pharmaceutical industry and also the health and well-being of the UK population. The mode of binding of CGRP and the way it activates its receptor is likely to be shared by other peptides in this family such as amylin (implicated in the control of eating) and calcitonin (well-established for the treatment of osteoporosis), further adding to the value of the project. The spectrum of disorders covered by the CGRP family of peptides include many which are common amongst elderly populations (e.g. heart failure, osteoporosis) and so this project is relevant to the BBSRC's initiative on lifelong health and well-being. More broadly, the challenges resulting from CLR modelling have serendipitously resulted in the generation of a helix alignment program that can work below the twilight zone (Vohra et al., J. Roy. Soc. 2013, Taddese et al., Plant Phys 2014) and we expect other methodologies to result from this challenging problem. Here, the way in which the modelling is closely integrated with experiment be applicable to a wide range of proteins of pharmaceutical or other interest. These include G-protein coupled receptors but extend far beyond those. In this respect, the project also addresses the BBSRC initiative on Technology development for the biosciences.