Addressing the architecture, dynamics and activation mechanism of the CGRP receptor

Lead Research Organisation: Aston University
Department Name: Sch of Life and Health Sciences

Abstract

G protein coupled receptors (GPCRs) are the largest family of proteins in the human genome and also the largest target for therapeutic drugs; thus they are of enormous scientific and practical interest. They are divided into a number of families. Of these, family-A is the best understood, but family-B includes receptors which are likely to be important in many disease states and so it is important to understand how these function, both to further our knowledge of fundamental biology and also for the design of new drugs.

Calcitonin gene-related peptide (CGRP) is found throughout the nervous system and is particularly important in regulating both the cardiovascular system (the heart and blood vessels) and also the immune system and inflammation. The receptor for CGRP is of special scientific interest as it involves a GPCR called CLR and also a second protein called RAMP1. RAMP1 is a member of a protein family that modulates a number of GPCRs of which the best characterised is CLR. CGRP is also likely to be important both in cardiovascular disorders and any disease that involves inflammation. The peptide is a major cause of migraine and drugs which block CGRP receptors have shown great promise in clinical trials; however, so far it has not been possible to use these clinically because of toxicity problems. Thus, there is an urgent need to develop new drugs that could act on CGRP receptors.

The CGRP receptor is made up of two parts. A portion called the transmembrane domain is found in the membranes of cells. This is connected to the extracellular domain, which is on the outside of cells. CGRP interacts with both parts of this structure and causes the transmembrane domain to change shape. This causes the receptor to interact with other proteins, leading to cell activation. We have a crystal structure of the part of the CGRP receptor that is on the outside of cells. Unfortunately, we do not know how CGRP binds to this, nor do we know how it binds to the transmembrane domain. This severely limits our understanding of the receptor and our ability to design drugs that could target it.

We have previously used experimental data from a technique known as site-directed mutagenesis to construct a computer model of the transmembrane domain of the CGRP receptor. This transmembrane domain is very similar to the transmembrane domains of two family-B GPCRs which were crystallised after our computer model was produced. This gives us confidence that our approach of combining experimental and computational methods is valuable. In this project, we intend to extend the approach to study how CGRP binds to both domains of the receptor and how this causes the receptor to become activated. We will use mutagenesis and also methods where we physically cross-link CGRP to the receptor to identify contact points. We will then use these to construct computational models, which we can refine through further experimentation. Using a computer, we can predict how the receptor shape will change when CGRP binds to it, so identifying the mechanism for receptor activation. This knowledge will be benefitial in the design of new drugs which can either block the receptor or promote its activation.

Technical Summary

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.

Planned Impact

The chief beneficiaries will include industrial scientists who are seeking to develop new drugs and the companies which employ them. In turn this would have economic benefits for the UK and also enhance the quality of life from anyone who might receive such drugs (which would further benefit the UK economy).

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" to align distantly related proteins (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, te 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.

Publications

10 25 50

publication icon
Uddin R (2018) Functional characterisation of G protein-coupled receptors. in Methods (San Diego, Calif.)

 
Description Wehave developed a method for photoaffinity labelling CGRP bound to the receptor. We have confirmed that a series of residues we predict that are close to the peptide binding site can cross-link to the ligand and have identified contacts within the probable binding pocket for CGRP in the transmembrane domain. We have confirmed the proximity of these reagents to the ligand binding site by changing these in the naturally occurring protein and shown that they disrupt the function of the receptor. We have also developed new computer methods to study how CGRP changes the shape of the protein to activate it.
Exploitation Route These findings open the way to the design of novel therapeutic agents, particularly for the treatment of migraine, hypertension and multiple sclerosis. The computer modelling methods we have developed may be of more general use in designing drugs specific for other receptors
Sectors Pharmaceuticals and Medical Biotechnology

URL https://research.aston.ac.uk/portal/en/persons/david-poyner(0bb31ffe-dec2-4b93-864b-7b6933d16fd8).html
 
Description We have obtained a BBSRC IPA to produce antibody to RAMP-receptor complexes, in collaboration with UCB. We are working on in-silico methods of protein modelling with MD Catapult
First Year Of Impact 2018
Sector Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description BBRSRC Responsive mode grant
Amount £27,991 (GBP)
Funding ID BB/R016755/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2018 
End 08/2021
 
Description BBSRC Pathfinder
Amount £12,000 (GBP)
Funding ID BB/S000100/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 06/2018 
End 09/2018
 
Description Internal PhD studentship
Amount £30,000 (GBP)
Organisation Aston University 
Department School of Life and Health Sciences
Sector Academic/University
Country United Kingdom
Start 09/2016 
End 08/2019
 
Description Pancreatic cancer UK
Amount £74,285 (GBP)
Funding ID RPG-2017-255 
Organisation Pancreatic Cancer UK 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2017 
End 02/2018
 
Description Royal Society Industrial Fellowships
Amount £151,861 (GBP)
Funding ID IF160090 
Organisation The Royal Society 
Sector Academic/University
Country United Kingdom
Start 09/2017 
 
Description Royal Society Summer Studentship
Amount £2,000 (GBP)
Organisation The Royal Society 
Sector Academic/University
Country United Kingdom
Start 08/2017 
End 09/2017
 
Title Method for generating GPCR models 
Description We have published a method for generating class B G protein-coupled receptor (GPCR) structures from X-ray, NMR or homology modelled sub-structures that is particularly suited to the 2 domain extracellular domain (ECD)/transmembrane (TM) structure of class B GPCRs. The essential feature is to generate partially overlapping fragments, which can be achieved through carefully docking of the full peptide ligand to both the ECD and the TM domain. The method also involves a two-step approach to handing photoaffinity labelling by first generating the model containing the labels in the presence of constraints and then translating the constraints into equivalent constraints for the wild-type receptor. The method is described in a series of 2016 publications, including: Wootten, D.; Reynolds, C. A.; Smith, K. J.; Mobarec, J. C.; Koole, C.; Savage, E. E.; Pabreja, K.; Simms, J.; Sridhar, R.; Furness, S. G.; Liu, M.; Thompson, P. E.; Miller, L. J.; Christopoulos, A.; Sexton, P. M. The Extracellular Surface of the GLP-1 Receptor Is a Molecular Trigger for Biased Agonism. Cell 2016, 165, 1632-43. Weston, C.; Winfield, I.; Harris, M.; Hodgson, R.; Shah, A.; Dowell, S. J.; Mobarec, J. C.; Woodlock, D. A.; Reynolds, C. A.; Poyner, D. R.; Watkins, H. A.; Ladds, G. Receptor Activity-modifying Protein-directed G Protein Signaling Specificity for the Calcitonin Gene-related Peptide Family of Receptors. J. Biol. Chem. 2016, 291, 21925-21944. 
Type Of Material Improvements to research infrastructure 
Year Produced 2016 
Provided To Others? Yes  
Impact The method was taken up by Heptares Therapeutics and was used to model a particular receptor (by Dr Conor Scully) as part of a drug design program. This enabled the programme to get off to a good start and the programme is progressing well. As a result, a REF impact case is being prepared. 
 
Title Transition state analysis 
Description We have developed an in-silico method to predict the pathway of GPCR activation; this resulted in a Pathfinder award to DRP 
Type Of Material Biological samples 
Year Produced 2018 
Provided To Others? No  
Impact We are currently working with MD Catapault to evaluate the utility of this method for drug discovery 
 
Title CGRP 
Description Computer models of the CGRP receptor have been deposited in the Essex Research Repository and are given a DOI from the relevant publications 
Type Of Material Computer model/algorithm 
Year Produced 2017 
Provided To Others? Yes  
Impact Deeper understanding into the structure and function of the CGRP receptor that may be relevant to drug design and heart disease/migraine. 
URL http://repository.essex.ac.uk
 
Title CTR/AMY 
Description Molecular models of the calcitonin receptor (CTR) and the Amylin Receptor (AMY1R, i.e. CTR in complex with a receptor activity modifying protein). These models are stored in the Essex Research Repository and are referenced from associated publications / articles submitted. 
Type Of Material Computer model/algorithm 
Year Produced 2018 
Provided To Others? No  
Impact Deeper understanding into the structure and dynamics of these calcitonin-based receptor models that are related to various diseases including osteoporosis, migraine and diabetes. 
URL http://repository.essex.ac.uk/
 
Title GLP-1R 
Description Computer models have been generated of the GLP-1 receptor, the adrenomedullin receptor and the PTH2 receptor; these have been validated by collaborative experimental studies. The models are available from ftp.essex.ac.uk/pub/oyster/ 
Type Of Material Database/Collection of data 
Year Produced 2016 
Provided To Others? Yes  
Impact The models have had a significant impact on explaining pharmacological data on important drug targets, as indicated in the following publications: 1. Wotten, D., Reynolds, C. A., Smith, K. J., Mobarec, J. C., Koole, C., Savage, E. E., Pabreja, K., Simms, J., Sridhar, R., and Furness, S. G., Miller, L. J., Christopoulos, A., and Sexton, P. M. (2016) The extracellular surface of the GLP-1 receptor is a molecular trigger for biased agonism. Cell 165,1632-1643. doi.org/10.1016/j.cell.2016.05.023 2. Wootten, D., Reynolds, C. A., Smith, K. J., Mobarec, J. C., Furness, S. G., Miller, L. J., Christopoulos, A., and Sexton, P. M. (2016) Key interactions by conserved polar amino acids located at the transmembrane helical boundaries in Class B GPCRs modulate activation, effector specificity and biased signalling in the glucagon-like peptide-1 receptor. Biochem. Pharmacol. 118,68-87. doi.org/10.1016/j.bcp.2016.08.015 3. Wootten, D., Reynolds, C. A., Koole, C., Smith, K. J., Mobarec, J. C., Simms, J., Quon, T., Coudrat, T., Furness, S. G., and Miller, L. J. (2016) A hydrogen-bonded polar network in the core of the glucagon-like peptide-1 receptor is a fulcrum for biased agonism: lessons from class B crystal structures. Mol. Pharmacol. 89,335-347. doi.org/10.1124/mol.115.101246 4. Weston, C., Winfield, I., Harris, M., Hodgson, R., Shah, A., Dowell, S. J., Mobarec, J. C., Woodlock, D. A., Reynolds, C. A., and Poyner, D. R. (2016) Receptor Activity-modifying Protein-directed G Protein Signaling Specificity for the Calcitonin Gene-related Peptide Family of Receptors. J. Biol. Chem. 291,21925-21944. doi.org/10.1074/jbc.M116.751362 5. Weaver, R. E., Mobarec, J. C., Wigglesworth, M. J., Reynolds, C. A., and Donnelly, D. (2016) High affinity binding of the peptide agonist TIP-39 to the parathyroid hormone 2 (PTH 2) receptor requires the hydroxyl group of Tyr-318 on transmembrane helix 5. Biochem. Pharmacol. doi.org/10.1016/j.bcp.2016.12.013 6. Watkins, H. A., Chakravarthy, M., Abhayawardana, R. S., Gingell, J. J., Garelja, M., Pardamwar, M., McElhinney, J. M., Lathbridge, A., Constantine, A., and Harris, P. W. (2016) Receptor Activity-modifying Proteins 2 and 3 Generate Adrenomedullin Receptor Subtypes with Distinct Molecular Properties. J. Biol. Chem. 291,11657-11675. doi.org/10.1074/jbc.M115.688218 
URL http://ftp.essex.ac.uk/pub/oyster/
 
Description CLR and congenital birth defects 
Organisation University of North Carolina at Chapel Hill
Country United States 
Sector Academic/University 
PI Contribution We have modelled a naturally occurring mutated form of the calcitonin receptor-like receptor which prevents its association with birth defects and leads to fatal birth defects
Collaborator Contribution Our partners identified the initial birth defect in clinical studies and have explored its phenotype in cellular and whole-organism models.
Impact Paper in preparation
Start Year 2017
 
Description Discovery Catapault 
Organisation Medicines Discovery Catapult
PI Contribution We will assess in-silico the activity of compounds where Discovery have details of activity
Collaborator Contribution Discovery hold experimental details of pharmacologically active compounds which we can assess using our software
Impact None so far
Start Year 2018
 
Description FCS to study SMALPed proteins 
Organisation University of Nottingham
Country United Kingdom 
Sector Academic/University 
PI Contribution We have supplied SMALP-purified adenosine 2a receptor for analysis by fluorescence correlation spectroscopy (FCS) by Dr Steve Briddon at Nottingham University
Collaborator Contribution Dr Briddon has performed and interpreted the FCS
Impact We have shown that it is possible to identify single molecules of the adenosine A2a receptor by FCS
Start Year 2017
 
Description GLP-1 signalling and bias (with Imperial) 
Organisation Imperial College London
Country United Kingdom 
Sector Academic/University 
PI Contribution Preliminary data on the structure of the GLP-1 receptor (GLP-1R)
Collaborator Contribution Preliminary data on identification of biased ligands for GLP-1R
Impact Award of an MRC grant entitled XXX on GLP-1R signalling and bias, (PI: , Co-Is: ); CAR was very pleased to supply a letter of support
Start Year 2017
 
Description Mass spectroscopy of GPCRs 
Organisation University of Oxford
Department Department of Chemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution We have supplied purified GPCRs
Collaborator Contribution The partners are analysing the GPCRs by mass spectroscopy
Impact This is an academic collaboration; we hope it will lead to new papers
Start Year 2015
 
Description Modelling remote homologues (with Johnson Matthey) 
Organisation Johnson Matthey
Country United Kingdom 
Sector Private 
PI Contribution Sequence alignment in the twilight zone in the context of modelling remote homologues for building models of enzymes required for carrying out chemical reactions
Collaborator Contribution Supplied sequences
Impact alignments
Start Year 2018
 
Description Modelling the Adenosine Receptor 
Organisation University of Cambridge
Department Department of Pharmacology
Country United Kingdom 
Sector Academic/University 
PI Contribution Molecular modelling of the adenosine A1 receptor
Collaborator Contribution Molecular pharmacology of the A1 receptor
Impact multi-disciplinary: molecular modelling and experimental pharmacology
Start Year 2017
 
Description Role of RCP in promoting signalling at GPCRs 
Organisation University of Rochester
Country United States 
Sector Academic/University 
PI Contribution We have been examining the ability of receptor component protein (RCP) to facilitate G-protein coupling to receptor/RAMP complexes using siRNA
Collaborator Contribution Dr Dickerson of Rochester has supplied us with his antibody directed against RCP
Impact None so far
Start Year 2016
 
Description Royal Society Industrial Fellowship: Markov State Modelling 
Organisation Heptares Therapeutics Ltd
Country United Kingdom 
Sector Private 
PI Contribution Expertise in modelling GPCRs
Collaborator Contribution Access to specialist software; specialist knowledge on GPCRs and drug design
Impact Summer student trained in bioinformatics
Start Year 2017
 
Description SMALPing of calcitonin receptor 
Organisation Monash University
Country Australia 
Sector Academic/University 
PI Contribution We have supplied reagents and provided training for a research student to purify the calcitonin receptor using SMALPs
Collaborator Contribution Provision of a cell line expressing the calcitonin receptor and a student to do the experimental work
Impact Production of the purified calcitonin receptor for structural studies
Start Year 2015
 
Description Article in community magazine describing recent research 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Some comments that the article was interesting

I was subsequently invited to speak about my work, to a community group
Year(s) Of Engagement Activity 2015
 
Description Article in community magazine on the importance of scientific evaluation 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact There were a few positive comments, but generally limited response

Difficult to say!
Year(s) Of Engagement Activity 2015
 
Description Drug Design Workshop for the giften and able 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact 8 Gifted and able students from Bromfords School, Essex, attended a week-long drug design workshop, which greatly increased the pupils enthusiasm for science.
Year(s) Of Engagement Activity 2016
 
Description Magazine article 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact I have written an article for a community newsletter about my research work.
Year(s) Of Engagement Activity 2019
 
Description Protein modelling master class 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Students from local schools visit the university and learn how to design drugs that bind to proteins using molecular modelling
Year(s) Of Engagement Activity 2016,2017
 
Description Protein modelling masterclass 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Hands-on demonstration of protein modelling and its application in drug discovery to 6th formers
Year(s) Of Engagement Activity 2017
 
Description Seminar at Department of Pharmacology, Cambridge 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact Talk in front of researchers and undergraduate students of pharmacology at Cambridge
Year(s) Of Engagement Activity 2017
 
Description Talk at village group 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Talk to around 15 people at group in the village of Highley about my work as scientist
Year(s) Of Engagement Activity 2017
 
Description Talk at village group 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact I spoke about my work as a scientist to "Catalyst", a social group at Highley, close to where I live
Year(s) Of Engagement Activity 2016
 
Description Talk to community group 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact There was a lot of discussion following the talk

I was asked if a 6th former could spend a day in my lab, by a the friend of his parent, who attended the talk. This visit took place on 2-11-15
Year(s) Of Engagement Activity 2015
 
Description Talks for ELRIG at Lab Innovations exhibition, Birmingham 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Significant discussions after the talk

Two new potential collaborations are being explored
Year(s) Of Engagement Activity 2015