THE ROLE OF RAMPS IN LIGAND-ENGENDERED SIGNAL BIAS OF SECRETIN-LIKE RECEPTORS

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

Abstract

Many hormones and neurotransmitters perform their function through a class of proteins called family B G protein-coupled receptors (GPCRs). These include hormones such as GLP-1 and glucagon which are relevant to diabetes and other metabolic disorders especially common in the elderly, corticotrophin releasing factor, involved in stress and anxiety and parathyroid hormone, involved in maintaining bones. It has been known for over 10 years that many of the receptors for these agents can interact with accessory proteins called receptor activity modifying proteins (RAMPs). RAMPs are found throughout the body. However, until recently, the consequences of RAMP-receptor interactions remained unknown. Recent studies of a small number of these family B GPCRs has shown that RAMPs have important consequences for function and so it is now timely to extend this study to all 15 family B GPCRs. We have discovered that this can be achieved quickly, cheaply and easily by analysing the behaviour of human receptors and RAMPs in yeast and we will use this method to comprehensively explore how all the family B GPCRs found in humans are influenced by RAMPs. We will extend our studies to determine the consequences of these interactions using human cells. The results we generate will enable more sophisticated experiments to be performed to determine the physiological consequences of these interactions in in-vivo models. This is important as our data indicates that RAMP association can radically change the properties of an individual receptor; experiments that neglect to consider the effects of RAMPs can give misleading impressions of the true function of a receptor. Furthermore, it is likely that the association of the RAMP with a receptor will create a structure that can be selectively targeted by drugs.

Technical Summary

Receptor activity modifying proteins (RAMPs) are essential partners of the calcitonin and calcitonin receptor-like receptors, allowing them to respond to amylin, calcitonin gene-related peptide and adrenomedullin. These receptors are members of the family B G protein-coupled receptors (GPCRs). It has been suggested that at least seven other family B GPCRs can interact with RAMPs, but the consequences of these interactions have not been explored except for the corticotrophin-releasing factor (CRF) type-1 receptor and the VPAC1 and VPAC 2 receptors, where RAMPs alter G protein specificity and agonist bias. For the CRFR1, RAMP2 association enhances the ability of the receptor to promote ACTH release in vivo. Thus, RAMP association with family B GPCRs is widespread and is physiologically important. This has created an urgent need to fully characterise the extent and consequences of these interactions and to properly understand the pharmacology of these important drug targets. The native pattern of GPCR coupling to G proteins is preserved in yeast engineered with mammalian-like G proteins and we have shown that this system can be modulated by RAMP co-expression. This provides the basis of a high-throughput assay to investigate the effects of RAMPs on G protein coupling and agonist bias. We will establish if RAMPs 1, 2 and 3 modulate receptor-G protein interactions for each of the 15 human family B GPCRs using multiple agonists for each receptor. This data will enable us to determine if each combination generates a specific signalling bias. We will continue these studies to investigate the consequences of the interactions using specific mammalian cell assays for RAMP-receptor interactions, agonist-stimulated G protein activation and second messenger regulation. Ultimately, the data we generate will form the basis of an extended study using animal models and will enable structural studies aimed at developing selective drugs for individual RAMP-receptor-G protein complexes.

Planned Impact

The research that we propose will have a broad range of impacts from advancement of fundamental scientific knowledge to training of a highly skilled workforce.

Advancement of scientific knowledge: Our research program will develop knowledge that will enhance our understanding of GPCR pharmacology. The secretin family GPCRs are important therapeutic targets for a host of disorders including diabetes, obesity, cardiovascular disease, neurodegenerative disorders. The incretins (GLP-1, glucagon and GIP) form a significant proportion of the secretin receptor family. These molecules have been targeted for treatments related to diabetes and obesity. To date, incretin-based therapeutics generates in excess of $1 billion annually and consequently, there is considerable interest in developing the next generation of diabetic drugs. Our investigations into RAMP association with secretin receptors will aid this research. Any novel biological data generated will yield new knowledge that will be disseminated through international journals, conference publications, seminars, collaborative contacts with other researchers and participation in professional body events.

Pharmaceutical and Biotechnology industries: The UK economy relies heavily on the Pharmaceutical and Biotechnology industry; it employs more than 250,000 people and generates billions of pounds annually. Drug discovery relies extensively on a strong basic-science background to provide insights into potential drug targets and the development of new assays and technologies. Our work, systematically analysing one entire family of GPCRs, will contribute to this knowledge base and is backed by industry. Our data will eventually aid in the design of more efficacious and effective drugs targeting the secretin receptor family. This will be highly relevant not only to "big pharma", but also to regional businesses in the Midlands.

Translational medicine: Our work has the potential to contribute to significantly improved efficacy and reduced treatment costs for diseases associated with many GPCR-based diseases. Thus our work is likely to impact positively on the economics of the health care sector over the longer-term.

Provision of skilled workforce: The PDRA (Dr Weston) and the research technician working on this project will be trained in a wide range of pharmacology techniques. These skills are highly specialised and valuable, and will equip them for careers in academia or industry. Dr Weston will be encourage to attend courses held at the University of Warwick's Centre for Lifelong Learning provides a range of courses to further aid members of staff in their professional development. Dr Weston will be encourage to attend these courses and also to enrol on the Postgraduate Certificate in Academic and Professional Practice (PCAPP). Further, Warwick and Aston provide a range of transferable skills courses designed to maximise individual potential and employability. Thus our work will contribute to the economic competitiveness of the UK through the provision of highly skilled labour.

The general public: We will disseminate our findings to the general public at the earliest juncture. WMS has an active public engagement policy and an excellent track record in outreach activities. We will host yearly public lectures for local secondary school teachers from around the Midlands at WMS. These events will be aimed at enthusing school children from aged 11-13 in the "Science of Cells". Public lectures on the importance and implications of our science will be given through Café de Scientifique a forum for debating science issues, promoting public engagement and making science accountable. Articles will also be produced for community newsletters. We will also develop a project webpage and podcasts aimed specifically at the lay audience to explain our science and its medical benefits.
 
Description We have shown that the properties of cell surface receptors are altered when they associate with accessory proteins known as receptor activity modifying proteins (RAMPs). Many of these receptors are important in diabetes; the association with RAMPs creates new drug targets for the treatment of this disease. We have also shown that RAMPs have profound influences on the properties of receptors that respond to calcitonin gene-related peptide and allied peptides. In this case, the RAMPs "steer" the receptor to different signalling systems within a cell, depending on how they are activated. This raises the possibility of being able to target only the signalling pathways relevant to disease, not those which might be important for other processes within the cell. Previously RAMPs were largely considered just to influence the way in which hormones and nerve transmitters bound to cells; we have shown that their predominant effect is on how these agents activate cells.
Exploitation Route These finding should eventually result in new drugs to treat diabetes and related diseases.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

URL https://research.aston.ac.uk/portal/en/persons/david-poyner(0bb31ffe-dec2-4b93-864b-7b6933d16fd8).html
 
Description The findings are being explored to develop new therapeutic antibodies that can target receptor-RAMP complexes. This has resulted in an BBSRC IPA to myself, Prof Mark Wheatley (Coventry University) and Prof Tim Dafforn (Birmingham University) and UCB.
Sector Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
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 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 Collaboration with UCB 
Organisation UCB Pharma
Department UCB Celltech
Country United Kingdom 
Sector Private 
PI Contribution We have supplied SMALP-solubilised GPCRs for antibody production; we are currently expanding this to RAMP-receptor complexes
Collaborator Contribution UCB are currently using our material to produce antibodies
Impact This lead to an application for a new project grant to the BBSRC and also resulted in the award of a university awarded PhD studentship.
Start Year 2015
 
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 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 New ways of looking at GPCRs 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Project website

This has lead to several enquiries for PhD places
Year(s) Of Engagement Activity 2013
URL http://www.aston.ac.uk/lhs/staff/az-index/poynerdr/
 
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 2016 SMALP users meeting 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact This was a 20 minute talk on the use of SMALPs for solubilising GPCRs
Year(s) Of Engagement Activity 2016
 
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 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 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