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

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.

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.

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-like family of GPCRs are important therapeutic targets for a host of disorders including diabetes, obesity and cardiovascular disease. Current sales of drugs targeting the incretins (3 members of the secretin-like receptor family) generate in excess of $1 billion. However these drugs can have serious adverse side effects. Consequently, there is considerable interest in developing the next generation of selective drugs. Our investigations into RAMP association with secretin-like 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. Further, at the conclusion of the project we will host a 1-day end of project workshop at Warwick where we will invite external collaborators.
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 (GSK, Heptares Therapeutics and Servier Laboratories). Our data will eventually aid in the design of more efficacious and effective drugs targeting the secretin-like receptor family.
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 it is likely to impact positively on the economics of the health care sector over the longer-term.
Provision of skilled workforce: The PDRA 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. Further, Warwick through its centre for Lifelong Learning provides a range of transferable skills courses designed to maximise individual potential and employability. Specifically, we will encourage the PDRA to enrol on the Postgraduate Certificate in Academic and Professional Practice. 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 opportunity. 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 in the "Science of Cells". Public lectures on the importance and implications of our science will be given through Café Scientifique, a forum for debating science issues, promoting public engagement and making science accountable. Articles will be produced for community newsletters. Further we will develop a project webpage and podcasts aimed specifically at the lay audience to explain our science and its medical benefits. Dr Ladds is currently generating a number of short, 5-minute open access podcasts showcasing his research to the general public. Annually members of Dr Ladds' laboratory partake in the Higher Education Access Programme for Schools and the "Big Bang" fair celebrating science, technology, engineering and mathematics for young people.