Investigating GPCR:RAMP interactions using nanobodies
Biotechnology and Biological Sciences Research Council's (BBSRC) Industrial Partnership Award (IPA)
Aston University, University of Birmingham, UCB
1 May 2019 to 30 April 2022
G-protein-coupled receptors are a family of receptor proteins that play a key physiological role by binding molecules released by cells to communicate with one another, such as hormones or neurotransmitters. On binding a hormone the GPCR is activated, which triggers a cascade of messages inside the cell known as a signalling pathway. This important function means that receptors are also of significant interest to the pharmaceutical industry – GPCRs are the largest family of proteins targeted by clinical drugs for conditions including high blood pressure, diabetes, osteoporosis and migraine. Understanding how GPCRs work at the molecular level and how they function at the cellular level is fundamentally important and is one of the 'big questions' in biology today.
The signalling pathways of one group of GPCRs - the family B GPCRs – can be modified by their association with an accessory protein known as a Receptor Activity Modifying Protein (RAMP) to form a GPCR:RAMP complex. It is well known that this is physiologically important, but there are many unanswered questions regarding how the proteins interact and where they are located within tissues.
Antibodies are powerful tools that can be used by researchers to understand the mechanisms and functions of proteins in human tissue. We will use a type of antibody, known as a nanobody, which binds specifically to the proteins that we are interested in. However, generating antibodies that recognise GPCRs in tissue is challenging as the receptors are unstable when extracted from the cell membrane by detergent during the purification process required to make antibodies. Recently, we have pioneered a way to 'solubilise' GPCRs without detergent, using a molecular 'pastry cutter' to generate GPCRs embedded in a miniscule disc of cell membrane (referred to as a 'SMALP'), thereby preserving the native environment to keep the GPCR stable. In collaboration with our Industrial Partner UCB, who are experts in antibody generation and engineering, we have already shown that GPCR-SMALP can be used to isolate GPCR nanobodies. We will use this method to generate nanobodies to two main family B receptors - the 'CGRP receptor' and the calcitonin receptor. We will also engineer 'designer' antibodies that bind to two targets simultaneously ('bi-specifics'), so that we can study GPCR:RAMP complexes.
The project is funded via BBSRC’s Industrial Partnership Award (IPA) scheme and will be carried out in collaboration with UCB, a global biopharmaceutical company, with Coventry University as the lead Institution.
These new tools will enable us to determine how RAMPs regulate GPCRs and to identify where this occurs in human tissue samples, thereby providing important physiological insights and new mechanistic understanding at the molecular level, that will be of interest to other researchers in academia and the pharmaceutical industry.
Routledge, SJ, Jamshad, M, Little, HA, Lin, Y-P, Simms, J, Thakker, A, Spickett, CM, Bill, RM, Dafforn, TR, Poyner, DR & Wheatley, M 2020, 'Ligand-Induced Conformational Changes in a SMALP-Encapsulated GPCR' BBA - Biomembranes, vol. (In-Press), 183235, pp. (In-Press). https://doi.org/10.1016/j.bbamem.2020.183235