Investigating GPCR:RAMP interactions using nanobodies

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The largest target for clinical drugs is a family of proteins called G-protein-coupled receptors (GPCRs). These are found on the surface of cells, where they bind molecules that cells release when they need to communicate with one another, such as hormones and neurotransmitters. On binding a hormone, the GPCR is activated, binds to another protein (often a G-protein, hence the name GPCR) and subsequently generates a chemical signal to the inside of the cell to bring about the required changes. GPCRs are thus of enormous interest, both in terms of basic biology but also for commercial exploitation by the pharmaceutical industry. Understanding how GPCRs work at the molecular level and function at the cellular level, is fundamentally important and is one of the 'big questions' in biology today. GPCRs have been classified into several families of structurally-related receptors. Family B GPCRs share a similar structure comprising a bundle of 7 transmembrane helices (7TM) linked by loops plus a large extracellular domain (ECD) and are important therapeutic targets for treatment of debilitating conditions such as obesity, diabetes, osteoporosis and migraine. They are activated by medium sized peptides which interact with both the ECD and the 7TM bundle although detailed understanding of the activation process is still limited. The pharmacology and signalling characteristics of family B GPCRs can be profoundly altered by association with a family of accessory membrane proteins referred to as Receptor Activity Modifying Proteins or RAMPs. The activation mechanisms of family B GPCRs and their interactions with RAMPs are poorly understood, although it is beyond question that the formation of these complexes is of profound pharmacological importance. Moreover, currently we do not know where these GPCR:RAMP complexes are localised in native human tissue. The main reason for this is the current lack of tools to properly localise or identify the GPCR:RAMP complexes in native tissues, or to selectively target them.Antibodies are powerful tools that will provide mechanistic and functional insights into family B GPCRs, their interactions with RAMP accessory proteins and localise GPCR:RAMP complexes in human tissue slices. Single domain antibodies (called nanobodies) made by llamas are particularly useful for this sort of task. However, generating antibodies that recognise native GPCRs in tissues has been a difficult challenge as GPCRs are unstable when extracted from the cell membrane by detergent as a pre-requisite for their purification. Recently, we have pioneered a way to 'solubilise' GPCRs without detergent, using a molecular 'pastry cutter' to generate GPCRs still in their native state, embedded in a miniscule disc of cell membrane (referred to as a 'SMALP'), thereby preserving the native environment. In collaboration with our Industrial Partner UCB, we have already shown that GPCR-SMALP can be used to isolate GPCR nanobodies. We will generate antibodies to two main family B receptors - the 'CGRP receptor' and the calcitonin receptor (which exists both alone and bound to RAMP). In addition to nanobodies that bind to only one target, we will engineer 'designer' antibodies that target two things simultaneously, so called 'bi-specifics'. These will target/bridge GPCRs:RAMP complexes, or will target two different domains in the same receptor, such as the ECD and 7TM, similar to natural activators (see above). We already have antibodies to the ECD, and loops, of a parathyroid receptor (PTH1R) suitable for making such 'designer' bi-specifics. Our nanobodies and 'bi-specifics' will be used to probe signalling by family B GPCRs, how it is regulated by RAMPs and to localise GPCR:RAMP complexes in tissue samples.Overall, this will provide important insights into how RAMPs regulate family B GPCRs and identify where this occurs in human tissue samples thereby providing physiological insights as well as defining underpinning mechanisms.