Investigating GPCR:RAMP interactions using nanobodies

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.

G-protein-coupled receptors (GPCRs) are central to cell signalling. Understanding how GPCRs function at the molecular/cellular level is fundamental to defining the under-pinning mechanisms of intracellular signalling. Family B GPCRs are therapeutically important but are far less well understood than family A GPCRs and often exist in a complex with an accessory protein referred to as a Receptor Activity Modifying Protein (RAMP). GPCR:RAMP complex formation profoundly affects the receptor pharmacology but this regulation is poorly understood in terms of both fundamental signalling mechanisms and also cellular location in native human tissues. 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. We will use llama single domain nanobodies recognising family B GPCRs and GPCR:RAMP complexes, generated in collaboration with our Industrial Partner UCB, using our pioneering detergent-free 'SMALP' methodology that preserves the native lipid environment and stability of purified GPCRs/GPCR:RAMP complexes. Nanobodies will be generated for the calcitonin receptor (CTR), CTR:RAMP1 complex and calcitonin gene-related receptor (CGRP-R; which is a calcitonin receptor-like receptor (CLR):RAMP1 complex). Individual nanobodies will be used to engineer 'designer' antibodies that incorporate two different binding domains (bi-specifics) to target/bridge GPCR:RAMP complexes, or to target two different domains in the same receptor. Using characterised anti-extracellular domain (ECD) plus anti-loop PTH1R antibodies that we have already, we will engineer bi-specifics targeting both the ECD plus extracellular loops of the PTH1R. Nanobodies and bi-specifics will be pharmacologically characterised, then used to probe signalling by family B GPCRs, how this is regulated by RAMPs and to localise GPCR:RAMP complexes in human tissue slices.

Family B GPCRs comprise important therapeutic targets for treatment of debilitating conditions such as obesity, diabetes, osteoporosis and migraine, directly relevant to BBSRC strategies on life-long health. Despite this therapeutic potential, drugs targeting family B GPCRs and especially their complexes with RAMPs are under-represented in the clinic. 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. The RAMP:GPCR interface represents an invaluable but unexploited target in family B GPCR therapeutics. This project will provide much-needed mechanistic insights into family B GPCR signalling and the functional modulation induced by RAMPs and, crucially, identify where GPCR:RAMP complexes are localised in human tissue slices. Until we know where these complexes exist, in native tissues, as opposed to recombinant cells, we cannot begin to understand their functional significance.

The immediate beneficiaries from this research will be those with a direct interest in family B GPCRs and the modulation of these receptors by complex formation with RAMP accessory proteins. This will include both academic and industrial researchers. Results generated in the course of this project will be directly relevant not only to academic researchers delineating the fundamental mechanisms of cell signalling by GPCRs but also to researchers in the pharmaceutical industry. All of our findings will be presented as 'open access' and made available to the wider scientific community, initially via conference presentations and then papers. In addition, the nanobodies and 'designer' bi-specific antibodies generated by this project will be made available to the wider scientific community, subject to the usual agreements for academic collaborations, once key output publications have been achieved. This meets the BBSRC strategic priority of 'technology development for biosciences'. In the industrial sector, the immediate beneficiary will be UCB who will have early access to our findings, but we anticipate many academic and pharma researchers will follow our various outputs.

There has been a growth in therapeutic antibodies in recent years but GPCR targets are under-represented. This lack is due in large part to the instability of GPCRs when removed from the cell membrane by detergent prior to purification. Our use in this project of purified GPCRs and GPCR:RAMP complexes encapsulated within a SMALP nanoscale membrane bilayer, in the total absence of detergent, circumvents the need for detergent and may promote the adoption of SMALPs in GPCR-directed antibody discovery in the future.

The project will impact on the career of the appointed PDRA as it will provide an extensive skill base in a wide range of diverse techniques. In particular the project gives experience of molecular pharmacology and biotechnology (protein production and engineering). The PDRA will also benefit from interacting with multiple research staff through formal collaborations between labs based in UoB (Wheatley/Dafforn) and Aston (Poyner). In addition, the PDRA will spend time hosted in the industrial research laboratories of UCB in Slough (UK). The diverse and extensive expertise involving both academic and industrial laboratories will make the PDRA very employable and will be an asset to their future career development, whatever their chosen arena.