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Understanding agonist bias at CGRP and adrenomedullin receptors

Primary Supervisor: Professor David Poyner, Life & Health Sciences

Secondary supervisor: John Simms

PhD project title: Understanding agonist bias at CGRP and adrenomedullin receptors

University of Registration: Aston University

Project outline:

Calcitonin gene-related peptide (CGRP) is an abundant sensory neuropeptide and its relations, adrenomedullin (AM) and AM2 are locally released hormone. All are potent vasodilators that are also cardioprotective; CGRP has protective effects in hypertension. CGRP is released in neurogenic inflammation and is implicated in migraine while AM and AM2 are important in angiogenesis and lymphangiogenesis.  All three peptides act at a common G-protein coupled receptor called calcitonin receptor-like receptor (CLR), but pharmacological specificity requires one of three accessory proteins called receptor activity modifying proteins (RAMPs). RAMP1 forms the canonical CGRP receptor whereas the complexes with RAMP2 and RAMP3 are considered to give receptors for AM; the AM1 and AM2 receptors1.

The best characterised response to CGRP and the AMs is an increase in cAMP, via stimulation of the G protein Gs. However, the peptides are also able to couple to the inhibitory G protein Gi to reduce cellular excitability and Gq to increase intracellular calcium. Recent work from ourselves and collaborators have shown when CGRP acts at the AM1 receptor, it is more potent than AM at activating Gi; the converse is seen with AM at the CGRP receptor. At both receptors, AM2 is the most potent peptide at activating Gq2. Thus the RAMP can “bias” CLR so that each peptide has a unique pharmacological profile. Ligand bias is an important concept in molecular pharmacology; it is clear that individual agonists can activate distinct signalling pathways but the RAMPs give an extra layer of complexity. Agonist bias opens the way to produce drugs that can act without deleterious side-effects, but the molecular basis for this remains unclear.

In this project, we use site-directed mutagenesis to understand the means by which CGRP, AM and AM2 show bias. We already have an extensive library of mutants of individual residues within CLR which we have used to understand how the peptides cause activation of cAMP3. Our first objective will be to use these to examine how they promote coupling to Gi (by measuring the ability of pertussis toxin to inhibit cAMP production) and Gq (by measuring intracellular calcium) at the CGRP and AM1 receptors. The receptors will be transiently transfected into Cos 7 cells and concentration response curves will be constructed to agonists, measuring the appropriate output by our established assays. Having identified the key amino acids within CLR, we will then turn to molecular modelling for our second objective, which will be to produce models of the CGRP and AM1 receptors bound to Gi and Gq with either CGRP or AM bound and compare these to the structures of CGRP and AM receptors with Gs, which have recently been solved by cryo-electon microscopy. Our third objective will be to undertake molecular dynamic simulations of the CLR-RAMP-G protein complexes, to understand the conformational changes that the individual agonists must bring about for these to form. This will allow a full description of biased signalling in these receptors and indicate the potential for the design of new drugs.

References:

  1. Hay DL, Garelja ML, Poyner DR, Walker CS. Br J Pharmacol. 2018;175(1):3-17.Update on the pharmacology of calcitonin/CGRP family of peptides: IUPHAR Review 25.
  2. Weston, C, Winfield, I, Harris, M, hodgson, R, Shah, A, Dowell, S J., Mobarec, JC, Woodcock, DA., Reynolds, C A., Poyner, D R., Watkins, H A. & Ladds, G. Receptor activity modifying protein-directed G protein signaling specificity for the calcitonin gene-related peptide family of receptors. 2016, J Biol Chem.
  3. Woolley MJ, Reynolds CA, Simms J, Walker CS, Mobarec JC, Garelja ML, Conner AC, Poyner DR, Hay DL. Biochem Pharmacol. 2017;142:96-110Receptor activity-modifying protein dependent and independent activation mechanisms in the coupling of calcitonin gene-related peptide and adrenomedullin receptors to Gs.

BBSRC Strategic Research Priority: Understanding the Rules of Life: Structural Biology

Techniques that will be undertaken during the project:

  • Cell culture
  • Site-directed mutagenesis
  • Assays of cAMP and intracellular calcium
  • Pharmacological analysis and curve fitting
  • Protein structure analysis
  • Homology modelling
  • Molecular dynamics

Contact: Professor David Poyner, Aston University