Molecular insights into the signalling of calcitonin receptor-like receptor:receptor activity-modifying protein complexes

Abstract

Adrenomedullin (AM) is a 52 amino acid peptide that belongs to the wider calcitonin peptide family. AM is expressed ubiquitously throughout the body, and plays a key role in the development and maintenance of the cardiovascular system. AM can activate three distinct receptors, all of which are comprised of a class B G protein-coupled receptor, the calcitonin receptor-like receptor (CLR), in complex with one of three receptor activity-modifying proteins (RAMPs). The different RAMPs exert influence over many aspects of CLR, ranging from defining its pharmacology to determining its fate following internalisation. These three receptors have an additional layer of complexity in that they can be activated by a number of related peptides, namely adrenomedullin 2 (AM2), α calcitonin gene-related peptide (αCGRP), and βCGRP. AM has shown promise as a therapeutic in the treatment of cardiovascular disorders, however at present AM has not been translated into a clinically useful drug. This failure to translate can be attributed to the negative side effects associated with AM administration. Developing an agonist based on AM which has high specificity for a single CLR:RAMP complex or which displays bias towards a clinically desired signalling pathway may improve its therapeutic utility by reducing side-effects. There are a number of areas that remain under-investigated in the field of AM pharmacology. Firstly, we lack information on the molecular determinants of AM activating its receptors. Secondly, we have little knowledge on how RAMPs affect the intracellular signalling cascades regulated by CLR. Lastly, we lack insight into how cellular contexts may affect the function of CLR. Thus, chapter 3 used alanine mutagenesis to investigate the role of an unexplored portion of CLR, the linker region. Chapter 4 investigated whether RAMPs influence the repertoire of signalling molecules that CLR can regulate. Chapter 5 explored the role of the AM N-terminus in receptor activation. Chapter 6 investigated whether findings from previous chapters were retained in a physiologically relevant setting by characterising peptides in a vascular endothelial cell-line which natively expresses AM-responsive receptors. Mutagenesis of the linker region indicated that the different RAMPs had a conserved effect on the CLR linker region. In contrast, RAMPs differentially influenced the suite of intracellular signalling pathways activated by CLR following receptor activation. The alanine scan of the AM N-terminus gave insight into mechanisms involved in receptor activation, highlighting peptide residues which were required for receptor activation, while also finding residues that could tolerate substitution (making them prime candidates for future modifications). Importantly, G19 was a key determinant of peptide activity; substituting this residue with alanine (as found natively in CGRP) creates an analogue which displays signalling abilities previously restricted to CGRP (such as the ability to stimulate inositol phosphate signalling). Pharmacological characterisation of endogenous ligands in the endothelial cell-line defined the functional AM-responsive receptor expressed by these cells as likely to be CLR:RAMP2. Alanine-substituted analogues retained their novel signalling profiles in endothelial cells, however signalling profiles were often exaggerated relative to the profiles seen in transfected cells, emphasising the value of translational studies. These findings will guide rational drug design, aiding the development of therapeutics based on AM for the treatment of cardiovascular disease.