The role of conserved residues in receptor activity-modifying protein 1 in the AMY1 receptor

Richard J Bailey1, Graham D Bailey1, Joshua WI Bradley1, Samuel D Robinson1, David R Poyner2, Debbie L Hay1. 1University of Auckland, Auckland, New Zealand, 2Aston University, Birmingham, United Kingdom.

The family of three receptor activity-modifying proteins (RAMPs) regulates the trafficking and function of several G protein-coupled receptors (GPCRs). For example RAMPs interact with the calcitonin receptor, producing subtypes of receptor with high affinity for the peptide hormone amylin (Amy); AMY1, AMY2 and AMY3, respectively. Without RAMP, the calcitonin receptor binds Amy with low affinity. Whether RAMPs modify the conformation of the calcitonin receptor to influence Amy binding or directly contribute points of contact to Amy is still an open question. It is also unclear whether there are shared roles for the individual amino acids in the RAMPs in the different GPCR/RAMP complexes or if they have a distinct contribution depending on the composition of the complex. To understand the role of conserved residues in the extracellular N-termini of RAMPs, we have mutated all 14 individually to alanine in human RAMP1 (apart from the conserved alanine). Mutant or wild-type mycRAMP1 and the insert negative isoform of the calcitonin receptor (AMY1(a) receptor complexes) were transfected into Cos-7 cells and cAMP measured following exposure to rat (r) Amy. The cell surface expression of receptor complexes was determined using a whole-cell ELISA, utilising the myc tag on RAMP1. As RAMP1 does not efficiently express at the cell surface in the absence of an interacting receptor partner, this assay can be used to indicate whether mutants affect RAMP1-calcitonin receptor association. All alanine mutants were detrimental to AMY1(a) receptor function, causing significant reductions in rAmy potency when compared to WT receptors. This varied from <10-fold (P115A) to >60-fold (N86A, C104A). Furthermore, substitution of any individual residue was sufficient to impair the cell surface expression of AMY1(a) receptors. However most mutants, with the exception of C57A, C72A and C104A enabled some association with CT(a) as determined by the presence of measurable mycRAMP1 at the cell surface. The cell surface expression of AMY1(a) receptors was least affected by alanine substitution at position P115, consistent with a lesser functional impact of this substitution. Mutation of the conserved residues probably causes structural perturbation of RAMP1, affecting receptor association and Amy interactions. This study has confirmed the importance of conserved residues in maintaining the functional integrity of RAMP1.