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A Critical Role for the Short Intracellular C Terminus in Receptor Activity-Modifying Protein Function

Drug Discovery Biology Laboratory, Department of Pharmacology, Monash University, Victoria, Australia (G.C., M.M., A.C., N.T., P.M.S.); and Howard Florey Institute (M.U., G.C., M.M., S.Y., P.M.S.) and Department of Pharmacology (M.U., M.M., A.C.), University of Melbourne, Victoria, Australia

Receptor activity-modifying proteins (RAMPs) interact with and modify the behavior of the calcitonin receptor (CTR) and calcitonin receptor-like receptor (CLR). We have examined the contribution of the short intracellular C terminus, using constructs that delete the last eight amino acids of each RAMP. C-Terminal deletion of individual RAMPs had little effect on the signaling profile induced when complexed with CLR in COS-7 or human embryonic kidney (HEK)293 cells. Likewise, confocal microscopy revealed each of the mutant RAMPs translocated hemagglutinin-tagged CLR to the cell surface. In contrast, a pronounced effect of RAMP C-terminal truncation was seen for RAMP/CTRa complexes, studied in COS-7 cells, with significant attenuation of amylin receptor phenotype induction that was stronger for RAMP1 and -2 than RAMP3. The loss of amylin binding upon C-terminal deletion could be partially recovered with overexpression of G_s, suggesting an impact of the RAMP C terminus on coupling of G proteins to the receptor complex. In HEK293 cells the c-Myc-RAMP1 C-terminal deletion mutant showed high receptor-independent cell surface expression; however, this construct showed low cell surface expression when expressed alone in COS-7 cells, indicating interaction of RAMPs with other cellular components via the C terminus. This mutant also had reduced cell surface expression when coexpressed with CTR. Thus, this study reveals important functionality of the RAMP C-terminal domain and identifies key differences in the role of the RAMP C terminus for CTR versus CLR-based receptors.

Address correspondence to: Dr. Patrick M. Sexton, Drug Discovery Biology Laboratory, Department of Pharmacology, Bldg. 13E, Monash University, Clayton, 3800 Victoria, Australia. E-mail: patrick.sexton{at}med.monash.edu.au

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