Abstract
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 Gαs, 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.
Footnotes
-
This work was supported by National Health and Medical Research Council (NHMRC) grant 299810 and the Ian Potter Neuropeptide laboratory. P.M.S. is a Principal Research Fellow of the NHMRC of Australia. A.C. is a Senior Research Fellow of the NHMRC.
-
M.U., G.C., and M.M. contributed equally to this work.
-
ABBREVIATIONS: Δ, deletion mutant; GPCR, G protein-coupled receptor; RAMP, receptor activity-modifying protein; CT, calcitonin; CTR, calcitonin receptor; CLR, calcitonin receptor-like receptor; AMY, amylin receptor phenotype; AM, adrenomedullin; CGRP, calcitonin gene-related peptide; CHO, Chinese hamster ovary; PDZ, postsynaptic density-95/Discs-large/ZO-1 homology; h, human; sCT, salmon calcitonin; rAmy, rat amylin; HA, hemagglutinin; WT, wild-type; DMEM, Dulbecco's modified Eagle's medium; Gpp(NH)p, guanosine 5′-(β,γ-imido)triphosphate; PBS, phosphate-buffered saline; RCP, receptor component protein.
- Received March 9, 2006.
- Accepted August 15, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
MolPharm articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|