Abstract
Receptor activity modifying proteins (RAMPs) constitute a group of three proteins, designated as RAMP1, 2, and 3, which are able to effect functional changes in some members of the G protein-coupled receptor family. Thus, RAMP1 or RAMP3 can modify the calcitonin receptor (CTR) to also function as a high-affinity amylin receptor-like phenotype. To examine the RAMP/CTR interaction, individual RAMPs were coexpressed with either of the two human CTR (hCTR) isoforms, the insert negative (hCTRI1−) or the insert positive (hCTRI1+), in Chinese hamster ovary (CHO-P) or African monkey kidney (COS-7) cells. CHO-P cells provide an environment conducive to a low, but significant, level of amylin binding with either hCTR isoform alone, unlike in COS-7, where RAMP coexpression is imperative for amylin binding. Also, in CHO-P, hCTRI1− induced amylin binding with all three RAMPs, in contrast to COS-7, where only RAMP1 or RAMP3 generate an amylin receptor phenotype. hCTRI1+ induced high-affinity amylin binding with any RAMP in either cell line. In COS-7 cells, hCTRI1+/RAMP-generated receptor displayed high- and low-affinity states, in contrast with the single-state binding seen with hCTRI1−/RAMP-generated receptor, whereas in CHO-P cells a two-affinity state receptor phenotype was evident with both hCTR isoforms. Endogenous RAMP expression is low and similar between cell lines. The results suggest that CTR/RAMP interaction in these cells is complex with other cellular factors such as the levels of different G proteins and/or receptor/RAMP stoichiometry following heterologous coexpression contributing to the ultimate receptor phenotype.
Footnotes
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Send reprint requests to: Dr. Patrick M. Sexton, Molecular Pharmacology Laboratory, Department of Pharmacology, The University of Melbourne, 3010, Victoria, Australia. E-mail:p.sexton{at}pharmacology.unimelb.edu.au
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↵1 This work was supported by grants from the National Health and Medical Research Council of Australia. P.M.S. is a Research Fellow of the National Health and Medical Research Council of Australia.
- Abbreviations:
- CTR
- calcitonin receptor
- CGRP
- calcitonin gene-related peptide
- CT
- calcitonin
- hCTR
- human calcitonin receptor
- RAMP
- receptor activity modifying protein
- CHO
- Chinese hamster ovary
- CHO-P cells
- a variant of CHO cells stably transfected with P-selectin
- CRLR
- calcitonin receptor-like receptor
- FBS
- fetal bovine serum
- sCT
- salmon calcitonin
- hCT
- human calcitonin
- DMEM
- Dulbecco's modified Eagle's medium
- RT-PCR
- reverse transcription-polymerase chain reaction
- RAEC
- rabbit arterial endothelial cells
- Received January 10, 2000.
- Accepted March 14, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
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