Amylin receptor phenotypes derived from human calcitonin receptor/RAMP coexpression exhibit pharmacological differences dependent on receptor isoform and host cell environment.
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 (hCTR(I1-)) or the insert positive (hCTR(I1+)), 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, hCTR(I1-) induced amylin binding with all three RAMPs, in contrast to COS-7, where only RAMP1 or RAMP3 generate an amylin receptor phenotype. hCTR(I1+) induced high-affinity amylin binding with any RAMP in either cell line. In COS-7 cells, hCTR(I1+)/RAMP-generated receptor displayed high- and low-affinity states, in contrast with the single-state binding seen with hCTR(I1-)/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.
Tilakaratne
N
N
Molecular Pharmacology Laboratory, Department of Pharmacology, The University of Melbourne, Victoria, Australia.
Christopoulos
G
G
Zumpe
E T
ET
Foord
S M
SM
Sexton
P M
PM
D000682:Amyloid / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D006224:Cricetinae; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D058228:Islet Amyloid Polypeptide; D008565:Membrane Proteins / Q000502:physiology; D010641:Phenotype; D020033:Protein Isoforms / Q000502:physiology; D058262:Receptor Activity-Modifying Protein 1; D058264:Receptor Activity-Modifying Protein 3; D058261:Receptor Activity-Modifying Proteins; D018003:Receptors, Calcitonin / Q000502:physiology*; D058287:Receptors, Islet Amyloid Polypeptide; D018000:Receptors, Peptide / Q000502:physiology*; D014162:Transfection
D000682:Amyloid; D047908:Intracellular Signaling Peptides and Proteins; D058228:Islet Amyloid Polypeptide; D008565:Membrane Proteins; D020033:Protein Isoforms; C545502:RAMP1 protein, human; C545504:RAMP3 protein, human; D058262:Receptor Activity-Modifying Protein 1; D058264:Receptor Activity-Modifying Protein 3; D058261:Receptor Activity-Modifying Proteins; D018003:Receptors, Calcitonin; D058287:Receptors, Islet Amyloid Polypeptide; D018000:Receptors, Peptide