An amylin receptor is revealed following co-transfection of a calcitonin receptor with receptor activity modifying proteins-1 or -3.
Human receptor activity modifying proteins (RAMP) regulate the ligand specificity of the calcitonin-receptor-like-receptor (McLatchie et al., Nature 393:333-339 (1998)). Here we have investigated binding of [125I]-labeled human (h) calcitonin ([125I]hCT) and rat amylin ([125I]amylin) to rabbit aortic endothelial cells (RAEC) co-transfected with the hCT receptor isotype 2 (hCTR2) and RAMP1, -2 or -3. Specific binding of 125 pM [125I]hCT to cells transfected with hCTR2 alone was 6.7 +/- 0.7 fmol/50,000 cells (n=5), and was reduced by 45 +/- 2% and 86 +/- 3% (P < 0.001) in the presence of RAMP1 and -3, but remained unchanged with RAMP2. In the absence and presence of individual RAMPs [125I]hCT binding inhibition occured with similar IC50 of between 6 nM and 11 nM hCT, and human amylin was 24- to 54-fold less potent. Specific binding of 125 pM [125I]amylin to cells transfected with hCTR2 alone was 0.9 +/- 0.2 fmol/50,000 cells (n=6), and was increased by 262 +/- 48% (P < 0.005), 73 +/- 26% (P < 0.05) and 338 +/- 57% (P < 0.005) with RAMP1, -2 or -3, respectively. [125I]amylin binding was inhibited with IC50 of 3.1 +/- 0.5 nM and 4.0 +/- 0.8 nM human amylin in cells co-transfected with RAMP1 or -3, respectively, and hCT was 45 +/- 2- and 126 +/- 3-fold less potent. In conclusion, RAMP1 and -3 decrease calcitonin receptor expression in RAEC transfected with hCTR2 encoding cDNA and simultanously reveal an amylin receptor.
Muff
R
R
Department for Orthopedic Surgery, University of Zurich, Switzerland. ramuz@balgrist.unizh.ch
Bühlmann
N
N
Fischer
J A
JA
Born
W
W
D000682:Amyloid / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D058228:Islet Amyloid Polypeptide; D008565:Membrane Proteins / Q000032:analysis / Q000502:physiology*; D011817:Rabbits; D058262:Receptor Activity-Modifying Protein 1; D058263:Receptor Activity-Modifying Protein 2; D058261:Receptor Activity-Modifying Proteins; D018003:Receptors, Calcitonin / Q000032:analysis / Q000502:physiology*; D058287:Receptors, Islet Amyloid Polypeptide; D018000:Receptors, Peptide / Q000032:analysis*; D014162:Transfection
D000682:Amyloid; D047908:Intracellular Signaling Peptides and Proteins; D058228:Islet Amyloid Polypeptide; D008565:Membrane Proteins; C545502:RAMP1 protein, human; C545503:RAMP2 protein, human; C545508:Ramp1 protein, rat; C545509:Ramp2 protein, rat; D058262:Receptor Activity-Modifying Protein 1; D058263:Receptor Activity-Modifying Protein 2; D058261:Receptor Activity-Modifying Proteins; D018003:Receptors, Calcitonin; D058287:Receptors, Islet Amyloid Polypeptide; D018000:Receptors, Peptide