An Amylin Receptor Is Revealed Following Co-Transfection of a Calcitonin Receptor with Receptor Activity Modifying Proteins-1 or -3

Abstract

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 occurred 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. [¹²⁵]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 simultaneously reveal an amylin receptor.