IDENTIFICATION OF MULTIPLE HUMAN CALCITONIN RECEPTOR ISOFORMS - HETEROLOGOUS EXPRESSION AND PHARMACOLOGICAL CHARACTERIZATION

The human breast carcinoma cell line T47D is known to express high-aff inity calcitonin receptors (CTRs). PCR amplification of the CTR cDNA f rom T47D mRNA resulted in the identification of two different cDNAs th at encode distinct receptor isoforms, h alpha CTR and h beta CTR. The two cDNAs are identical except that the h alpha CTR cDNA contains a 48 bp insert sequence that encodes a 16 amino acid domain in the first c ytosolic loop of the receptor. Stable transfection of each receptor cD NA into murine erythroleukaemia (MEL) cells resulted in the expression of receptors with high affinity for radiolabelled salmon calcitonin ( h alpha CTR K-d 0.09 nM, h beta CTR K-d 0.12 nM). Ligand competition b inding studies did not reveal any significant pharmacological differen ce between the receptor isoforms. In transfected MEL cells and COS-1 c ells the h beta CTR isoform was expressed at tenfold higher levels tha n the h alpha CTR. A reporter gene assay that monitored the coupling o f CTR to adenylate cyclase by increases in beta-galactosidase activity indicated that both receptors were able to stimulate cyclic AMP produ ction in response to ligand binding.