INHIBITION OF INOSITOL PHOSPHATE 2ND MESSENGER FORMATION BY INTRACELLULAR LOOP ONE OF A HUMAN CALCITONIN RECEPTOR - EXPRESSION AND MUTATIONAL ANALYSIS OF SYNTHETIC RECEPTOR GENES

Receptors for calcitonin (CTRs) have been cloned from several species, and two isoforms have been found to be expressed in human tissue. One human CTR isoform (hCTR-1) contains a 16-amino acid insertion in its first intracellular (I1) loop that is not present in porcine CTR (pCTR ), rat CTR, or the other human CTR (hCTR-2). To facilitate the study o f CTRs by mutational analysis, we have constructed synthetic hCTR-1 an d hCTR-2 genes. Activation of hCTR-1 expressed transiently in COS-I ce lls stimulates the formation of cAMP but not of inositol phosphates (I Ps) whereas pCTR, a chimeric CTR in which the I1 loop of pCTR was subs tituted for the I1 loop of hCTR-1, and hCTR-2 stimulate cAMP and IP fo rmation. A series of chimeric CTRs in which intracellular loops 1, 2, and 3 and the carboxyl tail of pCTR were substituted individually or i n combination for those of hCTR-1 were constructed. All chimeras stimu lated cAMP formation whereas chimeras containing the I1 loop of hCTR-1 with its Is-amino acid insertion were incapable of stimulating IP for mation. There was no correlation between maximal stimulation of cAMP a nd IP formation by these CTRs. Thus, an inserted sequence in the I1 lo op of hCTR-1 abolishes stimulation of the IP signal transduction pathw ay while allowing stimulation of the cAMP pathway.