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Two subtypes of the human calcitonin receptor (hCTR) have been described which differ from one another by the presence or absence of a 16-amino acid insert in the first intracellular loop. Both isoforms were stably expressed in baby hamster kidney cells to compare their ligand binding and second messenger coupling. The binding affinity and the on/off rate of binding for salmon CT were identical for the two receptor isoforms. However, the presence of the insert significantly reduced the ability of the receptor to couple to both adenylate cyclase and phospholipase C. Stimulation of a transient calcium response was only observed with the insert-negative receptor. Similarly, the ED50 for the cAMP response is 100-fold higher for the insert-positive form compared with the insert-negative form of the receptor. However, the maximal cAMP response was equivalent for both receptor isoforms. The rate of internalization of the insert-positive form of the receptor is significantly impaired relative to the insert-negative receptor, which suggests that this process may be dependent on the stimulation of a second messenger pathway. Cloning and characterization of the relevant portion of the hCTR gene revealed that these isoforms are generated by alternative splicing. We also discovered a third isoform of the hCTR, which can be generated by alternative splicing at the same position. The presence of a stop codon in this newly described alternative exon would lead to premature termination of the receptor at the C-terminal end of the first transmembrane domain.

We have cloned and characterized a second form of the human calcitonin receptor from T47D cells. It resembles the clone described by Gorn et al. [J. Clin. Invest. 90:1726-1735 (1992)] except that it lacks a 16-amino acid insert in the putative first intracellular loop. The insert-negative receptor appears to be the most abundant form, and it occurs at a relatively constant level in all expressing tissues. In contrast, the insert-positive receptor is found at low levels in most tissues but its expression levels appear to be much more variable. The insert-negative cDNA was stably expressed in baby hamster kidney cells. Like the endogenous T47D receptor, the recombinant receptor has an equally high affinity for salmon and porcine calcitonin but a 3-4-fold lower affinity for human calcitonin. High concentrations of calcitonin gene-related peptide, rat amylin, secretin, or vasoactive intestinal peptide do not significantly compete with calcitonin for binding to the recombinant receptor. Calcitonin stimulates a cAMP response in both T47D and transfected baby hamster kidney cells. Salmon calcitonin is more potent than human calcitonin for T47D cells, but the two are nearly equipotent for the transfectants. Furthermore, the ED50 for the cAMP response in the transfectants is 10-100-fold lower than in T47D cells. Calcitonin stimulates inositol phosphate turnover and elevates internal calcium levels in the transfectants. This response requires non-physiological levels of calcitonin and is directly correlated with the number of receptors. Lastly, by using a human/rodent somatic cell hybrid panel and in situ hybridization, we localized the human calcitonin receptor gene to chromosome 7.