Calcitonin receptor isoforms in mouse and rat osteoclasts

Abstract Calcitonin receptors (CTRs) from several species have recently been cloned and shown to belong to the 7 transmembrane domain class of receptor. We have identified two CTR isoforms in the rat, termed C1a and C1b, identical except for a 37-amino-acid insert in the putative second extracellular domain of C1b. To examine the CTR isoforms expressed in rat and mouse osteoclasts and the time course of their appearance in culture, bone marrow cells were cultured from C57/B16J mice and osteoclasts were isolated from newborn rat long bones. CTR-bearing cells were detected by autoradiography of 125I-salmon CT binding, and cultures were stained for tartrate-resistant acid phosphatase (TRAP). RNA was extracted from parallel cultures, and CTR mRNA was detected by Northern blot analysis, using a rat digoxigenin-labeled riboprobe. Characterization of mRNA for the CTR isoforms was by reverse transcription-polymerase chain reaction (RT-PCR) using primer sets and oligonucleotide probes specific for the two rat receptor isoforms. In mouse marrow cultures, TRAP positive mononucleated cells were present by day 2 of culture at which time CTR positive cells were few. Multinucleated cells with both these markers were seen only from day 4 and later. By Northern analysis of total RNA, a band of approximately 4 kb could be detected in day 4 and later cultures. RT-PCR showed that mouse homologs of both C1a and C1b mRNA species were expressed early in cultures of mouse osteoclasts, although at each time C1a appeared to predominate. RNA was also extracted from osteoclasts freshly isolated from neonatal rat long bone osteoclasts. Northern blot analysis suggested that these preparations contained less CTR mRNA than the developing mouse osteoclast preparations. RT-PCR indicated that both receptor isoforms were expressed in rat osteoclasts.