CALCITONIN RECEPTOR ISOFORMS IN MOUSE AND RAT OSTEOCLASTS

Calcitonin receptors (CTRs) from several species have recently been cl oned and shown to belong to the 7 transmembrane domain class of recept or. We have identified two CTR isoforms in the rat, termed C1a and G1b , identical except for a 37-amino-acid insert in the putative second e xtracellular domain of G1b. To examine the CTR isoforms expressed in r at and mouse osteoclasts and the time course of their appearance in cu lture, bone marrow cells were cultured from C57/B16J mice and osteocla sts were isolated from newborn rat long bones, CTR-bearing cells were detected by autoradiography of I-125-salmon CT binding, and cultures w ere stained for tartrate-resistant acid phosphatase (TRAP), RNA was ex tracted from parallel cultures, and CTR mRNA was detected by Northern blot analysis, using a rat digoxigenin-labeled riboprobe. Characteriza tion of mRNA for the CTR isoforms was by reverse transcription-polymer ase chain reaction (RT-PCR) using primer sets and oligonucleotide prob es specific for the two rat receptor isoforms. In mouse marrow culture s, TRAP positive mononucleated cells were present by day 2 of culture at which time CTR positive cells were few. Multinucleated cells with b oth these markers were seen only from day 4 and later. By Northern ana lysis 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 C1 a and C1b mRNA species were expressed early in cultures of mouse osteo clasts, although at each time C1a appeared to predominate. RNA was als o extracted from osteoclasts freshly isolated from neonatal rat long b one osteoclasts. Northern blot analysis suggested that these preparati ons contained less CTR mRNA than the developing mouse osteoclast prepa rations. RT-PCR indicated that both receptor isoforms were expressed i n rat osteoclasts.