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Gorn, AH, Rudolph, SM, Flannery, MR, Morton, CC, Weremowicz, S, Wang, TZ, Krane, SM and Goldring, SR (1995) Expression of two human skeletal calcitonin receptor isoforms cloned from a giant cell tumor of bone. The first intracellular domain modulates ligand binding and signal transduction. J. Clin. Invest. 95:2680-91
Two distinct calcitonin (CT) receptor (CTR)-encoding cDNAs (designated GC-2 and GC-10) were cloned and characterized from giant cell tumor of bone (GCT). Both GC-2 and GC-10 differ structurally from the human ovarian cell CTR (o-hCTR) that we cloned previously, but differ from each other only by the presence (GC-10) or absence (GC-2) of a predicted 16-amino acid insert in the putative first intracellular domain. Expression of all three CTR isoforms in COS cells demonstrated that GC-2 has a lower binding affinity for salmon (s) CT (Kd approximately 15 nM) than GC-10 or o-hCTR (Kd approximately 1.5 nM). Maximal stimulatory concentrations of CT resulted in a mean accumulation of cAMP in GC-2 transfected cells that was greater than eight times higher than in cells transfected with GC-10 after normalizing for the number of receptor-expressing cells. The marked difference in maximal cAMP response was also apparent after normalizing for receptor number. GC-2 also demonstrated a more potent ligand-mediated cAMP response compared with GC-10 for both human (h) and sCT (the EC50 values for GC-2 were approximately 0.2 nM for sCT and approximately 2 nM for hCT; EC50 values for GC-10 were approximately 6 nM for sCT and approximately 25 nM for hCT). Reverse transcriptase PCR of GCT RNA indicated that GC-2 transcripts are more abundant than those encoding for GC-10. In situ hybridization on GCT tissue sections demonstrated CTR mRNA expression in osteoclast-like cells. We localized the human CTR gene to chromosome 7 in band q22. The distinct functional characteristics of GC-2 and GC-10, which differ in structure only in the first intracellular domain, indicate that the first intracellular domain of the CTR plays a previously unidentified role in modulating ligand binding and signal transduction via the G protein/adenylate cyclase system.
Animals; Base Sequence; Bone Neoplasms/genetics; Calcitonin/metabolism; Cell Line; Cercopithecus aethiops; Chromosomes, Human, Pair 7; Cloning, Molecular; Cyclic AMP/metabolism; DNA Primers/chemistry; Gene Expression; Genes; Giant Cell Tumors/genetics; Humans; In Situ Hybridization; Ligands; Molecular Sequence Data; RNA, Messenger/genetics; Receptors, Calcitonin/genetics; Receptors, Calcitonin/metabolism; Signal Transduction; Structure-Activity Relationship; Transfection