title: 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.
abstract: 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.
authors.affiliation: Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: A H
authors.initials: AH
authors.lastname: Gorn
authors.forename: S M
authors.initials: SM
authors.lastname: Rudolph
authors.forename: M R
authors.initials: MR
authors.lastname: Flannery
authors.forename: C C
authors.initials: CC
authors.lastname: Morton
authors.forename: S
authors.initials: S
authors.lastname: Weremowicz
authors.forename: T Z
authors.initials: TZ
authors.lastname: Wang
authors.forename: S M
authors.initials: SM
authors.lastname: Krane
authors.forename: S R
authors.initials: SR
authors.lastname: Goldring
mesh_terms: D000818:Animals; D001483:Base Sequence; D001859:Bone Neoplasms / Q000235:genetics; D002116:Calcitonin / Q000378:metabolism*; D002460:Cell Line; D002522:Chlorocebus aethiops; D002897:Chromosomes, Human, Pair 7; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D015870:Gene Expression; D005796:Genes; D005870:Giant Cell Tumors / Q000235:genetics; D006801:Humans; D017403:In Situ Hybridization; D066298:In Vitro Techniques; D008024:Ligands; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics; D018003:Receptors, Calcitonin / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D014162:Transfection
chemical_list: D017931:DNA Primers; D008024:Ligands; D012333:RNA, Messenger; D018003:Receptors, Calcitonin; D002116:Calcitonin; D000242:Cyclic AMP