The deletion of 14 amino acids in the seventh transmembrane domain of a naturally occurring calcitonin receptor isoform alters ligand binding and selectively abolishes coupling to phospholipase C.
The cDNA that encodes the rabbit calcitonin receptor was cloned by screening a rabbit osteoclast library. Reverse transcription-polymerase chain reaction amplification of calcitonin receptor sequences from rabbit osteoclast RNA yielded cDNAs that encode two isoforms of the calcitonin receptor. One isoform is homologous to the C1a isoform previously identified in multiple cell types and species, while the second, designated CTRDeltae13, is a previously unidentified isoform that is apparently generated by alternative splicing during mRNA processing that deletes exon 13, resulting in the absence of 14 amino acids in the predicted seventh transmembrane domain. Expression of mRNA transcripts encoding the two isoforms varies in a tissue-specific manner, with CTRDeltae13 accounting for less than 15% of the total calcitonin receptor mRNA in osteoclasts, kidney, and brain, but comprising at least 50% of the transcripts in skeletal muscle and lung. The two isoforms were expressed, and the ligand binding and signal transduction properties were characterized. Deletion of the residues in the seventh transmembrane domain in CTRDeltae13 reduced the binding affinity for salmon and human calcitonin by more than 10-fold and approximately 2-fold, respectively, resulting in a receptor that failed to discriminate between the two forms of calcitonin. Both isoforms activated adenylyl cyclase, with EC50 values consistent with the difference in ligand affinities. In contrast, only the C1a isoform, but not the CTRDeltae13 isoform, activated phospholipase C. Thus, while the CTRDeltae13 remains active despite the deletion of a significant portion of its seventh transmembrane domain, it has significantly altered ligand recognition and signal transduction properties.
Shyu
J F
JF
Department of Cell Biology , Yale University School of Medicine, New Haven, Connecticut 06520-8044, USA.
Inoue
D
D
Baron
R
R
Horne
W C
WC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D019556:COS Cells; D002460:Cell Line; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010010:Osteoclasts / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011817:Rabbits; D018003:Receptors, Calcitonin / Q000737:chemistry* / Q000378:metabolism; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D014018:Tissue Distribution; D010738:Type C Phospholipases / Q000378:metabolism*
D018003:Receptors, Calcitonin; D000242:Cyclic AMP; D010738:Type C Phospholipases