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Characterization of the structural and functional properties of cloned calcitonin receptor cDNAs
S R Goldring1, A H Gorn, M Yamin
1Department of Medicine, Harvard Medical School, Massachusetts General Hospital, Boston.
Abstract:
We have recently cloned CTRs from cDNA libraries prepared from porcine renal and human ovarian cell lines. In situ hybridization and Northern analysis confirm the widespread distribution of CTR mRNA in numerous tissues. Hydropathy plots of the predicted amino acid sequence of the receptors demonstrate multiple hydrophobic regions that could generate 7 transmembrane spanning domains, similar to other G protein-coupled receptors. Searches of databanks for proteins with related amino acid sequences reveals that the CTRs are closely related to the receptors for parathyroid hormone/parathyroid hormone related peptide, secretin, vasoactive intestinal peptide, growth hormone releasing hormone, glucagon-like peptide-1 and glucagon. These receptors have no significant sequence homology to other G protein-coupled receptors, and therefore, appear to comprise a distinct receptor family. Expression of the hCTR or pCTR in COS cells results in expression of high affinity CTRs which are coupled to adenylate cyclase (AC). The hCTR, however, demonstrates higher affinity for human and salmon CT compared to the pCTR. Both CTRs demonstrate low affinity binding and AC activation in response to calcitonin gene related peptide, amylin or secretin, providing a possible explanation for the cross-reactivity among these peptides in vivo. Stable transfectants expressing the pCTR increase cAMP levels and increases in cytosolic free Ca2+ concentration consistent with dual coupling to AC and phospholipase C. Additional studies will help to establish the structural basis for this functional property as well as the evolutionary relationship of the members of this newly identified family of receptors.
Insights
We cloned calcitonin receptors (CTRs) and found they are a distinct G protein-coupled receptor family. These receptors are widely distributed and signal through adenylate cyclase and phospholipase C.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Calcitonin receptors (CTRs) are crucial for calcium homeostasis and are implicated in various physiological processes.
- Understanding the structural and functional characteristics of CTRs is essential for elucidating their roles in health and disease.
Purpose of the Study:
- To clone and characterize calcitonin receptors (CTRs) from porcine and human cell lines.
- To investigate the distribution, signaling pathways, and ligand-binding properties of CTRs.
- To determine if CTRs represent a distinct family of G protein-coupled receptors.
Main Methods:
- Cloning of CTRs from porcine renal and human ovarian cDNA libraries.
- In situ hybridization and Northern analysis to determine CTR mRNA distribution.
- Hydropathy plot analysis to predict transmembrane domains.
- Expression of CTRs in COS cells and stable transfectants for functional studies.
- Ligand-binding assays and measurement of adenylate cyclase (AC) and phospholipase C (PLC) activity.
Main Results:
- CTRs were successfully cloned and exhibit predicted 7 transmembrane domains, characteristic of G protein-coupled receptors.
- CTR mRNA is widely distributed across numerous tissues.
- CTRs show close sequence homology to parathyroid hormone and secretin receptor families, suggesting a distinct receptor family.
- Expressed CTRs bind calcitonin with high affinity and activate adenylate cyclase.
- Porcine CTR (pCTR) also activates phospholipase C, indicating dual signaling.
- Cross-reactivity with calcitonin gene-related peptide, amylin, and secretin was observed.
Conclusions:
- Calcitonin receptors (CTRs) represent a novel, distinct family of G protein-coupled receptors.
- CTRs are widely expressed and possess dual signaling capabilities (AC and PLC), particularly the pCTR.
- Further research is needed to elucidate the structural basis of CTR function and their evolutionary relationships.