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Cloning, characterization, and expression of a calcitonin receptor from guinea pig brain
1Department of Physiology and Biophysics, University of Miami School of Medicine, Florida 33101, USA.
Abstract:
A calcitonin receptor was cloned from guinea pig brain by using a degenerate reverse transcription-polymerase chain reaction (RT-PCR) strategy. When the cloned guinea pig calcitonin receptor was transfected into COS 1 cells, salmon calcitonin stimulated intracellular cyclic AMP accumulation with an EC50 of 0.1 nM, whereas human calcitonin was >250-fold less potent (EC50 27.6 nM). Related neuropeptides rat alphaCGRP and rat amylin did not activate the guinea pig calcitonin receptor at physiologic concentrations. Stimulation of the transfected guinea pig calcitonin receptor by salmon calcitonin also resulted in phosphatidylinositol hydrolysis with an EC50 of 2.5 nM. Expression of the calcitonin receptor was mapped by a combination of RT-PCR, northern analysis, and expression in Xenopus oocytes. The guinea pig calcitonin receptor was most highly expressed in diencephalon and a single subtype was detected.
Insights
Researchers cloned the guinea pig calcitonin receptor (GPCR) and found it highly responsive to salmon calcitonin. This calcitonin receptor clone is primarily expressed in the diencephalon region of the brain.
Area of Science:
- Neuroscience
- Molecular Biology
- Pharmacology
Background:
- Calcitonin receptors play crucial roles in calcium homeostasis and have been identified in various species.
- Understanding the specific characteristics of calcitonin receptors in different species, like the guinea pig, is essential for comparative pharmacology and neuroscience.
Purpose of the Study:
- To clone and characterize the calcitonin receptor from guinea pig brain.
- To investigate the functional responses of the cloned receptor to different calcitonin peptides and related neuropeptides.
- To determine the expression pattern of the calcitonin receptor within the guinea pig brain.
Main Methods:
- Degenerate reverse transcription-polymerase chain reaction (RT-PCR) was used to clone the guinea pig calcitonin receptor.
- The cloned receptor was transfected into COS-1 cells for functional assays.
- Cyclic AMP accumulation and phosphatidylinositol hydrolysis assays were performed to assess receptor activation.
- RT-PCR, northern analysis, and Xenopus oocyte expression were employed to map receptor expression.
Main Results:
- The guinea pig calcitonin receptor was successfully cloned.
- Salmon calcitonin potently stimulated cyclic AMP accumulation (EC50 = 0.1 nM) and phosphatidylinositol hydrolysis (EC50 = 2.5 nM) in transfected cells.
- Human calcitonin was significantly less potent (>250-fold) than salmon calcitonin.
- Rat alphaCGRP and rat amylin did not activate the receptor at physiological concentrations.
- The calcitonin receptor showed highest expression in the diencephalon, with a single detected subtype.
Conclusions:
- The cloned guinea pig calcitonin receptor exhibits distinct pharmacological properties, with a high affinity for salmon calcitonin.
- The receptor's signaling pathways include both cyclic AMP accumulation and phosphatidylinositol hydrolysis.
- The expression pattern suggests a specific role for the calcitonin receptor in the diencephalon of the guinea pig brain.