Title

Functional desensitization of calcitonin gene-related peptide (CGRP) receptors in SK-N-MC cells

Date of Completion

January 2007

Keywords

Health Sciences, Pharmacology

Degree

Ph.D.

Abstract

The receptor for calcitonin gene-related peptide (CGRP) has been cloned and well studied, but the mechanism of CGRP receptor desensitization has not been fully elucidated. This study is aimed at elucidating the desensitization of CGRP receptors by measuring agonist-mediated activation of adenylate cyclase in a model system employing human neuroblastoma-derived SK-N-MC cells. In these cells, pre-incubation with αCGRP or βCGRP induced a rapid desensitization of CGRP signaling. βCGRP was 4-600 times more potent at desensitizing the CAMP production as compared to the other receptor-activating ligands. The desensitized receptors exhibited slow re-sensitization upon removal of the pre-incubated ligand, with significant recovery of function achieved within 4 h after the washout procedure. Additional agonists within the calcitonin/CGRP family of peptides and the linear CGRP analogs were also compared to CGRP with regard to their ability to activate and desensitize CGRP receptors. Adrenomedullin and adrenomedullin2 produced desensitization but at a slower rate and with a weaker desensitization potency than CGRP-induced desensitization. Calcitonin and amylin did not produce desensitization. The two linear CGRP analogs Cys(Acm) 2,7αCGRP and cys(Et)2,7αCGRP have high binding affinity to CGRP receptors, but only cys(Et)2,7αCGRP could activate and desensitize the receptors. Cys(Et)2,7αCGRP produced desensitization at a slower rate and with much lower desensitization potency (DC50) than either αCGRP or βCGRP. Next it was tested whether the desensitizing effects of the distinct peptides involve protein kinase C (PKC) or protein kinase A (PKA). A PKC inhibitor, Ro 31-8220, concentration-dependently reduced the desensitization induced by αCGRP, βCGRP, cys(Et)2,7αCGRP, adrenomedullin, and adrenomedullin2, while having little effect on their desensitization potencies. PKA inhibitors, KT-5720 and H-89, on the other hand, showed little effect on the induced level of desensitization. Desensitization induced by all of the agonists tested were blocked by CGRP receptor antagonist CGRP8-37, indicating they were mediated through CGRP receptors. The findings indicate that functional desensitization is produced by distinct peptides acting through the active site of CGRP receptors, and involves the activation of PKC as a common component necessary to achieve maximal desensitization of receptor signaling. ^