Calcitonin gene-related peptide: Biosynthesis and receptor activation
Date of Award
Doctor of Philosophy (Ph.D.)
Biochemistry and Molecular Biology
First Committee Member
Ian Dickerson - Committee Chair
Neuropeptides are peptide hormones which are secreted by neurons and neuroendocrine cells. Neuropeptides are synthesized as large biologically inactive molecules which are cleaved posttranslationally to yield bioactive peptides. Calcitonin gene-related peptide (CGRP) is a 37 amino acid neuropeptide which is widely distributed and has been identified as a potent vasodilator, a central and peripheral neuromodulator, an immune effector, and a mediator of ocular neurogenic inflammation. Neuropeptides can be regulated at the level of biosynthesis and receptor activation. The biosynthesis of CGRP was investigated for specificity of cleavage at the tetrabasic site within the proCGRP molecule. Mutations were introduced into the proCGRP cDNA which altered the tetrabasic cleavage site. The mutated proCGRP cDNAs were transfected into AtT-20 cells which predominantly express the endoprotease prohormone convertase-1 (PC1/3), and into GH3 cells which predominantly express prohormone convertase-2 (PC2). ProCGRP containing each of the cleavage site permutations was efficiently cleaved in both AtT-20 and GH3 cells, even though similar sites were present but not cleaved in proopiomelanocortin, an endogenous peptide in AtT-20 cells. These data suggest that the specificity of cleavage at tetrabasic sites is not defined solely by the endoproteases expressed by the cell or by the amino acid sequence at the cleavage site, but is also dependent on the structure of the propeptide.The activation of CGRP receptors was also examined. Our laboratory has identified the CGRP-receptor component protein (RCP), a novel signal transduction protein which confers CGRP responsiveness to Xenopus laevis oocytes, and is co-localized with CGRP immunoreactive neurons in the cochlea and cerebellum. The cDNA for the rabbit ocular RCP was isolated and encodes a 148 amino acid protein which shares over 87% identity with the mouse RCP. The rabbit ocular RCP conferred responsiveness to CGRP in oocyte expression assays, as did rabbit ocular mRNA. RCP protein was localized by immunohistochemistry to ciliary body/iris blood vessels, ciliary epithelium layers, lens, and retina. These studies on rabbit ocular RCP provide further evidence for the critical function of the RCP in CGRP signal transduction as well as demonstrating the probable sites of CGRP's action as a mediator of a neurogenic inflammatory response.
Biology, Molecular; Biology, Neuroscience; Health Sciences, Ophthalmology
Rosenblatt, Mark Irvin, "Calcitonin gene-related peptide: Biosynthesis and receptor activation" (1999). Dissertations from ProQuest. 3751.