Date of Award
College of Natual Science and Mathematics
Robert M. Dores, Ph.D.
α-MSH, αTC1.9 cells, Endoproteolysis, Fusion protein, Joining peptide, Proopiomelanocortin
The maturation process of many prohormone molecules typically requires endoproteolytic cleavage C-terminal of dibasic residues, such as the K141R142↓ site N-terminal of the α-melanocyte stimulating hormone (α-MSH) sequence in Silurana tropicalis proopiomelanocortin (POMC). In order to determine the absolute requirement of basic amino acid residues in the cleavage process, site-directed mutagenesis was employed to substitute alanine for the wild-type residues in the frog POMC open reading frame. Specifically, the following underlined residues were individually targeted for alanine substitution: R 137 Q 138 E 139 N 140 K 141 R 142↓. The mouse pancreatic αTC1.9 cell line, which expresses prohormone convertase 2 (PC2), was selected as the model system for transfection studies. The processing of wild-type frog POMC was correctly predicted to mimic that observed in the intermediate pituitaries of the frog. Moreover, biochemical analysis of mutant frog constructs in the α-cells revealed that alanine substitutions of K141 and R142, but not of R137, disrupted endoproteolytic cleavage resulting in a JP-α-MSH mutant fusion protein. Quantitative real-time PCR analysis of the αTC1.9 cells detected the relative gene expression levels of mouse PC2, glucagon but not PC1/3; detection of S. tropicalis POMC was only evident in the transfected α-cells, which was consistent with immunocytochemical data.
Copyright is held by the author. User is responsible for all copyright compliance.
Quinn Kun Chen
Received from ProQuest
Chen, Quinn Kun, "Structure-Function Analysis of Endoproteolytic Cleavage: Site-Directed Mutagenesis Studies of the α-MSH Cleavage Site in Silurana tropicalis POMC" (2009). Electronic Theses and Dissertations. 779.