The human insulin receptor (HIR) mediate acute metabolic responses and growth potentiation of a cell to insulin. The plasma membrane-bound receptor is a glycoprotein that upon binding insulin undergoes an intramolecular event resulting in tyrosine phosphokinase activation. The biosynthetic processing of the HIR involves a number of steps that modify the single-chain nascent proreceptor to generate the mature alpha- and beta-subunits in a heterotetrameric configuration on the cell surface. These changes in biogenesis are Asparagine (N)-linked and Ester (O)- linked glycosylation, dimerization, proteolytic cleavage, and fatty acylation. The significance to receptor function of some of these structural modification are known, i.e., N-linked glycosylation is important for translocation of the receptor to the surface membrane. However, the role of the other changes to receptor function are unknown. This project is initiated to ultimately determine the significance of fatty acylation of the HIR, specifically myristylation.
The specific aims for this investigation are presented in a two-part effort to ascertain the role of fatty acylation in HIR function. the first part is a novel application of molecular biology methods to determine the site(s) of amide-linked myristylation of the proreceptor. The HIR cDNA is transcribed in vitro and mRNA translated in rabbit reticulocyte lysate to demonstrate [14C]-myristic acid labeling of the receptor precursor. Anti-sense oligodeoxynucleotides added to an in vitro translation reaction inhibits protein elongation. Oligonucleotides complementary to specific sequences of the HIR mRNA will be used to inhibit translation and produce peptides of pre-determined length. Systematic truncation of the proreceptor and differential identification of translated products with radioactive myristic acid and methionine will be used to determine the site(s) of myristylation. The second and most exciting part of the project is dependent on clear demonstration of the fatty acylation site(s). to determine the functional significance of covalently-linked fatty acids, a HIR deficient in this post-translational modification will be produced and its properties of insulin binding, tyrosine kinase activity, and the ability to mediate metabolic responses will be examined. by site-directed mutagenesis method, a HIR cDNA with the acylation site(s) substituted to produce a fatty acid deficient proreceptor will be constructed. This mutant receptor will then be inserted into a Bovine Papilloma Virus vector and expressed in NIH-3T3 cells. Functional studies will be performed in these cells and the results will be compared to studies of cells expressing the normal wild type receptor. Thus, the investigation in a cell-free system of proreceptor myristylation may provide the means to determine the role of fatty acylation of the HIR.
