Biosynthesis and Processing of the Igf-II Receptor

MacDonald, Richard

Abstract

The long-range goal of this project is to improve understanding of the mechanism and regulatory steps governing biosynthesis of plasma membrane polypeptide hormone receptors. The insulin-like growth factor II (IGF-II) receptor in H-35 hepatoma cells will be studied as a model for these receptors. The kinetics of biosynthesis and appearance of the IGF-II receptor will be measured in pulse-chase studies using tracer amino acids. Labeled receptors from the plasma membrane will be immunoprecipitated, and then quantitated by sodium dodecyl sulfate electrophoresis. The kinetics of transit of newly-synthesized receptor on its intracellular pathway toward the cell surface will be analyzed by labeling cells with radioactive or dense amino acids and then monitoring the appearance of labeled receptors with time in subcellular membrane fractions (e.g., microsomes, Golgi). Post-and co-translational proteolytic modifications of the IGF-II receptor will be examined. Possible leader sequences on the initially synthesized receptor will be analyzed by translation of receptor mRNA in vitro, immunoprecipitation and analysis of the N-terminal residues of the translation products using dansyl chloride. Nascent IGF-II receptor chains attached to tRNA will be labeled and fractionated by gel electrophoresis to determine if the receptor is translated as a single chain. Glycosylation of the receptor will be studied after metabolic labeling with 3H-sugars using inhibitors of cellular oligosaccharide side-chain processing (monensin, tunicamycin). Inhibitor-induced changes in incorporation of the labeled sugars will be measured after gel electrophoresis of the immunoprecipitated receptors isolated from subcellular membrane fractions. The extent of processing of the oligosaccharide side chains of the receptor will also be examined in these experiments by treating the labeled receptor with endoglycosidase H, which selectively remove """"""""high mannose"""""""" underprocessed N-linked oligosaccharides, and endoglycosidase F, which hydrolyzes both """"""""high mannose"""""""" and """"""""complex"""""""" (fully processed) side chains. Finally, the effects of the processing inhibitors on receptor properties i.e., binding of 125I-IGF-II and rate of insertion of new receptor into the plasma membrane, will be studied to determine the functional significance of receptor glycosylation.