BIOSYNTHESIS AND PROCESSING OF THE IGF-II RECEPTOR

Project: Research project

Description

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.
StatusFinished
Effective start/end date7/1/846/30/87

Funding

  • National Institutes of Health

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IGF Type 2 Receptor
Oligosaccharides
Electrophoresis
Subcellular Fractions
Mannose
Gels
Cell Membrane
Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase
Amino Acids
Tunicamycin
Monensin
Insulin-Like Growth Factor II
Membranes
Glycoside Hydrolases
Protein Biosynthesis
Microsomes
Transfer RNA
Glycosylation
Immunoprecipitation
Sodium Dodecyl Sulfate

ASJC

  • Medicine(all)