LATENT INSULIN RECEPTORS AND MODULATION OF EPIDERMAL GROWTH FACTOR RECEPTORS IN 3T3-L1 ADIPOCYTES
DEUTSCH, PAUL JAN
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Detergent-solubilized extracts of 3T3-L1 cells contained 40% more insulin binding activity than could be detected on the cell surface. The size of this cryptic pool was not altered when 95% of cell surface receptors were inactivated with chymotrypsin. The unexposed and cell surface receptors exhibited similar binding properties. However, the major binding polypeptide of the latent receptor identified by covalent cross-linking to ('125)I-insulin followed by immunoprecipitation and SDS-gel electrophoresis had an M(,r) of 200K as opposed to the major cell surface binding subunit of 130K. The existence of a precursor pool was also suggested by the recovery of 20% of external binding activity in cycloheximide following cell surface proteolysis.;Experiments involving labeling with ('35)S-methionine, wheat germ agglutinin affinity chromatography, immunoprecipitation, and SDS-gels reveal specific labeled polypetides in the following order of temporal appearance: a 180K species, then 200K, then two broad bands with M(,r) 85 to 90K and 120 to 130K. The two broad bands probably represent heterogeneous and maturing forms of the alpha and beta receptor subunits. The 200K species might be a proreceptor, a precursor of the two smaller subunits, and the 180K species might be the newly synthesized form of the proreceptor. Some 200K polypeptide appears to be not immediately processed but emerges on the cell surface in an orientation such that exogenous chymotrypsin can clip it to a 190K form. The other labeled species are either present in very small quantities at equilibrium and/or they cannot bind insulin.;The modulation of cell surface EGF receptors has been studied in the same cell line. Dexamethasone (Dex) treatment evokes the appearance of a class of EGF binding sites with 7-fold higher affinity than on untreated cells. Increased binding activity persists even when Dex is removed from the medium unless cells are refed on multiple days with serum. Under conditions where differentiation of the preadipocytes is promoted (Dex and methyl-isobutyl-xanthine treatment in the presence of fetal calf serum) capacity of EGF uptake but not binding per se transiently rises and then binding activity falls to well below the original level. Loss of EGF binding capacity may be part of the differentiation program.
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