Effect of alternative glycosylation on insulin receptor processing.
The mature insulin receptor is a cell surface heterotetrameric glycoprotein composed of two alpha- and two beta-subunits. In 3T3-L1 adipocytes as in other cell types, the receptor is synthesized as a single polypeptide consisting of uncleaved alpha- and beta-subunits, migrating as a 190-kDa glycoprotein. To examine the importance of N-linked glycosylation on insulin receptor processing, we have used glucose deprivation as a tool to alter protein glycosylation. Western blot analysis shows that glucose deprivation led to a time-dependent accumulation of an alternative proreceptor of 170 kDa in a subcellular fraction consistent with endoplasmic reticulum localization. Co-precipitation assays provide evidence that the alternative proreceptor bound GRP78, an endoplasmic reticulum molecular chaperone. N-Glycosidase F treatment shows that the alternative proreceptor contained N-linked oligosaccharides. Yet, endoglycosidase H insensitivity indicates an aberrant oligosaccharide structure. Using pulse-chase methodology, we show that the synthetic rate was similar between the normal and alternative proreceptor. However, the normal proreceptor was processed into alpha- and beta-subunits (t((1)/(2)) = 1.3 +/- 0.6 h), while the alternative proreceptor was degraded (t((1)/(2)) = 5.1 +/- 0.6 h). Upon refeeding cells that were initially deprived of glucose, the alternative proreceptor was processed to a higher molecular weight form and gained sensitivity to endoglycosidase H. This "intermediate" form of the proreceptor was also degraded, although a small fraction escaped degradation, resulting in cleavage to the alpha- and beta-subunits. These data provide evidence for the first time that glucose deprivation leads to the accumulation of an alternative proreceptor, which can be post-translationally glycosylated with the readdition of glucose inducing both accelerated degradation and maturation.
Hwang
J B
JB
Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Gainesville, Florida 32610, USA.
Frost
S C
SC
D016475:3T3 Cells; D000255:Adenosine Triphosphate / Q000494:pharmacology; D017667:Adipocytes; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D002451:Cell Compartmentation; D002458:Cell Fractionation; D015180:Electrophoresis, Gel, Two-Dimensional; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D000091342:Endoplasmic Reticulum Chaperone BiP; D005947:Glucose / Q000172:deficiency; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation / Q000187:drug effects; D006360:Heat-Shock Proteins; D006596:Hexosaminidases / Q000494:pharmacology; D051379:Mice; D008954:Models, Biological; D018832:Molecular Chaperones / Q000378:metabolism; D009844:Oligosaccharides / Q000737:chemistry; D011233:Precipitin Tests; D011485:Protein Binding / Q000187:drug effects; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011972:Receptor, Insulin / Q000737:chemistry / Q000378:metabolism*; D014415:Tunicamycin / Q000494:pharmacology
D002352:Carrier Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; D006023:Glycoproteins; D006360:Heat-Shock Proteins; C000717248:Hspa5 protein, mouse; D018832:Molecular Chaperones; D009844:Oligosaccharides; D011498:Protein Precursors; D014415:Tunicamycin; D000255:Adenosine Triphosphate; D011972:Receptor, Insulin; D006596:Hexosaminidases; D005947:Glucose