Folding of insulin receptor monomers is facilitated by the molecular chaperones calnexin and calreticulin and impaired by rapid dimerization.
Many complex membrane proteins undergo subunit folding and assembly in the ER before transport to the cell surface. Receptors for insulin and insulin-like growth factor I, both integral membrane proteins and members of the family of receptor tyrosine kinases (RTKs), are unusual in that they require homodimerization before export from the ER. To better understand chaperone mechanisms in endogenous membrane protein assembly in living cells, we have examined the folding, assembly, and transport of the human insulin receptor (HIR), a dimeric RTK. Using pulse-chase labeling and nonreducing SDS-PAGE analysis, we have explored the molecular basis of several sequential maturation steps during receptor biosynthesis. Under normal growth conditions, newly synthesized receptor monomers undergo disulfide bond formation while associated with the homologous chaperones calnexin (Cnx) and calreticulin (Crt). An inhibitor of glucose trimming, castanospermine (CST), abolished binding to Cnx/Crt but also unexpectedly accelerated receptor homodimerization resulting in misfolded oligomeric proreceptors whose processing was delayed and cell surface expression was also decreased by approximately 30%. Prematurely-dimerized receptors were retained in the ER and more avidly associated with the heat shock protein of 70 kD homologue binding protein. In CST-treated cells, receptor misfolding followed disordered oligomerization. Together, these studies demonstrate a chaperone function for Cnx/Crt in HIR folding in vivo and also provide evidence that folding efficiency and homodimerization are counterbalanced.
Bass
J
J
The Department of Medicine, The University of Chicago, Chicago, Illinois 60637, USA. jbass@midway.uchicago.edu
Chiu
G
G
Argon
Y
Y
Steiner
D F
DF
D000818:Animals; D001692:Biological Transport; D016466:CHO Cells; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D037281:Calnexin; D037282:Calreticulin; D006224:Cricetinae; D019281:Dimerization; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007212:Indolizines / Q000494:pharmacology; D037102:Lectins / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism*; D017510:Protein Folding; D011972:Receptor, Insulin / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000378:metabolism*; D001617:beta-Glucosidase / Q000037:antagonists & inhibitors; D054836:Indolizidines
D002135:Calcium-Binding Proteins; D037281:Calnexin; D037282:Calreticulin; D004791:Enzyme Inhibitors; D007212:Indolizines; D037102:Lectins; D018832:Molecular Chaperones; D011972:Receptor, Insulin; D012261:Ribonucleoproteins; D001617:beta-Glucosidase; C037806:castanospermine; D054836:Indolizidines