J. Cell Biol., Volume 141, Number 3, May 4, 1998 637-646

Folding of Insulin Receptor Monomers Is Facilitated by the Molecular Chaperones Calnexin and Calreticulin and Impaired by Rapid Dimerization

Joseph Bass,^* Gavin Chiu,^§ Yair Argon,^¶ and Donald F. Steiner^§

^* The Department of Medicine,  The Howard Hughes Medical Institute, ^§ The Department of Biochemistry and Molecular Biology,  The Committee on Immunology, and ^¶ The Department of Pathology, The University of Chicago, Chicago, Illinois 60637

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 ~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.

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