Vol. 62, Issue 4, 847-855, October 2002

Down-Regulation of Cell Surface Insulin Receptor and Insulin Receptor Substrate-1 Phosphorylation by Inhibitor of 90-kDa Heat-Shock Protein Family: Endoplasmic Reticulum Retention of Monomeric Insulin Receptor Precursor with Calnexin in Adrenal Chromaffin Cells

Tomokazu Saitoh, Toshihiko Yanagita, Seiji Shiraishi, Hiroki Yokoo, Hideyuki Kobayashi, Shin-ichi Minami, Toshio Onitsuka, and Akihiko Wada

Departments of Pharmacology (T.S., T.Y., S.S., H.Y., H.K., S.M., A.W.) and Surgery (T.S., T.O.), Miyazaki Medical College, Miyazaki, Japan

Treatment (6 h) of cultured bovine adrenal chromaffin cells with geldanamycin (GA) or herbimycin A (HA), an inhibitor of the 90-kDa heat-shock protein (Hsp90) family, decreased cell surface ¹²⁵I-insulin binding. The effect of GA was concentration (EC₅₀ = 84 nM)- and time (t_1/2 = 8.5 h)-dependent; GA (1 µM for 24 h) lowered the B_max value of ¹²⁵I-insulin binding by 80%, without changing the K_d value. Western blot analysis showed that GA (3 h) lowered insulin receptor (IR) level by 83% (t_1/2 = 7.4 h; EC₅₀ = 74 nM), while raising IR precursor level by 100% (t_1/2 = 7.9 h; EC₅₀ = 300 nM). Pulse-label followed by reducing and nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that monomeric IR precursor (~190 kDa) developed into the homodimeric IR precursor (~380 kDa) and the mature ₂₂ IR (~410 kDa) in nontreated cells, but not in GA-treated cells; in GA-treated cells, the homodimerization-incompetent form of monomeric IR precursor was degraded via endoplasmic reticulum (ER)-associated protein degradation. Immunoprecipitation followed by immunoblot analysis showed that IR precursor was associated with calnexin (CNX) to a greater extent in GA-treated cells, compared with nontreated cells. GA had no effect on IR mRNA levels and internalization rate of cell surface IRs. In GA-treated cells, insulin-induced tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) was attenuated by 77%, with no change in IRS-1 level. Thus, inhibition of the Hsp90 family by GA or HA interrupts homodimerization of monomeric IR precursor in the ER and increases retention of monomeric IR precursor with CNX; this event retards cell surface expression of IR and attenuates insulin-induced activation of IRS-1.