Abstract

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 surface¹²⁵I-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 theK _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 α₂β₂ 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.