Journal
MOLECULAR PHARMACOLOGY
Volume 62, Issue 4, Pages 847-855Publisher
AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS
DOI: 10.1124/mol.62.4.847
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Treatment (greater than or equal to6 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 I-125-insulin binding. The effect of GA was concentration (EC50 = 84 nM)- and time (t(1/2) = 8.5 h)-dependent; GA (1 muM for 24 h) lowered the B-max value of I-125-insulin binding by 80%, without changing the K-d value. Western blot analysis showed that GA (greater than or equal to3 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 (similar to190 kDa) developed into the homodimeric IR precursor (similar to380 kDa) and the mature alpha(2)beta(2) IR (similar to410 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.
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