title: 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.
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 surface (125)I-insulin binding. The effect of GA was concentration (EC(50) = 84 nM)- and time (t(1/2) = 8.5 h)-dependent; GA (1 microM for 24 h) lowered the B(max) value of (125)I-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; EC(50) = 74 nM), while raising IR precursor level by 100% (t(1/2) = 7.9 h; EC(50) = 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 (approximately 380 kDa) and the mature alpha(2)beta(2) 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.
authors.affiliation: Department of Pharmacology, Miyazaki Medical College, Miyazaki, Japan.
authors.forename: Tomokazu
authors.initials: T
authors.lastname: Saitoh
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Yanagita
authors.forename: Seiji
authors.initials: S
authors.lastname: Shiraishi
authors.forename: Hiroki
authors.initials: H
authors.lastname: Yokoo
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Kobayashi
authors.forename: Shin-Ichi
authors.initials: S
authors.lastname: Minami
authors.forename: Toshio
authors.initials: T
authors.lastname: Onitsuka
authors.forename: Akihiko
authors.initials: A
authors.lastname: Wada
mesh_terms: D000311:Adrenal Glands / Q000166:cytology; D000818:Animals; D016227:Benzoquinones; D002135:Calcium-Binding Proteins / Q000494:pharmacology*; D037281:Calnexin; D002417:Cattle; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000187:drug effects* / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D015536:Down-Regulation; D004721:Endoplasmic Reticulum; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D055504:Insulin Receptor Substrate Proteins; D007457:Iodine Radioisotopes; D047029:Lactams, Macrocyclic; D008715:Methionine / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011487:Protein Conformation; D011809:Quinones / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism*; D011956:Receptors, Cell Surface; D017828:Rifabutin / Q000031:analogs & derivatives; D013462:Sulfur Radioisotopes
chemical_list: D016227:Benzoquinones; D002135:Calcium-Binding Proteins; D004791:Enzyme Inhibitors; D018841:HSP90 Heat-Shock Proteins; D055504:Insulin Receptor Substrate Proteins; D007457:Iodine Radioisotopes; D047029:Lactams, Macrocyclic; D010750:Phosphoproteins; D011809:Quinones; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D013462:Sulfur Radioisotopes; D037281:Calnexin; D017828:Rifabutin; C020754:herbimycin; D008715:Methionine; D011972:Receptor, Insulin; D003545:Cysteine; C001277:geldanamycin