Intracellular Retention and Degradation of the Epidermal Growth Factor Receptor, Two Distinct Processes Mediated by Benzoquinone Ansamycins

Abstract

Epidermal growth factor (EGF) stimulates the growth of various types of cells via its cell surface tyrosine kinase receptor. The EGF receptor (EGF-R) has an oncogenic potential when overexpressed in a wide range of tumor cells. Geldanamycin (GA) and herbimycin (HA), specific inhibitors of the cytosolic chaperone HSP 90 and its endoplasmic reticulum homologue GRP 94, were shown to accelerate degradation of the EGF-R and of its homologue p185 c-erbB-2 . Here we compared the effects of GA and HA on intracellular degradation and maturation of EGF-R. By using an inhibitor of proteasomal degradation, we learned that GA, but not HA, blocks processing of newly synthesized EGF-R. The effects of GA and HA on receptor degradation are mediated by the cytosolic portion of EGF-R and could be conferred to the erythropoietin receptor (EPO-R), by employing the respective chimera. Neither HA nor GA affected stability of newly synthesized EGF-R lacking the cytosolic domain (Ex EGF-R), but GA caused intracellular retention of this mutant. Taken together, our results imply that GA has two distinct targets of action on the EGF-R, one for promoting its degradation and another for mediating its intracellular retention. Apparently, degradation of the EGF-R mediated by GA or HA requires the presence of the EGF-R cytosolic domain, whereas intracellular retention in the presence of GA is coupled to the extracellular domain of the EGF-R.Herbimycin A (HA) 1 and geldanamycin (GA) are benzoquinone ansamycins that specifically inhibit the cytosolic chaperone HSP 90 and its endoplasmic reticulum (ER) homologue GRP 94. These compounds have antiproliferative and antitumor effects as they bind to HSP 90, inhibit the HSP 90-mediated conformational maturation/refolding reaction, and thus promote degradation of HSP 90 substrates (reviewed in Ref. 1). The molecular basis underlying this inhibition is still under intensive investigation (2). GA was shown to accelerate the degradation of cell surface proteins including receptor Tyr kinases (3) and the cystic fibrosis transmembrane conductance regulator (CFTR) protein (4). Enhanced degradation in the presence of GA was also observed for non-membrane proteins including Ser/Thr kinases (5), Tyr kinases (6), and mutated p53 (7).The epidermal growth factor receptor (EGF-R) and its homologue p185 c-erbB-2 are well studied receptor Tyr kinases (8), which are degraded in the presence of GA and HA (9 -11), presumably via the proteasome (3, 12). The cytosolic domain of the EGF-R contains Tyr kinase activity and may also contain determinants that are required for GA-and HA-mediated degradation. This is suggested by evidence that a soluble EGF-R, an EGF-R derivative containing only the extracellular domain of the receptor (11, 13), and p185 c-erbB-2 lacking either the entire cytosolic domain or the kinase domain (10) were not degraded in the presence of HA (11, 13) or GA (10, 13). However, it is not clear whether Tyr kinase activity of the receptor or the presence of the entire kinase domain is required to confer GA...

Geldanamycin-associated Inhibition of Intracellular Trafficking Is Attributed to a Co-purified Activity

“…4, B and C, respectively). Newly synthesized Ex EGF-R was metabolically stable under all the treatments, because GA does not promote Ex EGF-R degradation (17). In GA(NCI)-treated and synthetic 17DMG-treated cells, as well as in control cells, two forms of Ex EGF-R were discerned after 4 and 6 h of chase (Fig.…”

“…GA(Invitrogen) however, does not inhibit transport of all proteins (5,17). Assuming that most proteins are delivered to the cell surface via a common pathway, the differential effect of GA(Invitrogen) on protein transport suggests that it alters specific sorting processes.…”

Aberrant expression and activation of insulin-like growth factor-1 receptor (IGF-1R) are mediated by an induction of IGF-1R promoter activity and stabilization of IGF-1R mRNA and contributes to growth factor independence and increased survival of the pancreatic cancer cell line MIA PaCa-2

“…Several endogenous proteins have been shown to regulate expression of growth factor receptors by degradation. A tyrosine kinase substrate, HRS, and some speci®c inhibitors of HSP 90 proteins have been shown to cause degradation of EGFR (Chin et al, 2000;Supino-Rosin et al, 2000). Similarly, degradation of the platelet derived growth factor receptor (PDGFR) is caused by a signal transduction adaptor molecule (STAM2) and by a cell adhesionmediated ubiquitin-dependent pathway (Baron and Schwartz, 2000).…”

Role of epidermal growth factor receptor degradation in gemcitabine-mediated cytotoxicity

“…For example, retinoic acid and histone deacetylase inhibitors downregulate EGFR via transcriptional inhibition (Rubin Grandis et al, 1996;Chinnaiyan et al, 2005). In addition, inhibitors of heat shock protein 90 promote EGFR degradation by affecting protein chaperoning and processing (Supino-Rosin et al, 2000). Recently, theaflavin-3,3 0 -digallate, a derivative from black tea, was demonstrated to trigger EGFR degradation along a proteosome/lysosome pathway, which is independent of receptor phosphorylation (Mizuno et al, 2005).…”