Treatment of IM-9 cells with human growth hormone (GH) promotes rapid disulfide linkage of the GH receptor.
Human GH (hGH) is believed to elicit its signal by promoting dimerization of the hGH receptor (hGHR). In this study, we examined a covalent linkage of receptors induced by hGH treatment of IM-9 cells. hGH induced a time- and concentration-dependent appearance of a disulfide-linked species of 215-230 kilodaltons, designated p215-230, that at 37 C was long-lived (> 1 h). p215-230 was confirmed to contain the hGHR (115-140 kilodaltons) as at least one of its constituents by two-dimensional diagonal sodium dodecyl sulfate-polyacrylamide gel electrophoresis. hGH induction of p215-230 required intact cells and was inhibitable by pretreatment of cells with N-ethylmaleimide (NEM), a sulfhydryl-reactive alkylating agent. NEM pretreatment did not, however, prevent hGH-dependent formation of a nondisulfide-linked p215-230 form, which was detected in NEM-pretreated hGH-stimulated cells by chemical cross-linking of detergent cell extracts. The disulfide-linked form of the hGHR accounted for a substantial fraction of the receptors that became tyrosine phosphorylated early into hGH treatment. However, formation of the disulfide-linked hGHR was not blocked by attenuation of tyrosine kinase activation, in that pretreatment of cells with staurosporine (1.25 microM) prevented detectable hGH-induced tyrosine phosphorylation without preventing the appearance of p215-230. These findings indicate that hGH induces its receptor to form a noncovalently associated complex, which then undergoes a rapid transition to a disulfide-linked form. These processes may have relevance to hGH signaling and/or hGHR trafficking.
Frank
S J
SJ
Department of Medicine, University of Alabama, Birmingham 35294.
Gilliland
G
G
Van Epps
C
C
D001402:B-Lymphocytes; D002478:Cells, Cultured; D004220:Disulfides / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology; D013006:Growth Hormone / Q000494:pharmacology*; D006801:Humans; D010766:Phosphorylation; D011986:Receptors, Somatotropin / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
D004220:Disulfides; D011986:Receptors, Somatotropin; D014443:Tyrosine; D013006:Growth Hormone; D005033:Ethylmaleimide