title: Phosphorylation, desensitization and internalization of human alpha1B-adrenoceptors induced by insulin-like growth factor-I.
abstract: The effect of insulin-like growth factor-I (IGF-I) on human alpha(1B)-adrenoceptor function, phosphorylation state and cellular location was studied. Rat-1 fibroblasts were transfected with a plasmid construction containing enhanced green fluorescent protein joined to the carboxyl terminus of the human alpha(1B)-adrenoceptor. Receptors were identified by radioligand binding and photoaffinity labeling, and were immunoprecipitated with an antiserum generated against the enhanced green fluorescent protein. The receptor was functional, as evidenced by noradrenaline action on intracellular calcium and inositol phosphate production. IGF-I had no significant effect by itself on these parameters but markedly reduced the effects of noradrenaline. IGF-I induced alpha(1B)-adrenoceptor phosphorylation, which was markedly reduced by the following agents: pertussis toxin, a metalloproteinase inhibitor, diphtheria toxin mutant CRM 197, an epidermal growth factor (EGF) receptor intrinsic kinase activity inhibitor, and by phosphoinositide 3-kinase and protein kinase C inhibitors. IGF-I action appears to involve activation of a pertussis toxin-sensitive G protein, shedding of heparin-binding EGF and autocrine activation of EGF receptors. G protein subunits and phosphotyrosine residues stimulate phosphoinositide 3-kinase activity leading to activation of protein kinase C, which in turn phosphorylates alpha(1B)-adrenoceptors. Confocal fluorescent microscopy showed that alpha(1B)-adrenoceptors fussed to the green fluorescent protein were located in plasma membrane and intracellular vesicles in the basal state. IGF-I induced receptor redistribution favoring the intracellular location; this effect was blocked by hypertonic sucrose and concanavalin A. Our data show that IGF-I induces alpha(1B)-adrenoceptor desensitization associated to receptor phosphorylation and internalization.
authors.affiliation: Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Apartado Postal 70-248, México D. F. 04510, Mexico.
authors.forename: Tzindilú
authors.initials: T
authors.lastname: Molina-Muñoz
authors.forename: M Teresa
authors.initials: MT
authors.lastname: Romero-Avila
authors.forename: S Eréndira
authors.initials: SE
authors.lastname: Avendaño-Vázquez
authors.forename: J Adolfo
authors.initials: JA
authors.lastname: García-Sáinz
mesh_terms: D000818:Animals; D002460:Cell Line; D066246:ErbB Receptors / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D066257:Heparin-binding EGF-like Growth Factor; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000502:physiology*; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D037342:Pertussis Toxin / Q000494:pharmacology; D010766:Phosphorylation; D019920:Photoaffinity Labels; D011869:Radioligand Assay; D051381:Rats; D018340:Receptors, Adrenergic, alpha-1 / Q000378:metabolism*
chemical_list: C547716:ADRA1B protein, human; C547718:Adra1b protein, rat; C587883:HBEGF protein, human; C587885:Hbegf protein, rat; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D019920:Photoaffinity Labels; D018340:Receptors, Adrenergic, alpha-1; D049452:Green Fluorescent Proteins; D007334:Insulin-Like Growth Factor I; D037342:Pertussis Toxin; D066246:ErbB Receptors; D019204:GTP-Binding Proteins