title: Epidermal growth factor (urogastrone)-mediated phosphorylation of a 35-kDa substrate in human placental membranes: relationship to the beta subunit of the guanine nucleotide regulatory complex.
abstract: We have identified a component of about 35 kDa (pp35), present in human placental membrane preparations, that is a substrate for epidermal growth factor (urogastrone) [EGF(Uro)]-mediated phosphorylation. The EGF(Uro)-stimulated phosphorylation of pp35 was calcium-dependent and was markedly enhanced in membranes prepared in the presence (but not in the absence) of calcium. The phosphate incorporated into pp35 in the presence of EGF(Uro) was alkali-stable and was present as O4-phosphotyrosine. Under identical conditions, insulin did not stimulate pp35 phosphorylation. Either in its native or in its phosphorylated form, pp35 could be released from the membranes in the presence of calcium-chelating agents (EDTA/EGTA); and EGF(Uro)-stimulated phosphorylation was reconstituted by adding back EDTA/EGTA eluates to EDTA/EGTA-washed membranes in the presence of calcium. The properties of pp35 were similar if not identical to those of beta-35, a 35-kDa polypeptide similar to the beta subunit of the guanine nucleotide-binding oligomers that stimulate (Gs) or inhibit (Gi) the adenylate cyclase system. As with pp35, EGF(Uro)-stimulated phosphorylation of isolated rabbit liver beta-35 was observed in a reconstituted system using either EDTA/EGTA-washed placental membranes or solubilized EGF(Uro) receptor immobilized on concanavalin A-agarose. In contrast, the addition of beta subunits derived from rabbit liver Gi or bovine transducin did not result in phosphorylation of a 35-kDa substrate in the reconstituted system. Further, a 35-kDa protein released from placental membranes crossreacted with an anti-transducin antibody that can recognize the beta subunit isolated from a variety of sources. We conclude that the human placental pp35 substrate likely represents the placental equivalent of the beta-35 protein. Our data point to a possible link between those receptors involved in growth-factor action and the regulatory systems that utilize GTP-binding proteins as transducing elements.
authors.forename: K A
authors.initials: KA
authors.lastname: Valentine-Braun
authors.forename: J K
authors.initials: JK
authors.lastname: Northup
authors.forename: M D
authors.initials: MD
authors.lastname: Hollenberg
mesh_terms: D002118:Calcium / Q000502:physiology; D004815:Epidermal Growth Factor / Q000378:metabolism*; D005260:Female; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000378:metabolism*; D008970:Molecular Weight; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D010920:Placenta / Q000378:metabolism*; D011247:Pregnancy; D011505:Protein-Tyrosine Kinases / Q000378:metabolism
chemical_list: D046911:Macromolecular Substances; D008565:Membrane Proteins; D010750:Phosphoproteins; D004815:Epidermal Growth Factor; D011505:Protein-Tyrosine Kinases; D019204:GTP-Binding Proteins; D002118:Calcium