Epidermal Growth Factor (Urogastrone)-Mediated Phosphorylation of a 35-kDa Substrate in Human Placental Membranes: Relationship to the β Subunit of the Guanine Nucleotide Regulatory Complex

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 β -35, a 35-kDa polypeptide similar to the β 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 β -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 β 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 β subunit isolated from a variety of sources. We conclude that the human placental pp35 substrate likely represents the placental equivalent of the β -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.