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

The authors 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 [32P]-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 (G/sub s/) or inhibit (G/sub i/) the adenylate cyclase system. In contrast, the addition of β subunits derived from rabbit liver G/sub i/ or bovine transducin did not result in phosphorylation of a 35-kDa substrate in the reconstituted system. They conclude that the human placental pp35 substrate likely represents the placental equivalent of the β-35 protein. The 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