The p185(neu)-containing glycoprotein complex of a microfilament-associated signal transduction particle - Purification, reconstitution, and molecular associations with p58(gag) and actin

Microfilaments associate with the microvillar membrane of 13762 ascites mam mary adenocarcinoma cells via a large transmembrane complex (TMC) comprisin g the major glycoproteins TMC-gp120, -110, -80, -65, and -55, the receptor kinase p185(neu), and the cytoplasmic proteins actin and p58(gag), linking the receptor with microfilaments in a signal transduction particle. Immunob lot screening with polyclonal antisera to TMC glycoproteins showed selectiv e epithelial expression in normal rat tissues and epithelially derived tumo r cells. The TMC glycoproteins were isolated by solubilization of microfila ment core preparations in SDS, dilution, and separation on a concanavalin A -agarose affinity column. The large p185(neu)-containing complex was recons tituted from the column eluate after displacement of SDS with nonionic dete rgent, demonstrated by gel filtration and co-immunoprecipitation of the gly coproteins with anti-gp55 or anti-p185(neu). Exhaustive biotinylation of th e glycoproteins gave a stoichiometry of gp120:gp110:gp80: gp65:gp55 of appr oximately 1:1:1:0.5:1, Overlay blots with biotinylated actin and in vitro t ranslated, [S-35]methionine-labeled p58(gag), respectively, showed specific interactions of actin with gp55 and gp120 and of p58(gag) with gp65 and gp 55, These results provide evidence for a specific complex of microfilament- associated glycoproteins containing p185(neu) and p58(gag) and suggest a ro le for the complex in signal transduction scaffolding.