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 mammary adenocarcinoma cells via a large transmembrane complex (TMC) comprising 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. Immunoblot screening with polyclonal antisera to TMC glycoproteins showed selective epithelial expression in normal rat tissues and epithelially derived tumor cells. The TMC glycoproteins were isolated by solubilization of microfilament core preparations in SDS, dilution, and separation on a concanavalin A-agarose affinity column. The large p185(neu)-containing complex was reconstituted from the column eluate after displacement of SDS with nonionic detergent, demonstrated by gel filtration and co-immunoprecipitation of the glycoproteins with anti-gp55 or anti-p185(neu). Exhaustive biotinylation of the glycoproteins gave a stoichiometry of gp120:gp110:gp80:gp65:gp55 of approximately 1:1:1:0.5:1. Overlay blots with biotinylated actin and in vitro translated, [(35)S]methionine-labeled p58(gag), respectively, showed specific interactions of actin with gp55 and gp120 and of p58(gag) with gp65 and gp55. These results provide evidence for a specific complex of microfilament-associated glycoproteins containing p185(neu) and p58(gag) and suggest a role for the complex in signal transduction scaffolding.
Li
Y
Y
Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, Florida 33101, USA.
Hua
F
F
Carraway
K L
KL
Carraway
C A
CA
D008841:Actin Cytoskeleton / Q000378:metabolism*; D000199:Actins / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D051381:Rats; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000737:chemistry* / Q000378:metabolism*; D015398:Signal Transduction
D000199:Actins; D006023:Glycoproteins; D008565:Membrane Proteins; D018719:Erb-b2 Receptor Tyrosine Kinases; C500813:membrane and microfilament-associated protein p58, rat