Purification and characterization of p185(neu) receptor-associated transmembrane complex glycoproteins (TMC-gp\u27s) from the 13762 ascites tumor cell microvilli

Microfilaments are associated with microvillar membrane of 13762 rat mammary adenocarcinoma C1 cells via a transmembrane complex (TMC) (Carraway et al., 1983) which contains actin, p58 and at least five glycoproteins, named the transmembrane complex glycoproteins (TMC-gp\u27s) (Carraway et al., 1993). The association of p185$\sp{\rm neu}$ with microfilament core (MF) was mediated by its stable interaction with TMC. The goal for the Ph.D. thesis was to understand interactions among the components of TMC, characteristics of the TMC-gp\u27s and the role that the TMC-gp\u27s play in signal transduction.By Concanavalin A affinity chromatography, the TMC-gp\u27s, comprised minimally of five glycoproteins (M$\sb{\rm r}$ 120, 110, 80, 65, 55 kDa) associated in a large, stable multimer (Carraway et al., 1991), were purified from SDS-solubilized MF or rTMC. Displacement of SDS with a 5-fold excess of Triton X-100 resulted in reconstitution of glycoproteins into a complex which contained p185$\sp{\rm neu}$. The TMC-gp\u27s and p185$\sp{\rm neu}$ were coimmunoprecipitated by anti-p185$\sp{\rm neu}$ antibody from the reconstituted complex but not from a control, unreconstituted preparation in SDS. Sedimentation analysis showed that, under actin polymerization conditions, actin bound to the reconstituted p185$\sp{\rm neu}$-containing TMC-gp\u27s; TMC-gp55 was shown by biotinylated actin overlay to be the major actin-binding protein in the complex. In vitro translated, $\sp{35}$S-methionine labelled p58 was co-immunoprecipitated with the TMC-gp\u27s by anti-TMC-gp55 antibody, and TMC-gp65 and 55 were identified as p58-binding proteins by the $\sp{35}$S-methionine-labelled p58 overlay. Stoichiometry by biotinylation showed that the ratio of TMC-gp120:110:80:65:55, in the TMC, is 1:1:1:0.5:1. N-terminal protein sequence of TMC-gp55, G-D-F-E-Y-A-H-Q, was obtained from purified TMC-gp\u27s.Studies of phosphorylation showed that TMC-gp120, 80 and 55 were phosphorylated in vitro in permeabilized microvilli or in TMC-containing microvillar fractions, and TMC-gp55 was auto-phosphorylated as a kinase by renaturation of kinase activity on a blot after SDS PAGE. Src homology region 2 (SH2) domain affinity chromatography showed that the TMC-gp55 bound to the recombinant SH2 domains of Src and PLC$\sb{\gamma}$ but not p85. Thus this glycoprotein kinase may bind Src and PLC$\sb{\gamma}$ through their SH2 domains and play a role in the dynamics of microfilaments.Immunoblot analyses with anti-TMC-gp55 antibody showed that TMC-gp55 was found in epithelial tissues and cultured mammary carcinoma cells but not in fibroblasts. These observations suggest roles for TMC-gp55 in establishing or maintaining cell polarity in the tissues and signal transduction in the cells.These studies clearly demonstrate that the transmembrane complex glycoproteins, serving as a core for the signal transduction particle in MAT-C1 cell, directly associate with growth factor receptor p185$\sp{\rm neu}$, with actin and p58 of the transmembrane complex