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J Biol Chem, Vol. 274, Issue 36, 25651-25658, September 3, 1999

The p185^neu-containing Glycoprotein Complex of a Microfilament-associated Signal Transduction Particle
PURIFICATION, RECONSTITUTION, AND MOLECULAR ASSOCIATIONS WITH p58^gag AND ACTIN^*

Yongqing Li, Fang Hua, Kermit L. Carraway^§, and Coralie A. Carothers Carraway^¶

From the Departments of  Biochemistry and Molecular Biology and ^§ Cell Biology and Anatomy, University of Miami School of Medicine, Miami, Florida 33101

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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, [³⁵S]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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The organization and reorganization of cell surface proteins play critical roles in a multitude of cellular activities. These include initiation of membrane polarity and cell morphogenesis, motility, endocytosis, exocytosis, immune recognition and response, and cell-cell and cell-matrix interactions. Surface protein organization can be controlled by interactions of membrane proteins with extracellular components and with cytoplasmic cytoskeletal elements, particularly microfilaments (1, 2). Membrane-microfilament interaction sites play important roles both in anchorage for the filaments (reviewed in Refs. 3 and 4) and in the transduction of extracellular signals to the cytoplasm (5, 6). An emerging paradigm for sites of membrane-microfilament interaction describes these sites as complexes of membrane and cytoplasmic proteins, which are assembled and associated, at least transiently, with cytoskeletal and signaling proteins (6). The sizes and relative stabilities of the complexes depend in part on the receptor and may be transient, as with growth factor receptor stimulation, or more stable, as in cell-cell interaction sites.

The integral membrane component is in some cases relatively simple, as for the integrins, heterodimeric - and -receptors that link cells to extracellular matrix and to microfilaments (7-10). Through their cytoplasmic domains, integrins provide the organizing site for membrane cytoplasmic complexes, which contain numerous peripheral membrane components, including several that interact with microfilaments (9, 11, 12). Adherens junctions contain cadherin-catenin complexes (13, 14) linked to microfilaments. Cadherin is involved in the intercellular interactions at cell junctions through Ca²⁺-dependent homophilic associations (13, 15, 16). Its cytoplasmic domain is associated with a complex of -, -, and -catenins (17, 18), the last of which binds to microfilaments (19). In both of these cases tyrosine kinases and other signaling proteins have been localized to these membrane-microfilament interaction sites.

In other cases, glycoprotein multimers provide the docking sites for cytoskeletal or membrane skeletal proteins. For example, in the neuromuscular junction a major membrane-microfilament interaction site is the acetylcholine receptor, a multimeric integral membrane protein complex that forms a ligand-activated ion channel (20-23). The multimeric dystrophin-associated glycoprotein complex in muscle cells (24) provides a site for interaction with microfilaments (25). The dystrophin-glycoprotein complex containing four integral mem- brane glycoproteins is also linked to the ion channel to form more complex structures in neuromuscular junctions (26). This complex is associated with a peripheral membrane glycoprotein -dystroglycan (27), which in the neuromuscular junction is a receptor for agrin, an extracellular matrix protein that promotes clustering of the acetylcholine receptor (28-30). Multisubunit receptors of the immune system, including the T cell antigen receptor, the B cell antigen receptor, and the high affinity receptor for immunoglobulin E, form associations with the cytoskeletal matrix upon cell activation (reviewed in Ref. 31). In each of these examples, microfilaments are attached to a transmembrane complex containing multiple integral membrane protein subunits.

In microvilli (32) from the aggressive 13762 ascites rat mammary adenocarcinoma (33), a large, multimeric glycoprotein-containing TMC¹ (34, 35) provides a docking site for microfilaments and other cytoplasmic proteins. The TMC is a major membrane-microfilament interaction site in the abundant microvilli of the ascites cells (34). The core of the complex is a large transmembrane glycoprotein complex (35) stably associated with actin and a 58-kDa cytoplasmic protein (p58) previously implicated in cell surface stability (36, 37) and xenotransplantability (33). Purified p58 bound both to microfilaments in the manner of a capping protein and to phospholipids (38). Because the cytoplasmic protein was not required for the binding of the glycoproteins to actin, we postulated that the glycoprotein-actin interaction is direct (34, 35). Complete cDNA sequencing of p58 characterized it as a truncated retroviral Gag-like protein lacking the nucleic acid-binding domain (39). Transfection of p58^gag into COS-7 cells disrupted microfilament organization (39), consistent with its proposed role in altering morphology. p58^gag contains PXXP motifs implicated in Src homology 3 domain binding (40), binds to and activates c-Src via its Src homology 3 domain and is phosphorylated by c-Src (41).

The TMC glycoprotein complex, which comprises the core or scaffolding of the membrane-microfilament interaction site in the ascites microvilli, contains at least five major membrane glycoproteins, with molecular masses of 120, 110, 80, 65, and 55 kDa (35). Additionally, it contains the (proto)oncogene receptor p185^neu/ErbB2 (42), which has been implicated in both mitogenesis and differentiation (reviewed in Ref. 43) in epithelial cells and is overexpressed in many human breast cancers (44). The presence of the receptor kinase as well as the cytoplasmic tyrosine kinases p60^c-src and c-Abl (45) in stable association with microvillar microfilaments via interaction with the TMC suggested that this large complex serves as a microfilament-linked, plasma membrane signal transduction particle (42). The TMC glycoproteins have been purified by gel filtration as a high M_r glycoprotein complex from detergent lysates of either microvillar membranes or microfilaments extracted under high pH, high salt conditions (35). In the present work, we report a new isolation method for the TMC-gps in which the glycoproteins of the complex are separated by ConA affinity chromatography after complete dissociation under denaturing conditions from p60^src, c-Abl (45), and other cytoplasmic membrane skeletal and signaling proteins (57). We demonstrate that the p185^neu-containing glycoprotein complex can be reconstituted and use this preparation for reconstitution studies to analyze specific interactions of the TMC-gps with the other major components of the TMC, actin and p58^gag. The results are consistent with our hypothesis that the TMC-gps are the core of a stable organization site for microfilaments at the membrane in the microvilli (34). As such, the glycoprotein complex can be viewed as another class of scaffolding protein (46) involved in the organization of signaling proteins. Further, the following paper (57) describes the association of the components of the Ras to mitogen-activated protein kinase mitogenic pathway with this p185^neu-containing TMC, supporting our suggestion that this major membrane-microfilament interaction site is a signal transduction particle (42) in the constitutively activated 13762 ascites mammary tumor cells (45).

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Growth and Isolation of Microvilli and Microvillar Fractions-- MAT-C1 subline cells of the 13762 ascites rat mammary adenocarcinoma were grown by intraperitoneal injection of 1.0-1.5 × 10⁶ cells in 0.1-0.3 ml of 0.9% NaCl into 60-90-day-old female Fischer 344 rats (36). Cells were collected from the peritoneal cavity after 7 days, washed with ice-cold PBS, and resuspended in PBS. Microvilli were isolated by gently shearing the cells through a 14-gauge needle, and the cell bodies were removed by centrifugation at 2,500 × g for 5 min (32, 47). The microvilli in the supernatant were obtained by differential centrifugation at 20,000 × g for 30 min and washed twice with PBS (47). Microfilament cores were prepared by extraction of microvilli in a microfilament-stabilizing buffer (TPK buffer; 0.2% Triton, 100 mM KCl, 2 mM MgCl₂, 0.1 mM phenylmethylsulfonyl fluoride, and 20 mM PIPES, pH 6.8) for 15 min and centrifugation at 15,000 × g for 15 min (48). Reconstituted TMC (rTMC) was prepared from microfilament cores as described previously (35, 38). Briefly, microfilament cores were solubilized in high salt (1 M KCl) buffer at 4 °C for 2.5 h to depolymerize actin and release actin-associated proteins. The extract was centrifuged at 100,000 × g, and the supernatant was dialyzed against isotonic (100 mM KCl) buffer overnight at 4 °C. The resulting reassociated TMC complex was pelleted by centrifugation at 100,000 × g for 1.5 h to yield a sedimentable complex containing predominantly actin, p58^gag, and the TMC glycoproteins.

Preparation of Polyclonal Antisera to TMC-gp Complex and TMC-gp55 and Antibody Screening of Cells and Tissues-- Antisera were prepared using two different sources of TMC glycoprotein. The first was the early TMC-gp complex peak from Sephacryl S-1000 gel filtration (42), which was solubilized in SDS, lyophilized, and reconstituted in Freund's complete adjuvant prior to injection into rabbits. The animals were boosted four times at 2-3-week intervals with 150-200 µg of protein prior to screening against TMC-gp-enriched microvillar fractions. In the second method, ConA affinity-purified TMC-gps from the glycoprotein-enriched rTMC were fractionated by SDS-PAGE. The gel was stained briefly with Coomassie Blue, and the bands were excised and eluted from the gel, mixed with Freund's complete adjuvant, and injected into mice. After four or five boosts at 2-3-week intervals, immunoblotting on microvilli and microvillar fractions was performed with the resulting mouse polyclonal antiserum to determine the titer and specificity of the antibody. For production of mouse polyclonal antisera to TMC-gp55 the ConA-agarose-purified TMC-gps were separated on SDS-PAGE, and TMC-gp55 was excised from the gel. The TMC-gp55 gel band (10-40 µg) was homogenized in 0.9% NaCl and injected into the peritoneal cavity of BALB/c mice, and booster injections were made at 2-week intervals. After the fifth boost (total of ~150 µg of TMC-gp55 injected), the mice were bled, and anti-TMC-gp55 antibody was detected by screening against the ConA-Sepharose-purified TMC-gps. For screening by immunoblot, cells and cell fractions were solubilized in electrophoresis buffer and separated by SDS-PAGE. For tissue screening, rat tissues were removed and frozen in liquid nitrogen prior to homogenization in buffer containing protease inhibitor mixture (42). The homogenates were solubilized by boiling in SDS sample buffer, and the proteins were separated on SDS-PAGE and transferred to nitrocellulose membranes for immunoblotting, as described previously (42, 49).

Purification of TMC-gps by Concanavalin A Affinity Chromatography-- Microfilament core or reconstituted TMC was solubilized in 2% SDS, boiled for 5 min, diluted to 0.05% SDS, and incubated with ConA-Sepharose (Sigma) overnight at 4 °C. The Sepharose suspension was packed into a column and washed with 20 bed volumes of 0.05% SDS in 20 mM TES, pH 7.8. The glycoproteins were eluted from the column with 1 ml of the same buffer containing 0.3 M -methylmannoside.

Reconstitution of TMC-gp Complex and Analysis of Components-- For reconstitution of the complex, PBS containing Triton X-100 was added to give a final Triton concentration 5 times the concentration of SDS. After incubation at 4 °C overnight, the TMC-gp complex (about 80 µg of protein) was analyzed by Sephadex G-500 gel filtration in PBS or by immunoprecipitation with anti-p185^neu. For analysis by gel filtration, the ascites cells were initially metabolically labeled with [¹⁴C]glucosamine as described previously for the detection of glycosylated proteins (35). The bound fraction from the ConA column containing TMC-gp's from either microfilament core or rTMC was incubated in PBS with or without Triton-100 and loaded onto a Sephadex G-500 column (1.0 × 95 cm) equilibrated with 0.05% Triton in PBS. Fractions were eluted with the same buffer. Aliquots of column fractions were assayed for the glycoproteins by scintillation counting for radioactivity. The ¹⁴C-containing fractions were subjected to SDS-PAGE and transferred to nitrocellulose for analysis by ConA blotting (35, 50). For immunoprecipitation with anti-p185^neu, aliquots of reconstituted or unreconstituted TMC-gp complex from ConA-Sepharose were mixed with protein A-Sepharose beads and either 1 µg of anti-p185^neu (Ab 3, Oncogene Research Products, Cambridge, MA) or 1.5-3 µg of normal mouse serum and incubated overnight at 4 °C. Immunoprecipitates were washed with PBS and eluted from the beads with SDS electrophoresis sample buffer. The immunoprecipitates were analyzed by anti-p185^neu, anti-gp55, and ConA blots.

Stoichiometric Analysis of TMC Glycoproteins by Biotinylation-- Reconstituted TMC in SDS was biotinylated by treatment with 0.1 mM N-hydroxysuccinimidylbiotin (water-soluble biotin; Pierce) for 2 h before loading onto the ConA affinity column. The separated flow-through and bound fractions were fractionated by SDS-PAGE, transferred to nitrocellulose membranes, and blocked with 5% skim milk. The membranes were incubated with avidin-horseradish peroxidase, and stained bands were detected using the Renaissance^TM chemiluminescence assay kit (NEN Life Science Products).

Analysis of Actin Binding Proteins by Blot Overlay with Biotinylated Actin (50, 51)-- Actin was purified from rabbit muscle by the method of Pardee and Spudich (52). Biotinylated actin was prepared by the method of Okabe and Hirokawa (53). Actin (25 mg) polymerized in 100 mM KCl, 2 mM MgCl₂, 1 mM ATP, 0.1 mM CaCl₂, 10 mM Tris-HCl, pH 7.5, at 25 °C for 10 min was incubated with 8 mg of N-hydroxysuccinimidylbiotin (at a concentration of 100 mg/ml in Me₂SO) for 10 min. The labeling reaction was quenched by the addition of 100 mg of sodium glutamate, and the biotinylated F-actin was isolated by centrifugation at 150,000 × g for 90 min at 4 °C. The pellet was resuspended and depolymerized in 2 ml of 2 mM Tris-HCl, pH 7.5, 0.1 mM CaCl₂, 0.5 mM ATP, 0.2 mM dithiothreitol. After two cycles of polymerization-depolymerization, the biotinylated actin pellet was resuspended in 2 mM Tris-HCl, pH 7.5, 0.1 mM ATP and dialyzed against the same buffer overnight. The G-actin was clarified by centrifugation at 100,000 × g for 5 min, frozen in aliquots in liquid nitrogen, and stored at 80 °C. TMC-gp and flow-through fractions from the ConA affinity fractionation of microfilament cores were subjected to SDS-PAGE and transferred to an Immobilon polyvinylidene difluoride membrane. The membrane was incubated at room temperature with gentle rocking in denaturation buffer containing 7 M guanidine-HCl, 50 mM TES (pH 8.3), 2 mM EDTA, 50 mM dithiothreitol for 1 h and in renaturation buffer (50 mM TES, 100 mM KCl, 0.5 mM dithiothreitol, 2 mM EDTA, 1% bovine serum albumin, 0.1% Nonidet P-40, pH 7.5) at 4 °C overnight. The membrane was blocked with 5% bovine serum albumin in 30 mM TES, pH 7.5, 120 mM KCl at room temperature for 1 h and incubated with biotinylated actin in actin polymerization buffer (plus 0.02% Triton) at 4 °C overnight. After three washes in 30 mM Tris (pH 7.5), 120 mM NaCl, 0.05% Tween 20 and two washes in 30 mM Tris (pH 7.5), 120 mM NaCl, the blot was incubated with avidin conjugated to horseradish peroxidase at room temperature for 2 h and developed with 4-chloro-1-naphthol.

Binding of [³⁵S]Methionine-labeled, in Vitro Translated p58^gag to TMC Glycoproteins-- In vitro translation of p58^gag was performed as described previously (39) using the TNT Coupled Reticulocyte Lysate System (Promega, Madison, WI) according to the manufacturer's instructions. Briefly, DNA template of p58^gag (2 µg/50-µl reaction) was mixed with 25 µl of reticulocyte lysate, 40 units of RNasin inhibitor, 40 µCi of [³⁵S]methionine (Amersham Pharmacia Biotech), amino acid mixture minus methionine, and T7 RNA polymerase. The reaction mixture was incubated at 30 °C for 2 h, and the product was analyzed by SDS-PAGE and autoradiography. Reconstituted TMC-gp, obtained by Triton treatment of the eluted fraction from the ConA affinity column, was incubated with the in vitro-translated p58^gag in PBS containing 0.05% Triton at 4 °C overnight. After centrifugation at 10,000 × g for 10 min, the supernatant of the mixture was incubated with protein A-Sepharose beads and either 1.5-2 µg of anti-gp55 antibody or 1.5-3 µg of normal mouse serum at 4 °C overnight. Immunoprecipitates were washed with PBS and analyzed by SDS-PAGE and autoradiography.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Preparation of TMC-gps by ConA Affinity Chromatography-- As a first approach toward understanding the structure and interactions of proteins in the large, microfilament-associated transmembrane complex, we initiated studies to dissociate and reconstitute the signal transduction particle components. This approach was complicated by the major difficulty that the stability of the complex resulted in the co-purification of cytoplasmic proteins, including c-Src and c-Abl (45) and sometimes actin and associated proteins (35). Attempts to purify the glycoproteins under various conditions short of denaturing conditions were unsuccessful. To assure the removal of actin and other tightly associated components, the starting material (microfilament cores or other TMC-enriched fraction) was first solubilized in SDS. Since the TMC-gps are ConA-binding proteins (35), ConA affinity chromatography was then used in a one-step purification method, which greatly improved the yield. The SDS concentration after solubilization was diluted to 0.05%, which prevents reassociation of the glycoproteins and other components and does not interfere with glycoprotein binding to the ConA-agarose.

For these preparations, any TMC-enriched fraction can serve as starting material (35, 42). Fig. 1 shows a comparison of the results starting with the largest TMC-containing fraction, the microfilament core, and the rTMC, a more highly purified fraction obtained by high salt extraction of the microfilament core (35, 38). Analysis by SDS-PAGE shows that all of the proteins readily detected by Coomassie Blue were eluted in the flow-through, while the entire complement of TMC-gps, which stain poorly with protein stains (35), was eluted by -methylmannoside from the bound fraction. When ConA-binding proteins from unfractionated microvilli were affinity-purified by this method, the bound fraction contained glycoproteins other than those associated with microfilaments, including the major ascites cell ConA-binding membrane protein ASGP-2 (37). Immunoblots of the bound fractions with antisera to purified TMC-gp55 showed a band having the same electrophoretic mobility as the ConA-staining band previously identified as TMC-gp55. Anti-p185^neu immunoblotting (Fig. 1, last panel) showed that the TMC-gp-associated (proto)oncogene product tyrosine kinase receptor p185^neu (45) is also found in the ConA-bound fraction from SDS-solubilized STP-containing fractions, consistent with the observation by Lin and Clinton (54) that this growth factor receptor is a ConA-binding glycoprotein.

View larger version (62K): [in this window] [in a new window]   Fig. 1.   Isolation of TMC-gps from reconstituted TMC by ConA affinity chromatography in SDS. Microfilament cores and rTMC were solubilized in SDS, diluted, and applied to ConA affinity columns as described under "Experimental Procedures." Starting material, unbound (flow-through) fraction, and bound fraction eluted with -methylmannoside were assayed by Coomassie Blue staining and ConA blotting. Immunoblotting with antibodies to p185neu and TMC-gp55 was also performed on rTMC and its unbound and bound fractions.

Antibodies against TMC-gps-- For investigations of this glycoprotein complex and its functions, we have prepared polyclonal antibodies to different glycoprotein preparations. Antibodies were made in rabbits to the entire TMC-gp complex using TMC-gp complex prepared by the high salt, high pH gel filtration method (35). A comparison of the immunoblot analysis with the ConA blot of TMC-gp complex purified on ConA-agarose (Fig. 2A) showed that these antisera bound primarily to TMC-gp55 and TMC-gp65, the only proteins detected by the antisera in isolated microvilli (data not shown). The other TMC-gps of the complex elicited a weak immunogenic response, which required further boosts to detect (data not shown). Immunoblot analyses of several rat tissues with the anti-TMC-gp complex antisera (Fig. 2B and Table I) and with antisera made in mice against blot-purified TMC-gp-65 and -55 (Table I) showed the preferential expression of these two glycoproteins in epithelial tissues. Further, they were also found in varying levels in tumor cell lines derived from epithelia, but not in fibroblasts (Table I). An apical localization was suggested by their abundant expression in brush borders isolated from rat intestinal preparations (data not shown). These results indicate that at least TMC-gp65 and TMC-gp55 are expressed in normal epithelial tissues and suggest that the TMC may play a role in epithelial cell organization.

View larger version (49K): [in this window] [in a new window]   Fig. 2.   Reactivity of purified TMC-gps and normal rat tissues with TMC-gp polyclonal antisera. A, immunoblot analysis of purified TMC-gps from the MAT-C1 adenocarcinoma purified on ConA-agarose. B, immunoblot analysis of normal rat tissue extracts.

Stoichiometry of the Major Glycoproteins in the TMC and TMC-gp Complex-- One of the problems in analyzing the TMC and TMC-gps has been that the glycoproteins stain very poorly with Coomassie Blue (Ref. 35; Fig. 1). Coomassie staining of gels of TMC isolated from Triton-solubilized microvillar membranes by velocity sedimentation sucrose density gradient centrifugation showed predominantly actin and p58^gag. Metabolic labeling with leucine, on the other hand, showed a more nearly stoichiometric relationship between actin, p58^gag, and glycoprotein (34). Metabolic labeling with [¹⁴C]glucosamine (35), as well as ConA blots from the same preparations (35), detected five glycoproteins in the complex. Several approaches were attempted to establish the stoichiometric relationships among the major components of the TMC. Other protein stains were less effective than Coomassie Blue in staining the glycoproteins. Silver staining resulted in extraction of the glycoproteins from the gels, as previously noted (35). The metabolic labeling procedure used in the early studies suffered from the drawback that incorporation was low and varied among the glycoproteins with time of incorporation of label (data not shown). Biotinylation gave the most reproducible results. ConA-agarose-purified TMC-gps from microfilament cores were biotinylated and analyzed by Coomassie Blue gel, ConA blot, and a blot of the biotinylated glycoproteins detected with avidin-horseradish peroxidase (Fig. 3). The glycoproteins stained poorly with the protein stain (Fig. 3A) but were readily visualized with ConA (Fig. 3B). A comparison of the ConA blot with the biotinylation blot (Fig. 3C) showed that gp55 and gp45 were detected more efficiently by ConA than by biotinylation, probably because of the differences in amounts of N-linked carbohydrate among the glycoproteins. Notably, p185^neu was readily detected by biotinylation, although only weakly detected by ConA. Because a significant amount of ASGP-2, the transmembrane subunit of rat sialomucin complex (Muc-4) (120-140 kDa), remains associated with microfilament cores (35, 49), a more highly purified TMC-gp complex preparation was treated in the same manner for quantification. A TMC-gp-enriched fraction reconstituted after a high salt extraction of microfilament cores (rTMC; Refs. 35 and 38) was analyzed to determine the relative amounts of the individual TMC-gps. The approximate ratios obtained from these analyses were as follows: gp120/110, 1; gp80, 1; gp65, 0.5; gp55, 1. Quantification of an equivalent amount of biotinylated flow-through material (data not shown) gave a relative value of 1 for p58^gag. Actin was variable in the rTMC, ranging from 2 to 6 in different rTMC preparations. Quantification from several experiments gave approximately the same ratios, with some variation in the relative amounts of gp80 and gp65 retained after the high salt extraction procedure. Notably, the relative amount of p185^neu remaining associated with the TMC-gps was 0.8-1.5. The biotinylation method shows clearly that the TMC contains similar amounts of p58^gag and the TMC-gps rather than the much greater amount of p58^gag shown by protein staining. Thus, the TMC contains sufficient amounts of the TMC-gps to serve as a scaffolding with which other components of the TMC can associate.

View larger version (78K): [in this window] [in a new window]   Fig. 3.   Analysis of purified TMC glycoproteins by biotinylation. Microfilament (MF) cores and rTMC were fractionated by ConA affinity chromatography, and the bound fractions were biotinylated and analyzed as described under "Experimental Procedures."

Reassociation of Purified TMC-gps and p185^neu-- One of the aims in generating a highly purified TMC-gp preparation was to obtain the glycoprotein complex for reconstitution studies to generate the putative signal transduction particle. Such reconstitution studies require reassociation of the TMC-gps, the putative core of the signal transduction particle, and their formation of a complex with p185^neu. The glycoprotein fraction eluted from the ConA column was subjected to treatment with buffers containing nonionic detergent to displace SDS bound to the proteins. The optimal reconstitution conditions consisted of overnight treatment with PBS containing a 5-fold excess of Triton X-100 to displace SDS. Two types of analyses were used to demonstrate reconstitution. In the first, reconstituted and unreconstituted metabolically labeled TMC-gp complex were compared by gel filtration. The column profiles of the glucosamine-labeled glycoproteins showed that reconstituted TMC-gp complex eluted as a discrete peak near the void volume of the Sephacryl S-500 column, while the unreconstituted glycoproteins eluted much later (Fig. 4A). ConA blots and anti-gp55 blots of reconstituted (Fig. 4B) and unreconstituted (Fig. 4C) TMC-gps revealed the individual glycoproteins eluting with each.

View larger version (37K): [in this window] [in a new window]   Fig. 4.   Sephacryl S-500 analysis of unreconstituted and reconstituted ConA-purified TMC-gps. p185neu-containing TMC-gps were isolated from the microvillar microfilament core from [14C]glucosamine-labeled ascites cells by ConA affinity chromatography as in Fig. 1. Aliquots of the eluted bound fraction were dialyzed against 0.05% SDS and incubated without or with a 5-fold excess of Triton X-100 in isoionic buffer. The unreconstituted and reconstituted TMC-gp complex-containing samples were fractionated on a Sephacryl S-500 column and analyzed by scintillation counting. A, [14C]glucosamine profiles; B, ConA blot of reconstituted complex; C, ConA blot of unreconstituted complex.

In the second type of analysis, the reconstituted TMC-gp complex was immunoprecipitated with anti-p185^neu, and the immunoprecipitates were analyzed by SDS-PAGE and immunoblotting with anti-p185^neu, anti-gp55, and ConA. As shown in Fig. 5, all of the glycoprotein components in the reconstituted TMC-gp complex were immunoprecipitated with anti-p185^neu, indicating that they had reassociated into a complex that binds p185^neu. In the unreconstituted purified TMC-gps, the TMC-gps were not co-precipitated. None of the glycoproteins in either mixture was immunoprecipitated by nonimmune mouse serum. Co-immunoprecipitation of p185^neu with the reconstituted glycoprotein complex was also observed if immunoprecipitation was performed with anti-gp55, and the immunoprecipitate was assayed by immunoblotting with anti-p185^neu (data not shown). These results indicate that p185^neu binds directly to the TMC-gp complex without a requirement for actin, although they do not rule out additional direct binding of p185^neu to microfilaments.

View larger version (39K): [in this window] [in a new window]   Fig. 5.   Immunoprecipitation of reconstituted TMC-gp complex with anti-p185neu. Reconstituted complex prepared as described in the legend to Fig. 3 was immunoprecipitated with anti-p185neu or nonimmune serum. The immunoprecipitates were assayed by immunoblotting with anti-p185 and anti-gp55 and ConA blotting. Lanes 1, immunoprecipitation with nonimmune serum; lanes 2, immunoprecipitation with anti-p185neu.

Association of Actin with TMC-gps-- One of the primary proposed functions of the TMC-gp complex is the association with microfilaments. We have previously shown that the complex partially purified by high salt, high pH gel filtration reassociates with actin during polymerization. To determine whether the reconstituted purified TMC-gp complex will bind microfilaments, Triton-treated fractions from the ConA column were incubated with actin under polymerizing conditions and sedimented under conditions that sediment microfilaments associated with the glycoprotein complex but not unassociated microfilaments or glycoprotein unattached to the microfilaments (35). The pellet was analyzed by SDS-PAGE and blotting with ConA and anti-p185^neu. Actin co-sedimented with the glycoprotein complex and p185^neu when it was polymerized in the presence of the complex but not in its absence (Fig. 6). Likewise, the glycoprotein complex, including p185^neu, was sedimented with polymerized actin, but not in its absence.

View larger version (43K): [in this window] [in a new window]   Fig. 6.   Association of reconstituted TMC-gp complex with polymerized actin. TMC-gp glycoproteins eluted from ConA-agarose were reconstituted into a complex as described. The reconstituted complex was incubated with actin under polymerizing conditions and centrifuged as described under "Experimental Procedures." The pellets were analyzed by SDS-PAGE followed by Coomassie Blue staining and blotting with ConA and anti-p185neu.

An overlay procedure with biotinylated actin was used to investigate the association of specific glycoprotein(s) with actin. By this method, the predominant actin-binding protein observed was gp55 (Fig. 7). Lesser binding to gp120 and to a band of about 180 kDa was observed. Binding to all was abolished in the presence of excess unlabeled actin. These results suggest that gp55 and perhaps other members of the TMC-gp complex may serve as binding sites for microfilaments at the membrane. The identity of the ~180-kDa protein is not known with certainty, but preliminary studies suggest that it may be p185^neu.²

View larger version (47K): [in this window] [in a new window]   Fig. 7.   Detection of actin binding to specific TMC glycoproteins by biotinylated actin overlay analysis. TMC-gp complex from microfilament (MF) core, eluted from ConA-agarose, was run on SDS-PAGE and assayed by Coomassie staining. A second set of samples from the same gel were transferred to nitrocellulose for actin overlay assay. Biotinylated actin was incubated with the blot in the absence or presence of excess unlabeled actin. The biotinylated actin bound to the blot was visualized with streptavidin-horseradish peroxidase as described.

Association of p58^gag with TMC-gps-- In previous studies, we have shown that in vitro translated p58^gag associates with the TMC during reconstitution from high salt buffer (42). To determine whether p58^gag binds directly to the TMC-gps as well as to microfilaments, ConA-purified TMC-gps were reconstituted into a complex in Triton and incubated with the ³⁵S-labeled p58^gag translation mixture (39). Immunoprecipitation of the incubation mixture with anti-TMC-gp55, but not nonimmune serum, and assay by immunoblotting with anti-p58 and anti-p185^neu demonstrated the association of the p58^gag with the TMC-gps (Fig. 8). Overlay analysis of the purified TMC-gps using ³⁵S-labeled p58^gag showed that p58^gag associated with TMC-gp65 and TMC-gp55 (Fig. 9). Binding was competed by an excess of unlabeled p58^gag.

View larger version (31K): [in this window] [in a new window]   Fig. 8.   Association of in vitro translated p58gag with TMC-gps. Reconstituted TMC-gp complex was incubated with in vitro-translated, 35S-labeled p58gag prepared as described previously (39). The incubation mixture was immunoprecipitated with anti-gp55, and the immunoprecipitates (imppts.) were analyzed for p58gag by fluorography and by immunoblotting with anti-p185neu and ConA blotting for the TMC-gps.

View larger version (37K): [in this window] [in a new window]   Fig. 9.   Overlay analysis of binding of p58gag to TMC-gps. A blot of ConA-agarose-purified TMC-gps was incubated with in vitro translated, 35S-labeled p58gag in the absence and presence of excess purified p58gag.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Early studies on microvilli of the MAT-C1 ascites subline of the 13762 rat mammary adenocarcinoma focused on delineating the molecular mechanisms for interactions of microfilaments with the membrane. Subsequent studies identified a large, stable, microfilament-associated glycoprotein complex that was characterized as a major membrane-microfilament interaction site in the microvilli (34, 35, 42, 45, 47). It became clear upon further characterization of the complex (35, 41, 42) that it was a signaling complex as well as a membrane-microfilament interaction site. The ultimate objective was to characterize the components and interactions of a "minimal" TMC, taking the approach of systematically increasing the stringency of the extraction buffers. High salt treatment removed many of the membrane skeletal proteins but was inadequate for completely removing membrane (nonfilamentous) actin and other cytoplasmic proteins, including c-Src (35). Ultimately, denaturing conditions were required for complete dissociation of the complex.

The present study was directed toward a basic understanding of the interactions among the major components of this critical interaction site: the TMC-gps, p58^gag, and actin (see Fig. 10). To address the question of the organization of the TMC, we have begun a series of studies to reconstitute the TMC-gp as a core on which to build a functional complex. For reconstitution, the most important step was the purification of the core glycoproteins. The use of ConA affinity chromatography in SDS is ideal because it combines complete dissociation of the glycoproteins from associated components with a rapid, specific, and high yield method of isolation. Most importantly, the glycoproteins reform the complex after displacement of SDS by nonionic detergent, and p185^neu reassociates with the reconstituted glycoprotein complex. The composition and stoichiometry of the reconstituted complex are not discernibly different from that isolated from microvillar fractions. This reconstitution study provides further evidence for the TMC-gp complex as a discrete entity and for the avidity of its association with p185^neu. Further, we have demonstrated functionality of the reconstituted complex in two ways. 1) The reconstituted TMC-gp complex binds F-actin in a polymerization/sedimentation assay, as previously shown for the less highly purified complex isolated by gel filtration (35). These results provide further support for our proposal that the TMC-gp complex is a microfilament association site that may be involved in the dynamics of cellular actin. 2) The reconstituted TMC-gp complex binds in vitro translated p58^gag, as previously demonstrated for p58^gag displaced from the TMC and reconstituted from high salt buffers (38). Blot overlay studies define the major components of actin binding/polymerization in the TMC-gp to be TMC-gp120 and TMC-gp55, with other possible associations, including p185^neu. In some TMC-gp preparations, a large glycoprotein having an M_r in the tyrosine kinase receptor range also exhibits actin binding. Previous studies have shown that F-actin binds to the epidermal growth factor receptor at a site in its cytoplasmic domain similar to a peptide of profilin (56). Since the cytoplasmic domain of p185^neu contains a peptide of similar sequence, this epidermal growth factor receptor family member may also bind F-actin. Studies are under way to determine whether this protein is actually p185^neu and, if so, whether the variability in binding may be a function of the phosphorylation state of the receptor. Multiple associations of membrane components of complexes with microfilaments have also been observed in focal adhesion complexes (9), which may also be sites of integration of signals.

View larger version (29K): [in this window] [in a new window]   Fig. 10.   Model for p185neu-containing TMC-gp complex association with actin and p58gag. MF, microfilament.

However, the TMC is more than just a microfilament attachment site at the membrane, as indicated by its strong association with the receptor kinase p185^neu (42) and with p60^src and c-Abl (45). The observation that the cells contain constitutively tyrosine-phosphorylated proteins, including p58^gag, suggested that the growth factor receptor is constitutively activated (45). This activation has been proposed to occur by an ascites cell integral membrane ligand ASGP-2, which selectively binds ErB2 and modulates ErbB2/ErbB3 responsiveness to NDF/heregulin (55). From these collective studies, we have proposed that the TMC acts as a signal transduction particle that can regulate the association of microfilaments at the membrane and integrate signals from different pathways.

These findings and proposals raise two major questions. 1) Are the TMC glycoproteins components of normal cells? 2) How is the TMC organized to allow it to function as a regulatory site in the cell membrane? Immunoblot analyses on normal rat tissues with antibodies to the TMC-gps clearly show the presence of TMC-gp55 and TMC-gp65, apparently the most immunogenic of the TMC-gps, in tissues that contain epithelia, but not in nonepithelial tissues. Further, they are found in intestinal brush border preparations, suggesting an apical localization. These results, in combination with our studies on the tumor cells, suggest that the TMC-gp complex may play a role in the generation and/or maintenance of polarity in epithelial cells.

A major question largely unelucidated in studies of cell regulation is that of the molecular mechanisms involved in mediating the pleiotypic effects on a cell as a consequence of growth factor binding, e.g. cytoskeletal reorganization. Localization of signaling elements at the membrane-microfilament interface would position them strategically for integrating changes in gene expression with the global structural alterations in the cell that must occur prior to and during mitogenesis. We envision that the TMC glycoproteins serve as a microfilament-associated scaffolding that can integrate signals impinging on the normal epithelial cell. Major signals include growth modulators and interactions of the cell with both matrix components and with adjacent cells. We propose that signals mediated by integrins, cadherin, and growth factor receptors, all of which associate with microfilaments, may be integrated via selective associations with the TMCs. In malignantly transformed cells, the pleiotypic alterations observed on cell properties encompass numerous pathways, many known to involve interactions with microfilaments. We further hypothesize that in tumor cells disruption of signal integration involves a reorganization of the TMC's and their associated microfilaments, permitting re-localization of receptors. This redistribution of signaling proteins and their associated microfilament assemblies contribute to the coordinated loss of normal epithelial polarity, cell-cell and cell-matrix interactions, gene transcription, and metabolic functions of all types.

Recent experiments have shown that several of the TMC-gps, including gp55, can be phosphorylated.² Thus, microfilament dynamics may be directly regulated by phosphorylation of the glycoproteins. Tyrosine phosphorylation site(s) may also recruit kinases that have Src homology 2 domains, such as Src, to the TMC-gp, where they could phosphorylate the glycoproteins. The association of phosphorylated p185^neu and other phosphorylated components in the TMC leads to its second potential function, as a signal transduction particle. We have recently demonstrated the association with the TMC of all of the components of the Ras/mitogen-activated protein kinase mitogenesis pathway (companion paper). In addition to p60^c-src and c-Abl (45), other signaling elements, such as protein kinase C, phospholipase C, and the p85 subunit of phosphatidylinositol 3-kinase³ are also associated with the TMC. These results suggest that the TMC glycoproteins may serve a scaffolding role, not only in the organization of individual signal transduction pathways, such as the mitogenesis pathway, but also in the integration of different pathways. The isolation of the TMC-gp-p185^neu complex provides a starting point for biochemical and molecular biological approaches to elucidating mechanisms for the formation and functions of some of these regulatory complexes.

	FOOTNOTES

^* This work was supported in part by National Institutes of Health Grants GM 33795 and CA72577.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology R-629, University of Miami School of Medicine, Miami, FL 33101. Tel.: 305-243-5759; Fax: 305-243-4431; E-mail: ccarrawa@mednet.med.miami.edu.

² Y. Li, unpublished observation.

³ M. E. Carvajal, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: TMC, transmembrane complex; TMC-gp, transmembrane complex glycoprotein; ConA, concanavalin A; PBS, phosphate-buffered saline (Dulbecco's, no Ca²⁺); PIPES, piperazine-N,N'-bis(2-ethanesulfonic acid); rTMC, reconstituted transmembrane complex; PAGE, polyacrylamide gel electrophoresis; STP, signal transduction particle; TES, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid.

	REFERENCES

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