CaCo-2 cells treated with Clostridium perfringens enterotoxin form multiple large complex species, one of which contains the tight junction protein occludin

The previous model for the action of Clostridium perfringens enterotoxin (CPE) proposed that (i) CPE binds to host cell receptor(s), forming a small ( approximately 90 kDa) complex, (ii) the small complex interacts with other eucaryotic protein(s), forming a large ( approximately 160 kDa) complex, and (iii) the large complex triggers massive permeability changes, thereby inducing enterocyte death. In the current study, Western immunoblot analysis demonstrated that CPE bound to CaCo-2 human intestinal cells at 37 degrees C forms multiple large complex species, with apparent sizes of approximately 200, approximately 155, and approximately 135 kDa. These immunoblot experiments also revealed that occludin, an approximately 65-kDa tight junction protein, is present in the approximately 200-kDa large complex but absent from the other large complex species. Immunoprecipitation studies confirmed that occludin physically associates with CPE in large complex material and also indicated that occludin is absent from small complex. These results strongly suggest that occludin becomes associated with CPE during formation of the approximately 200-kDa large complex. A postbinding association between CPE and occludin is consistent with the failure of rat fibroblast transfectants expressing occludin to bind CPE in the current study. Those occludin transfectants were also insensitive to CPE, strongly suggesting that occludin expression is not sufficient to confer CPE sensitivity. However, the occludin-containing, approximately 200-kDa large complex may contribute to CPE-induced cytotoxicity, because nontoxic CPE point mutants did not form any large complex species. By showing that large complex material is comprised of several species (one containing occludin), the current studies indicate that CPE action is more complicated than previously appreciated and also provide additional evidence for CPE interactions with tight junction proteins, which could be important for CPE-induced pathophysiology.     

Comparative biochemical and immunocytochemical studies reveal differences in the effects of Clostridium perfringens enterotoxin on polarized CaCo-2 cells versus Vero cells.

Since most in vitro studies exploring the action of Clostridium perfringens enterotoxin (CPE) utilize either Vero or CaCo-2 cells, the current study directly compared the CPE responsiveness of those two cell lines. When CPE-treated in suspension, both CaCo-2 and Vero cells formed SDS-resistant, CPE-containing complexes of approximately 135, approximately 155, and approximately 200 kDa. However, confluent Transwell cultures of either cell line CPE-treated for 20 min formed only the approximately 155-kDa complex. Since those Transwell cultures also exhibited significant (86)Rb release, approximately 155-kDa complex formation is sufficient for CPE-induced cytotoxicity. Several differences in CPE responsiveness between the two cell lines were also detected. (i) CaCo-2 cells were more sensitive when CPE-treated on their basal surface, whereas Vero cells were more sensitive when CPE-treated on their apical surface; those sensitivity differences correlated with CPE binding the apical versus basolateral surfaces of these two cell lines. (ii) CPE-treated Vero cells released (86)Rb into both Transwell chambers, whereas CaCo-2 cells released (86)Rb only into the CPE-containing Transwell chamber. (iii) Vero cells express the tight junction (TJ) protein occludin but (unlike CaCo-2 cells) cannot form TJs. The ability of TJs to affect CPE responsiveness is supported by the similar effects of CPE on Transwell cultures of CaCo-2 cells and Madin-Darby canine kidney cells, another polarized cell forming TJs. Confluent CaCo-2 Transwell cultures CPE-treated for >1 h formed the approximately 200-kDa CPE complex (which also contains occludin), exhibited morphologic damage, and had occludin removed from their TJs. Collectively, these results identify CPE as a bifunctional toxin that, in confluent polarized cells, first exerts a cytotoxic effect mediated by the approximately 155-kDa complex. Resultant damage then provides CPE access to TJs, leading to approximately 200-kDa complex formation, internalization of some TJ proteins, and TJ damage that may increase paracellular permeability and thereby contribute to the diarrhea of CPE-induced gastrointestinal disease.     

New insights into the cytotoxic mechanisms of Clostridium perfringens enterotoxin

Clostridium perfringens type A isolates producing the 35 kDa enterotoxin (CPE) are an important cause of food poisoning, human non-foodborne gastrointestinal (GI) disease, and some veterinary GI diseases. Studies using CPE knock-out mutants confirmed the importance of enterotoxin expression for the enteric virulence of CPE-positive type A isolates. CPE action involves formation of a series of complexes in mammalian plasma membranes. One such CPE-containing complex (of approximately 155 kDa) is important for the induction of plasma membrane permeability alterations, which are responsible for killing enterotoxin-treated mammalian cells. Those membrane permeability changes damage the epithelium, allowing the enterotoxin to interact with the tight junction (TJ) protein occludin. CPE:occludin interactions result in formation of an approximately 200 kDa CPE complex and internalization of occludin into the cytoplasm. That removal of occludin (and possibly other proteins) damages TJs and disrupts the normal paracellular permeability barrier of the intestinal epithelium, which may contribute to CPE-induced diarrhea. Recent studies demonstrated that low CPE doses kill mammalian cells by inducing a classic apoptotic pathway involving mitochondrial membrane depolarization, cytochrome C release, and caspase 3/7 activation. In contrast, high enterotoxin doses induce oncosis, a proinflammatory event. Thus, inflammation may also contribute to the GI symptoms of patients whose intestines contain high CPE levels. In summary, CPE is a unique, multifunctional toxin with cytotoxic, TJ-damaging, and (probably) proinflammatory activities.     

Comparative characterization of mouse rectum CMT93-I and -II cells by expression of claudin isoforms and tight junction morphology and function

Recent studies suggest that the morphological and physiological properties of tight junctions (TJs) are determined by the combination and mixing ratios of claudin isoforms. In this study, we tried to characterize mouse cell lines by expression of claudin isoforms to use for studying epithelial TJs by overexpression or suppression of claudin(s) in the cells and found that claudin-2 was expressed in a few mouse rectum carcinoma cells, CMT93 cells. We have isolated CMT93-I and -II cells from CMT93 cells by immunohistochemical screening for the presence or absence of claudin-2 expression. Immunofluorescence and RT-PCR analyses showed that expression of claudin-4, -6, -7 and -12 was detected in both cell lines, but claudin-2 was only expressed in CMT93-II cells. There were no differences in paracellular permeability between CMT93-I and -II cells examined by 4 kDa FITC-dextran and fluorescein sodium, or in the number of TJ strands examined by freeze-fracture electron microscopy. However, the transepithelial electrical resistance (TER) of CMT93-I cells was approximately 6.5 times higher than that of CMT93-II cells, suggesting that expression of claudin-2 may be related to decreased TER. Comparative examinations of CMT93-I and -II cells provide a clue how the combination and mixing ratios of claudin isoforms regulate the paracellular permeability.     

Co-expressed recombinant human Translin-Trax complex binds DNA

Trax, expressed alone aggregates into insoluble complexes, whereas upon co-expression with Translin becomes readily soluble and forms a stable heteromeric complex ( approximately 430 kDa) containing both proteins at nearly equimolar ratio. Based on the subunit molecular weights, estimated by MALDI-TOF-MS, the purified complex appears to comprise of either an octameric Translin plus a hexameric Trax (calculated MW 420 kDa) or a heptamer each of Trax and Translin (calculated MW 425 kDa) or a hexameric Translin plus an octameric Trax (calculated MW 431 kDa). The complex binds single-stranded/double-stranded DNA. ssDNA gel-shifted complex shows both proteins at nearly equimolar ratio, suggesting that Translin "chaperones" Trax and forms heteromeric complex that is DNA binding competent.     

Caspase-3 cleaves Apaf-1 into an approximately 30 kDa fragment that associates with an inappropriately oligomerized and biologically inactive approximately 1.4 MDa apoptosome complex

Cytochrome c and dATP/ATP induce oligomerization of Apaf-1 into two distinct apoptosome complexes: an approximately 700 kDa complex, which recruits and activates caspases-9, -3 and -7, and an approximately 1.4 MDa complex, which recruits and processes caspase-9, but does not efficiently activate effector caspases. While searching for potential inhibitors of the approximately 1.4 MDa apoptosome complex, we observed an approximately 30 kDa Apaf-1 immunoreactive fragment that was associated exclusively with the inactive complex. We subsequently determined that caspase-3 cleaved Apaf-1 within its CED-4 domain (SVTD(271) downward arrowS) in both dATP-activated lysates and apoptotic cells to form a prominent approximately 30 kDa (p30) N-terminal fragment. Purified recombinant Apaf-1 p30 fragment weakly inhibited dATP-dependent activation of caspase-3 in vitro. However, more importantly, prevention of endogenous formation of the p30 fragment did not stimulate latent effector caspase processing activity in the large complex. Similarly, the possibility that XIAP, an inhibitor of apoptosis protein (IAP), was responsible for the inactivity of the approximately 1.4 MDa complex was excluded as immunodepletion of this caspase inhibitor failed to relieve the inhibition. However, selective proteolytic digestion of the approximately 1.4 MDa and approximately 700 kDa complexes showed that Apaf-1 was present in conformationally distinct forms in these two complexes. Therefore, the inability of the approximately 1.4 MDa apoptosome complex to process effector caspases most likely results from inappropriately folded or oligomerized Apaf-1.     

Structure of the food-poisoning Clostridium perfringens enterotoxin reveals similarity to the aerolysin-like pore-forming toxins

Clostridium perfringens enterotoxin (CPE) is a major cause of food poisoning and antibiotic-associated diarrhea. Upon its release from C. perfringens spores, CPE binds to its receptor, claudin, at the tight junctions between the epithelial cells of the gut wall and subsequently forms pores in the cell membranes. A number of different complexes between CPE and claudin have been observed, and the process of pore formation has not been fully elucidated. We have determined the three-dimensional structure of the soluble form of CPE in two crystal forms by X-ray crystallography, to a resolution of 2.7 and 4.0 Å, respectively, and found that the N-terminal domain shows structural homology with the aerolysin-like β-pore-forming family of proteins. We show that CPE forms a trimer in both crystal forms and that this trimer is likely to be biologically relevant but is not the active pore form. We use these data to discuss models of pore formation.     

Subcellular localization, oligomerization, and ATP-binding of Caenorhabditis elegans CED-4.

Caspase family cell death proteases are activated during apoptosis through the oligomerization of caspase-binding "adapter" proteins. In the nematode Caenorhabditis elegans one adapter protein, CED-4, exists. Here we report an analysis of CED-4 protein expressed in insect Sf9 cells by infection with recombinant baculovirus. During expression, CED-4 assumed a perinuclear spherical or reticular localization where it was partly resistant to extraction with nonionic detergents. Both purified FLAG-CED-4 and GST-FLAG-CED-4 proteins were present in solution as large complexes. FLAG-CED-4 complexes were estimated by gel filtration to have a molecular weight of approximately 500 kDa to >1.2 MDa, while GST-FLAG-CED-4 complexes appeared somewhat smaller. Unlike its mammalian homologue Apaf-1, CED-4 exhibited a marked preference for ATP over dATP in filter binding studies and in competition experiments. ATP hydrolysis was required neither for complex stability nor for binding of CED-3. These features are likely to be relevant for CED-4's function as a caspase adapter.     

Clostridium perfringens Enterotoxin Interacts with Claudins via Electrostatic Attraction

Clostridium perfringens enterotoxin (CPE), a causative agent of food poisoning, is a pore-forming toxin disrupting the selective permeability of the plasma membrane of target cells, resulting in cell death. We previously identified claudin as the cell surface receptor for CPE. Claudin, a component of tight junctions, is a tetratransmembrane protein and constitutes a large family of more than 20 members, not all of which serve as the receptor for CPE. The mechanism by which the toxin distinguishes the sensitive claudins is unknown. In this study, we localized the region of claudin responsible for interaction with CPE to the C-terminal part of the second extracellular loop and found that the isoelectric point of this region in sensitive claudins was higher than insensitive claudins. Amino acid substitutions to lower the pI resulted in reduced sensitivity to CPE among sensitive claudins, whereas substitutions to raise the pI endowed CPE-insensitive claudins with sensitivity. The steric structure of the claudin-binding domain of CPE reveals an acidic cleft surrounded by Tyr³⁰⁶, Tyr³¹⁰, Tyr³¹², and Leu³¹⁵, which were reported to be essential for interaction with the sensitive claudins. These results imply that an electrostatic attraction between the basic claudin region and the acidic CPE cleft is involved in their interaction.     

The cellular level of PR500, a protein complex related to the 19S regulatory particle of the proteasome, is regulated in response to stresses in plants

In Arabidopsis seedlings and cauliflower florets, Rpn6 (a proteasome non-ATPase regulatory subunit) was found in two distinct protein complexes of approximately 800 and 500 kDa, respectively. The large complex likely represents the proteasome 19S regulator particle (RP) because it displays the expected subunit composition and all characteristics. The small complex, designated PR500, shares at least three subunits with the "lid" subcomplex of 19S RP and is loosely associated with an hsp70 protein. In Arabidopsis COP9 signalosome mutants, PR500 was specifically absent or reduced to an extent that correlates with the severity of the mutations. Furthermore, PR500 was also diminished in response to potential protein-misfolding stresses caused by the heat shock and canavanine treatment. Immunofluorescence studies suggest that PR500 has a distinct localization pattern and is enriched in specific nuclear foci. We propose that PR500 may be evolved in higher plants to cope with the frequently encountered environmental stresses.     

The Apaf-1 apoptosome: a large caspase-activating complex

It is increasingly recognized that many key biological processes, including apoptosis, are carried out within very large multi-protein complexes. Apoptosis can be initiated by activation of death receptors or perturbation of the mitochondria causing the release of apoptogenic proteins, which result in the activation of caspases which are responsible for most of the biochemical and morphological changes observed during apoptosis. Caspases are normally inactive and require proteolytic processing for activity and this is achieved by the formation of large protein complexes known as the DISC (death inducing signalling complex) and the apoptosome. In the case of the latter complex, the central scaffold protein is a mammalian CED-4 homologue known as Apaf-1. This is an approximately 130 kDa protein, which in the presence of cytochrome c and dATP oligomerizes to form a very large (approximately 700-1400 kDa) apoptosome complex. The apoptosome recruits and processes caspase-9 to form a holoenzyme complex, which in turn recruits and activates the effector caspases. The apoptosome has been described in cells undergoing apoptosis, in dATP activated cell lysates and in reconstitution studies with recombinant proteins. Recent studies show that formation and function of the apoptosome can be regulated by a variety of factors including intracellular levels of K(+), inhibitor of apoptosis proteins (IAPs), heat shock proteins and Smac/Diablo. These various factors thus ensure that the apoptosome complex is only fully assembled and functional when the cell is irrevocably destined to die.     

Identification of multiprotein complexes containing DNA replication factors by native immunoblotting of HeLa cell protein preparations with T-antigen-dependent SV40 DNA replication activity

Increasing evidence has supported the concept that many of the enzymes and factors involved in the replication of mammalian DNA function together as a multiprotein complex. We have previously reported on the partial purification of a multiprotein form of DNA polymerase from human HeLa cells shown to be fully competent to support origin-specific large T-antigen-dependent simian virus 40 (SV40) DNA replication in vitro. In an attempt to more definitively identify the complex or complexes responsible for DNA replication in vitro, partially purified human HeLa cell protein preparations competent to replicate DNA in vitro were subjected to native polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose. The Native Western blots were probed with a panel of antibodies directed against proteins believed to be required for DNA replication in vitro. Apparent complexes of 620 kDa and 500 kDa were identified by monoclonal antibodies directed against DNA polymerase α and DNA polymerase δ, respectively. To detect epitopes possibly unexposed within the native multiprotein complexes, blots were also analyzed following denaturation in situ following treatment with detergent and reducing agent. The epitope or access to the epitope recognized by the monoclonal antibody against DNA polymerase α was destroyed by exposure of the blots to denaturing conditions. In contrast, an epitope present on a very large complex of approximately 1000 kDa was recognized by a monoclonal antibody against proliferating cell nuclear antigen only following treatment of the native immunoblots with denaturing agents. Identification of these complexes will allow their further purification, characterization, and elucidation of their role in the replication of DNA. © 1996 Wiley-Liss, Inc.     

Formation of membrane-bound ring complexes by prohibitins in mitochondria

Prohibitins comprise a remarkably conserved protein family in eukaryotic cells with proposed functions in cell cycle progression, senescence, apoptosis, and the regulation of mitochondrial activities. Two prohibitin homologues, Phb1 and Phb2, assemble into a high molecular weight complex of approximately 1.2 MDa in the mitochondrial inner membrane, but a nuclear localization of Phb1 and Phb2 also has been reported. Here, we have analyzed the biogenesis and structure of the prohibitin complex in Saccharomyces cerevisiae. Both Phb1 and Phb2 subunits are targeted to mitochondria by unconventional noncleavable targeting sequences at their amino terminal end. Membrane insertion involves binding of newly imported Phb1 to Tim8/13 complexes in the intermembrane space and is mediated by the TIM23-translocase. Assembly occurs via intermediate-sized complexes of approximately 120 kDa containing both Phb1 and Phb2. Conserved carboxy-terminal coiled-coil regions in both subunits mediate the formation of large assemblies in the inner membrane. Single particle electron microscopy of purified prohibitin complexes identifies diverse ring-shaped structures with outer dimensions of approximately 270 x 200 angstroms. Implications of these findings for proposed cellular activities of prohibitins are discussed.     

Characterization of the soluble human granulocyte-macrophage colony-stimulating factor receptor complex.

The human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GM-R) is expressed on both hematopoietic and non-hematopoietic tissues. Although the receptor has been identified by cross-linking studies as an 84,000-dalton protein, very little is known about its biochemistry. In this report, we describe a soluble binding assay for the human GM-R which allowed us to characterize the receptor complex from various sources, including plasma membranes of placenta, neutrophils, and human myeloid leukemia cell lines. Preparation of membranes as well as solubilization by Triton X-100 and N-octylglucoside resulted in a 5-10-fold lower affinity of the receptor for GM-CSF. The Kd decreased from 20 to 80 pM in intact cells to 200-500 pM in both intact and solubilized membranes. Binding in solution was rapid, specific for GM-CSF, and best fit a "one-site" model with an approximate Kd of 500 pM. The dissociation rate constant for the soluble GM-R was very similar to that of intact cells (k2 = 0.013 min-1 versus 0.017 min-1, respectively). As expected, solubilized membranes obtained from those cells expressing the highest number of GM-R (neutrophils and dimethyl sulfoxide-induced HL-60 cells; approximately 500-800 sites/cell) possessed the highest concentration of soluble GM-R (approximately 2-3 x 10(8) GM-R/micrograms). Cross-linking of 125I-GM-CSF to soluble GM-R resulted in the appearance of two specifically labeled complexes. A major 110-kDa receptor-ligand complex is found when cross-linking is performed with intact cells; both 110- and 200-kDa species are seen when cross-linking is performed with either intact membranes or soluble GM-R. These studies define methods by which intact GM-R can be solubilized and measured in solution, permitting a more complete biochemical characterization of the intact GM-R complex.     

Mutations in subunits of the Escherichia coli twin-arginine translocase block function via differing effects on translocation activity or tat complex structure

We have used a combination of blue-native (BN) gel electrophoresis and protein purification to analyze the effects of TatA or TatC mutations on the structures of the primary TatABC and multimeric TatA complexes in Escherichia coli. Expression of wild-type TatABC leads to the production of a single major TatABC complex of 370 kDa and a heterogeneous set of TatA complexes of <100 kDa to approximately 500 kDa. Two TatC mutations that block translocation have different effects on complex structures. P48A causes massive defects in TatABC assembly, including a marked separation of the TatBC subunits and the production of TatB and TatC aggregates. In contrast, TatABC complexes from the inactive TatC F94A mutant are structurally intact, suggesting that this mutation affects translocation activity rather than assembly. Neither TatC mutation affects the separate TatA complexes, showing that assembly of the TatA complexes is independent of TatABC assembly or activity. In contrast, three TatA mutations affect both the TatA and TatABC complexes. F39A assembles into smaller, incorrectly organized TatA complexes and the TatABC complexes contain an incorrect TatB:TatC ratio and unusually large amounts of TatA. A triple mutant in the amphipathic region forms slightly larger TatA complexes that are likewise disorganized, and a mutant containing three glycine substitutions in the transmembrane (TM) span assembles as grossly affected TatA complexes that are much larger than wild-type complexes. These mutants lead to a partial failure of TatB to assemble correctly. The data show that the amphipathic and TM regions play critical roles in TatA complex assembly. All of the TatA mutations lead to partial or substantial defects in TatABC complex formation, demonstrating that the properties of TatA can have a marked influence on the TatABC complex.     

Transition-state analogue gamma-secretase inhibitors stabilize a 900 kDa presenilin/nicastrin complex

Gamma-secretase mediates the final step, which generates Alzheimer's disease Abeta amyloid protein, by cleaving the transmembrane domain of the amyloid-beta protein precursor. Four gene products, presenilin, nicastrin, APH-1, and PEN-2, are required for gamma-secretase activity that is contained within a high molecular mass complex. To further characterize gamma-secretase, we probed membranes from human neuroblastoma SH-SY5Y cells with gamma-secretase inhibitor biotin derivatives of L-685,458, pepstatin A, and the difluoro alcohol 1-Bt. These inhibitor derivatives bound and precipitated PS1 fragments from membrane CHAPSO extracts. Analysis of PS1 complexes by blue native gel electrophoresis and western blotting indicated that the CHAPSO extracts contained complexes of approximately 900, 500, and 400 kDa. With this technique, derivatives of the three inhibitors were detected only in association with the 900 kDa species. Size-exclusion chromatography showed that 13% of PS1 immunoreactivity extracted with CHAPSO was comprised within a >or=900 kDa species with the remaining eluting in fractions of 669-250 kDa but that most enzymatic activity was associated with the 900 kDa fractions. After treatment with L-685,458 inhibitor, 49% PS1 immunoreactivity was eluted in the 900 kDa fraction, supporting evidence that the inhibitor stabilized this complex. Subcellular fractionation of SH-SY5Y cells indicated that the 900 kDa complex was formed as PS1 and NCT matured through the secretory pathway and that enzymatic activity correlated with complex maturation. From these observations, we propose a model for the structure of active gamma-secretase that would consist of dimerization of 400-500 kDa subunits and be consistent with the apparent molecular mass of the complex.     

Endosome-associated complex,ESCRT-II,recruits transport machinery for protein sorting at the multivesicular body

Sorting of ubiquitinated endosomal membrane proteins into the MVB pathway is executed by the class E Vps protein complexes ESCRT-I, -II, and -III, and the AAA-type ATPase Vps4. This study characterizes ESCRT-II, a soluble approximately 155 kDa protein complex formed by the class E Vps proteins Vps22, Vps25, and Vps36. This protein complex transiently associates with the endosomal membrane and thereby initiates the formation of ESCRT-III, a membrane-associated protein complex that functions immediately downstream of ESCRT-II during sorting of MVB cargo. ESCRT-II in turn functions downstream of ESCRT-I, a protein complex that binds to ubiquitinated endosomal cargo. We propose that the ESCRT complexes perform a coordinated cascade of events to select and sort MVB cargoes for delivery to the lumen of the vacuole/lysosome.     

Clostridium perfringens epsilon-toxin acts on MDCK cells by forming a large membrane complex.

Epsilon-toxin is produced by Clostridium perfringens types B and D and is responsible for a rapidly fatal enterotoxemia in animals, which is characterized by edema in several organs due to an increase in blood vessel permeability. The Madin-Darby canine kidney (MDCK) cell line has been found to be susceptible to epsilon-toxin (D. W. Payne, E. D. Williamson, H. Havard, N. Modi, and J. Brown, FEMS Microbiol. Lett. 116:161-168, 1994). Here we present evidence that epsilon-toxin cytotoxic activity is correlated with the formation of a large membrane complex (about 155 kDa) and efflux of intracellular K+ without entry of the toxin into the cytosol. Epsilon-toxin induced swelling, blebbing, and lysis of MDCK cells. Iodolabeled epsilon-toxin bound specifically to MDCK cell membranes at 4 and 37 labeled C and was associated with a large complex (about 155 kDa). The binding of epsilon-toxin to the cell surface was corroborated by immunofluorescence staining. The complex formed at 37 degrees C was more stable than that formed at 4 degrees C, since it was not dissociated by 5% sodium dodecyl sulfate and boiling.     

Identification of a Sam68 Ribonucleoprotein Complex Regulated by Epidermal Growth Factor

Sam68, Src associated in mitosis of 68 kDa, is a known RNA-binding protein and a signaling adaptor protein for tyrosine kinases. However, the proteins associated with Sam68 and the existence of a Sam68 complex, its mass, and regulation are, however, unknown. Herein we identify a large Sam68 complex with a mass >1 MDa in HeLa cells that is composed of ∼40 proteins using an immunoprecipitation followed by a mass spectrometry approach. Many of the proteins identified are RNA-binding proteins and are known components of a previously identified structure termed the spreading initiation center. The large Sam68 complex is a ribonucleoprotein complex, as treatment with RNases caused a shift in the molecular mass of the complex to 200–450 kDa. Moreover, treatment of HeLa cells with phorbol 12-myristate 13-acetate or epidermal growth factor induced the disassociation of Sam68 from the large complex and the appearance of Sam68 within the smaller complex. Actually, in certain cell lines such as breast cancer cell lines MCF-7 and BT-20, Sam68 exists in equilibrium between a large and a small complex. The appearance of the small Sam68 complex in cells correlates with the ability of Sam68 to promote the alternative splicing of CD44 and cell migration. Our findings show that Sam68 exists in equilibrium in transformed cells between two complexes and that extracellular signals, such as epidermal growth factor stimulation, promote alternative splicing by modulating the composition of the Sam68 complex.     

Characterization of heparin-binding growth-associated factor receptor on NIH 3T3 cells.

Scatchard plot analysis of the binding of 125I-labeled heparin binding cell growth-associated factor (125I-HBGAF) to NIH 3T3 cells revealed a single class of high affinity receptors (-5000/cell) with kd of -0.6 nM. 125I-HBGAF was covalently cross-linked to the cell surface receptor on NIH 3T3 cells with disuccinimidyl suberate (DSS). Two 125I-HBGAF-cross-linked complexes of 170 kDa and 142 kDa were observed on SDS-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. The 125I-HBGAF-cross-linked complex formation was completely abolished in the presence of greater than or equal to 100-fold excess of unlabeled HBGAF but not PDGF, EGF, aFGF, bFGF, or insulin. 125I-HBGAF appeared to undergo rapid internalization and relatively slow degradation following binding to the HBGAF receptor on NIH 3T3 cells. These results suggest that NIH 3T3 cells express a high affinity HBGAF receptor which shows two different estimated molecular masses of -155 kDa and -127 kDa. This high affinity HBGAF receptor was also found to express in other cell types.