Identification of a cyclase-associated protein (CAP) homologue in Dictyostelium discoideum and characterization of its interaction with actin

In search for novel actin binding proteins in Dictyostelium discoideum we have isolated a cDNA clone coding for a protein of approximately 50 kDa that is highly homologous to the class of adenylyl cyclase-associated proteins (CAP). In Saccharomyces cerevisiae the amino-terminal part of CAP is involved in the regulation of the adenylyl cyclase whereas the loss of the carboxyl-terminal domain results in morphological and nutritional defects. To study the interaction of Dictyostelium CAP with actin, the complete protein and its amino-terminal and carboxyl-terminal domains were expressed in Escherichia coli and used in actin binding assays. CAP sequestered actin in a Ca2+ independent way. This activity was localized to the carboxyl-terminal domain. CAP and its carboxyl-terminal domain led to a fluorescence enhancement of pyrene-labeled G-actin up to 50% indicating a direct interaction, whereas the amino-terminal domain did not enhance. In polymerization as well as in viscometric assays the ability of the carboxyl-terminal domain to sequester actin and to prevent F-actin formation was approximately two times higher than that of intact CAP. The sequestering activity of full length CAP could be inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2), whereas the activity of the carboxyl-terminal domain alone was not influenced, suggesting that the amino-terminal half of the protein is required for the PIP2 modulation of the CAP function. In profilin-minus cells the CAP concentration is increased by approximately 73%, indicating that CAP may compensate some profilin functions in vivo. In migrating D. discoideum cells CAP was enriched at anterior and posterior plasma membrane regions. Only a weak staining of the cytoplasm was observed. In chemotactically stimulated cells the protein was very prominent in leading fronts. The data suggest an involvement of D. discoideum CAP in microfilament reorganization near the plasma membrane in a PIP2-regulated manner.     

Identification of EBP50: A PDZ-containing phosphoprotein that associates with members of the ezrin-radixin-moesin family

Members of the ezrin-radixin-moesin (ERM) family of membrane-cytoskeletal linking proteins have NH2- and COOH-terminal domains that associate with the plasma membrane and the actin cytoskeleton, respectively. To search for ERM binding partners potentially involved in membrane association, tissue lysates were subjected to affinity chromatography on the immobilized NH2-terminal domains of ezrin and moesin, which comprise the ezrin-radixin-moesin-association domain (N-ERMAD). A collection of polypeptides at 50-53 kD from human placenta and at 58-59 kD from bovine brain bound directly to both N-ERMADs. The 50-53-kD placental proteins migrated as a major 50-kD species after phosphatase treatment, indicating that the heterogeneity is due to different phosphorylation states. We refer to these polypeptides as ERM-binding phosphoprotein 50 (EBP50). Sequence analysis of human EBP50 was used to identify an approximately 2-kb human cDNA that encodes a 357-residue polypeptide. Recombinant EBP50 binds tightly to the N-ERMADs of ezrin and moesin. Peptide sequences from the brain candidate indicated that it is closely related to EBP50. EBP50 has two PSD-95/DlgA/ZO-1-like (PDZ) domains and is most likely a homologue of rabbit protein cofactor, which is involved in the protein kinase A regulation of the renal brush border Na+/H+ exchanger. EBP50 is widely distributed in tissues, and is particularly enriched in those containing polarized epithelia. Immunofluorescence microscopy of cultured cells and tissues revealed that EBP50 colocalizes with actin and ezrin in the apical microvilli of epithelial cells, and immunoelectron microscopy demonstrated that it is specifically associated with the microvilli of the placental syncytiotrophoblast. Moreover, EBP50 and ezrin can be coimmunoprecipitated as a complex from isolated human placental microvilli. These findings show that EBP50 is a physiologically relevant ezrin binding protein. Since PDZ domains are known to mediate associations with integral membrane proteins, one mode of membrane attachment of ezrin is likely to be mediated through EBP50.     

Molecular dissection of radixin: distinct and interdependent functions of the amino- and carboxy-terminal domains

The ERM proteins--ezrin, radixin, and moesin--occur in particular cortical cytoskeletal structures. Several lines of evidence suggest that they interact with both cytoskeletal elements and plasma membrane components. Here we described the properties of full-length and truncated radixin polypeptides expressed in transfected cells. In stable transfectants, exogenous full-length radixin behaves much like endogenous ERM proteins, localizing to the same cortical structures. However, the presence of full-length radixin or its carboxy-terminal domain in cortical structures correlates with greatly diminished staining of endogenous moesin in those structures, suggesting that radixin and moesin compete for a limiting factor required for normal associations in the cell. The results also reveal distinct roles for the amino- and carboxy-terminal domains. At low levels relative to endogenous radixin, the carboxy-terminal polypeptide is associated with most of the correct cortical targets except cleavage furrows. In contrast, the amino-terminal polypeptide is diffusely localized throughout the cell. Low level expression of full-length radixin or either of the truncated polypeptides has no detectable effect on cell physiology. However, high level expression of the carboxy-terminal domain dramatically disrupts normal cytoskeletal structures and functions. At these high levels, the amino-terminal polypeptide does localize to cortical structures, but does not affect the cells. We conclude that the behavior of radixin in cells depends upon activities contributed by separate domains of the protein, but also requires modulating interactions between those domains.     

Association of ezrin with intercellular adhesion molecule-1 and -2 (ICAM-1 and ICAM-2). Regulation by phosphatidylinositol 4, 5-bisphosphate

Ezrin is a cytoplasmic linker molecule between plasma membrane components and the actin-containing cytoskeleton. We studied whether ezrin is associated with intercellular adhesion molecule (ICAM)-1, -2, and -3. In transfected cells, ICAM-1 and ICAM-2 colocalized with ezrin in microvillar projections, whereas an ICAM-1 construct attached to cell membrane via a glycophosphatidylinositol anchor was uniformly distributed on the cell surface. An interaction of ICAM-2 and ezrin was seen by affinity precipitation, microtiter binding assay, coimmunoprecipitation, and surface plasmon resonance methods. The calculated KD value was 3.3 x 10(-7) M. Phosphatidylinositol 4, 5-bisphosphate (PtdIns(4,5)P2) induced an interaction of ezrin and ICAM-1 and enhanced the interaction of ezrin and ICAM-2, but ICAM-3 did not bind ezrin even in the presence of PtdIns(4,5)P2. PtdIns(4, 5)P2 was shown to bind to cytoplasmic tails of ICAM-1 and ICAM-2, which are the first adhesion proteins demonstrated to interact with PtdIns(4,5)P2. The results indicate an interaction of ezrin with ICAM-1 and ICAM-2 and suggest a regulatory role of phosphoinositide signaling pathways in regulation of ICAM-ezrin interaction.     

Rho-kinase phosphorylates COOH-terminal threonines of ezrin/radixin/moesin (ERM) proteins and regulates their head-to-tail association

The ezrin/radixin/moesin (ERM) proteins are involved in actin filament/plasma membrane interaction that is regulated by Rho. We examined whether ERM proteins are directly phosphorylated by Rho-associated kinase (Rho-kinase), a direct target of Rho. Recombinant full-length and COOH-terminal half radixin were incubated with constitutively active catalytic domain of Rho-kinase, and approximately 30 and approximately 100% of these molecules, respectively, were phosphorylated mainly at the COOH-terminal threonine (T564). Next, to detect Rho-kinase-dependent phosphorylation of ERM proteins in vivo, we raised a mAb that recognized the T564-phosphorylated radixin as well as ezrin and moesin phosphorylated at the corresponding threonine residue (T567 and T558, respectively). Immunoblotting of serum-starved Swiss 3T3 cells with this mAb revealed that after LPA stimulation ERM proteins were rapidly phosphorylated at T567 (ezrin), T564 (radixin), and T558 (moesin) in a Rho-dependent manner and then dephosphorylated within 2 min. Furthermore, the T564 phosphorylation of recombinant COOH-terminal half radixin did not affect its ability to bind to actin filaments in vitro but significantly suppressed its direct interaction with the NH2-terminal half of radixin. These observations indicate that the Rho-kinase-dependent phosphorylation interferes with the intramolecular and/ or intermolecular head-to-tail association of ERM proteins, which is an important mechanism of regulation of their activity as actin filament/plasma membrane cross-linkers.     

Suppression of radixin and moesin alters growth cone morphology, motility, and process formation in primary cultured neurons

The Rho-type GTPase Cdc42p has been implicated in diverse cellular functions including cell shape, cell motility, and cytokinesis, all of which involve the reorganization of the actin cytoskeleton. Targets of Cdc42p that interface the actin cytoskeleton are likely candidates for mediating cellular activities. In this report, we identify and characterize a yeast homologue for the mammalian IQGAP, a cytoskeletal target for Cdc42p. The yeast IQGAP homologue, designated Iqg1p, displays a two-hybrid interaction with activated Cdc42p and coimmunoprecipitates with actin filaments. Deletion of IQG1 results in a temperature-sensitive lethality and causes aberrant morphologies including elongated and round multinucleated cells. This together with its localization at the mother-bud neck, suggest that Iqg1p promotes budding and cytokinesis. At restrictive temperatures, the vacuoles of the mutant cells enlarge and vesicles accumulate in the bud. Interestingly, Iqg1p shows two-hybrid interactions with the ankyrin repeat-containing protein, Akr1p (Kao, L.-R., J. Peterson, J. Ruiru, L. Bender, and A. Bender. 1996. Mol. Cell. Biol. 16:168-178), which inhibits pheromone signaling and appears to promote cytokinesis and/or trafficking. We also show two-hybrid interactions between Iqg1p and Afr1p, a septin-binding protein involved in projection formation (Konopka, J.B., C. DeMattei, and C. Davis. 1995. Mol. Cell. Biol. 15:723-730). We propose that Iqg1p acts as a scaffold to recruit and localize a protein complex involved in actin-based cellular functions and thus mediates the regulatory effects of Cdc42p on the actin cytoskeleton.     

Model for the alignment of actin filaments in endothelial cells subjected to fluid shear stress

Cultured vascular endothelial cells undergo significant morphological changes when subjected to sustained fluid shear stress. The cells elongate and align in the direction of applied flow. Accompanying this shape change is a reorganization at the intracellular level. The cytoskeletal actin filaments reorient in the direction of the cells' long axis. How this external stimulus is transmitted to the endothelial cytoskeleton still remains unclear. In this article, we present a theoretical model accounting for the cytoskeletal reorganization under the influence of fluid shear stress. We develop a system of integro-partial-differential equations describing the dynamics of actin filaments, the actin-binding proteins, and the drift of transmembrane proteins due to the fluid shear forces applied on the plasma membrane. Numerical simulations of the equations show that under certain conditions, initially randomly oriented cytoskeletal actin filaments reorient in structures parallel to the externally applied fluid shear forces. Thus, the model suggests a mechanism by which shear forces acting on the cell membrane can be transmitted to the entire cytoskeleton via molecular interactions alone.     

The bacterial actin nucleator protein ActA of Listeria monocytogenes contains multiple binding sites for host microfilament proteins

BACKGROUND: Several intracellular pathogens, including Listeria monocytogenes, use components of the host actin-based cytoskeleton for intracellular movement and for cell-to-cell spread. These bacterial systems provide relatively simple model systems with which to study actin-based motility. Genetic analysis of L. monocytogenes led to the identification of the 90 kD surface-bound ActA polypeptide as the sole bacterial factor required for the initiation of recruitment of host actin filaments. Numerous host actin-binding proteins have been localized within the actin-based cytoskeleton that surrounds Listeria once it is inside a mammalian cell, including alpha-actinin, fimbrin, filamin, villin, ezrin/radixin, profilin and the vasodilator-stimulated phosphoprotein, VASP. Only VASP is known to bind directly to ActA. We sought to determine which regions of the ActA molecule interact with VASP and other components of the host microfilament system. RESULTS: We used the previously developed mitochondrial targeting assay to determine regions of the ActA protein that are involved in the recruitment of the host actin-based cytoskeleton. By examining amino-terminally truncated ActA derivatives for their ability to recruit cytoskeletal proteins, an essential element for actin filament nucleation was identified between amino acids 128 and 151 of ActA. An ActA derivative from which the central proline-rich repeats were deleted retained its ability to recruit filamentous actin, albeit poorly, but was unable to bind VASP. CONCLUSIONS: Our studies reveal the initial interactions that take place between invading Listeria and host microfilament proteins. The listerial ActA polypeptide contains at least two essential sites that are required for efficient microfilament assembly: an amino-terminal 23 amino-acid region for actin filament nucleation, and VASP-binding proline-rich repeats. Hence, ActA represents a prototype actin filament nucleator. We suggest that host cell analogues of ActA exist and are important components of structures involved in cell motility.     

Regulation of cytoskeleton by myelin components: studies on shiverer oligodendrocytes carrying an Mbp transgene

Oligodendrocytes from the shiverer mutant mouse are missing most of the myelin basic protein (Mbp) gene. In axon-free cultures, they produce membrane sheets with abnormally assembled microtubule and actin-based structures. This suggests that an Mbp gene product may have an important role in regulating the organization and stability of the wild-type oligodendrocyte cytoskeleton. We now present evidence extending these observations, using cultured oligodendrocytes that carry both the shiverer mutation and the Mbp1 transgene which partially corrects their deficit. Shiverer oligodendrocytes that carry one dose of the Mbp1 transgene abnormally express MBP along major cytoskeletal vein-like structures in processes and sheets. Shiverer oligodendrocytes that carry two doses of the Mbp1 transgene contain two types of membrane sheet regions, i.e. regions filled with aberrant punctate foci of MBP, and regions with normal domains of MBP. Immunocytochemical staining data show that the distribution of cytoskeleton and associated 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) is dependent upon how MBP is organized. Bundling of actin filaments occurs only around MBP domains, and the colocalization of CNPase along microtubular structures also appears to be regulated by MBP domains in sheets. Multinucleated oligodendrocytes are observed, a likely result of the inability of dividing pro-oligodendrocytes to bundle actin filaments. In addition, the ability of MBP to mediate extracellular signals that modulate cytoskeleton appears to be dependent upon MBP's organization. Transduction of the galactocerebroside signaling pathway, which results in the destabilization of microtubules but not actin filaments, occurs only in sheets containing MBP domains. The distribution of MBP, however, does not affect the myelin/oligodendrocyte-specific protein signaling pathway, which results in growth of microtubular structures and extensive destabilization of the actin cytoskeleton.     

Identification of a novel cortactin SH3 domain-binding protein and its localization to growth cones of cultured neurons

Cortactin is an actin-binding protein that contains several potential signaling motifs including a Src homology 3 (SH3) domain at the distal C terminus. Translocation of cortactin to specific cortical actin structures and hyperphosphorylation of cortactin on tyrosine have been associated with the cortical cytoskeleton reorganization induced by a variety of cellular stimuli. The function of cortactin in these processes is largely unknown in part due to the lack of information about cellular binding partners for cortactin. Here we report the identification of a novel cortactin-binding protein of approximately 180 kDa by yeast two-hybrid interaction screening. The interaction of cortactin with this 180-kDa protein was confirmed by both in vitro and in vivo methods, and the SH3 domain of cortactin was found to direct this interaction. Since this protein represents the first reported natural ligand for the cortactin SH3 domain, we designated it CortBP1 for cortactin-binding protein 1. CortBP1 contains two recognizable sequence motifs within its C-terminal region, including a consensus sequence for cortactin SH3 domain-binding peptides and a sterile alpha motif. Northern and Western blot analysis indicated that CortBP1 is expressed predominately in brain tissue. Immunofluorescence studies revealed colocalization of CortBP1 with cortactin and cortical actin filaments in lamellipodia and membrane ruffles in fibroblasts expressing CortBP1. Colocalization of endogenous CortBP1 and cortactin was also observed in growth cones of developing hippocampal neurons, implicating CortBP1 and cortactin in cytoskeleton reorganization during neurite outgrowth.     

Concentration of an integral membrane protein, CD43 (leukosialin, sialophorin), in the cleavage furrow through the interaction of its cytoplasmic domain with actin-based cytoskeletons

In leukocytes such as thymocytes and basophilic leukemia cells, a glycosilated integral membrane protein called CD43 (leukosialin or sialophorin), which is defective in patients with Wiskott-Aldrich syndrome, was highly concentrated in the cleavage furrow during cytokinesis. Not only at the mitotic phase but also at interphase, CD43 was precisely colocalized with ezrin-radixin-moesin family members. (ERM), which were previously reported to play an important role in the plasma membrane-actin filament association in general. At the electron microscopic level, throughout the cell cycle, both CD43 and ERM were tightly associated with microvilli, providing membrane attachment sites for actin filaments. We constructed a cDNA encoding a chimeric molecule consisting of the extracellular domain of mouse E-cadherin and the transmembrane/cytoplasmic domain of rat CD43, and introduced it into mouse L fibroblasts lacking both endogenous CD43 and E-cadherin. In dividing transfectants, the chimeric molecules were concentrated in the cleavage furrow together with ERM, and both proteins were precisely colocalized throughout the cell cycle. Furthermore, using this transfection system, we narrowed down the domain responsible for the CD43-concentration in the cleavage furrow. Based on these findings, we conclude that CD43 is concentrated in the cleavage furrow through the direct or indirect interaction of its cytoplasmic domain with ERM and actin filaments.     

Participation of serotonin in capsaicin-induced mouse ear edema

Microfilaments were localised by immunofluorescence and immunogold cytochemistry to examine their distribution in granular cells of the isolated frog skin epithelium. Strongly fluorescent bundles of actin were observed beneath the plasma membrane with little evidence for actin in the central regions. Higher resolution offered by cytochemistry revealed that bundles of actin filaments comprised a substantial portion of the cortical cytoskeleton. Quantitative analysis of the frequency of gold label revealed an extremely rich array of filaments beneath the apical membrane of granular cells, with markedly less label along the basolateral membrane and in the central cytoplasm. Treating cells with cytochalasin B or arginine vasopressin caused an apparent disruption of the apical actin fibres, concurrent with a decrease in gold label density. Assumably these signs are indicative of depolymerization of the filaments. Although the significance of this distribution is unknown, the apical polarisation of actin is consistent with a role in regulating the Na+ permeability of the apical membrane. The data are discussed in relation to possible roles of the cytoskeleton in the regulation of transepithelial sodium transport by vasopressin.     

Functional analysis of a rickettsial OmpA homology domain of Shigella flexneri icsA

Shigella flexneri is a gram-negative bacterium that causes diarrhea and dysentery by invasion and spread through the colonic epithelium. Bacteria spread by assembling actin and other cytoskeletal proteins of the host into "actin tails" at the bacterial pole; actin tail assembly provides the force required to move bacteria through the cell cytoplasm and into adjacent cells. The 120-kDa S. flexneri outer membrane protein IcsA is essential for actin assembly. IcsA is anchored in the outer membrane by a carboxy-terminal domain (the beta domain), such that the amino-terminal 706 amino acid residues (the alpha domain) are exposed on the exterior of the bacillus. The alpha domain is therefore likely to contain the domains that are important to interactions with host factors. We identify and characterize a domain of IcsA within the alpha domain that bears significant sequence similarity to two repeated domains of rickettsial OmpA, which has been implicated in rickettsial actin tail formation. Strains of S. flexneri and Escherichia coli that carry derivatives of IcsA containing deletions within this domain display loss of actin recruitment and increased accessibility to IcsA-specific antibody on the surface of intracytoplasmic bacteria. However, site-directed mutagenesis of charged residues within this domain results in actin assembly that is indistinguishable from that of the wild type, and in vitro competition of a polypeptide of this domain fused to glutathione S-transferase did not alter the motility of the wild-type construct. Taken together, our data suggest that the rickettsial homology domain of IcsA is required for the proper conformation of IcsA and that its disruption leads to loss of interactions of other IcsA domains within the amino terminus with host cytoskeletal proteins.     

Performance status and comorbidity in elderly cancer patients compared with young patients with neoplasia and elderly patients without neoplastic conditions

MARCKS, the major protein kinase C substrate in various cells and tissues, binds to calmodulin, acidic membrane phospholipids, and actin filaments, and these interactions are regulated by protein phosphorylation. We have previously analyzed MARCKS purified from bovine brain using capillary liquid chromatography/electrospray mass spectrometry and found that the protein structure differed significantly from that deduced from cDNA sequences [Taniguchi, H., Manenti, S., Suzuki, M., and Titani, K. (1994) J. Biol. Chem. 269, 18299-18302]. Moreover, the alignment of the protein from various species showed a lack of any conserved sequences in the C-terminal half of the molecule. This prompted us to reexamine the C-terminal amino-acid sequence of bovine MARCKS. The purified protein was digested with lysyl endoprotease, and the obtained C-terminal peptide was further digested with either Staphylococcus V8 protease or NTCB. The small peptides thus obtained were analyzed by liquid chromatography/electrospray/tandem mass spectrometry. This combined with gas-phase Edman sequencing allowed us to determine the C-terminal primary structure. The sequence obtained differed significantly from that reported previously, and the comparison with other species revealed the presence of a novel conserved domain in the C-terminal region of MARCKS.     

Role of the ninaC proteins in photoreceptor cell structure: ultrastructure of ninaC deletion mutants and binding to actin filaments

The ninaC proteins are found in Drosophila photoreceptor cells. Their primary sequences suggest they are kinase/myosin chimeras, but their myosin head-like domain is the most divergent amongst all the myosin-like proteins described to date. To investigate possible roles of the ninaC proteins in cell structure, we examined the ultrastructure of the photoreceptor cells in various ninaC mutants, and tested the ability of the proteins to interact with actin filaments in a myosin-like manner. In flies lacking the larger ninaC protein, p174, an ultrastructural phenotype was evident before eclosion. The axial actin cytoskeleton of the rhabdomeral microvilli appeared either fragmented or as an isolated structure, without linkage to the microvillar membrane. Deletion of the myosin head-like domain or the calmodulin-binding domain of p174 resulted in a similar abnormal cytoskeleton. Breakdown of the rhabdomeres followed, although at different rates depending on the deletion. Lack of the smaller protein, p132, per se did not result in photoreceptor degeneration, but in older flies there was an abnormal accumulation of multivesicular bodies. Moreover, the presence of p132 retarded the degeneration that occurs in the absence of p174, even though the p132 remained outside the rhabdomere. Biochemical studies showed that both ninaC proteins bind actin filaments and cosediment with actin filaments in an ATP-sensitive manner. These results outline structural roles for the ninaC proteins, and are consistent with the notion, suggested by their amino acid sequences, that the proteins are actin-based mechanoenzymes.     

The proton motive force, acting on acidic residues, promotes translocation of amino-terminal domains of membrane proteins when the hydrophobicity of the translocation signal is low

We have shown previously that the first transmembrane segment of leader peptidase can function to translocate the polar amino-terminal Pf3 domain across the membrane into the periplasm independently of the proton motive force (pmf) (Lee, J. I., Kuhn, A., and Dalbey, R. E. (1992) J. Biol. Chem. 267, 938-943). We now show that when the first transmembrane segment lacks a strong hydrophobic character, the pmf is required for translocation. In addition, we find that the amino-terminal acidic residue proximal to the transmembrane domain plays a critical role in pmf-dependent amino-terminal translocation. Moreover, the pmf is required to hold the amino-terminal domain in the periplasm to prevent it from slipping such that the amino terminus is no longer exposed to the periplasm. In all cases, translocation occurs under conditions in which the function of the Sec machinery is impaired. These studies show that the low hydrophobicity of the first apolar domain (the translocation signal) can be compensated for by a negative charge in the amino-terminal region, upon which the pmf acts.     

Role of adhesion molecule cytoplasmic domains in mediating interactions with the cytoskeleton

The past ten years have seen significant progress in cell biology research aimed at understanding how cytoskeletal filaments interact with the plasma membrane. Considerable evidence suggests that both actin microfilaments and intermediate filaments attach to the membrane via the cytoplasmic domains of various membrane proteins including adhesion molecules. Interactions between the cytoskeleton and adhesion molecules appear to be essential for a variety of cellular functions, including cell-cell and cell-extracellular matrix (ECM) interactions, cell motility, receptor-ligand interactions, and receptor internalization. Recently, many of the detailed molecular mechanisms which mediate the associations between actin filaments and adhesion molecules have been identified. Among adhesion molecules that support the attachment of cytoskeletal filaments to their cytoplasmic domains are members of the integrin and cadherin families, the intracellular adhesion molecule-1 (ICAM-1, an immunoglobulin family member), and the glycoprotein Ib/IX complex in platelets. A general conclusion emerging from these studies is that physical associations between cytoskeletal filaments and transmembrane glycoproteins do not occur directly between the filaments and the cytoplasmic tails of adhesion molecules. Instead, these interactions appear to be indirect and involve a complex ensemble of intermediary linker proteins. The severe effects of cytoplasmic domain deletion and mutagenesis on adhesion-dependent functions support the view that receptor cytoplasmic domains play a vital role in regulating receptor function and in mediating communication across the membrane. Transfection studies with mutant and chimeric adhesion molecules, along with protein-binding studies, are clarifying the mechanisms which physically link the cytoskeleton to transmembrane proteins, regulate cytoskeletal organization, mediate signaling across the cell membrane, and regulate the ligand specificity and binding affinity of surface receptors.     

Characterization of the association of the actin-binding protein, IQGAP, and activated Cdc42 with Golgi membranes

IQGAP is a recently identified actin-binding protein, which is a putative target for the Cdc42 and Rac GTP-binding proteins. Cdc42 was localized to the Golgi (Erickson, J. W., Zhang, C., Kahn, R. A., Evans, T., and Cerione, R. A. (1996) J. Biol. Chem. 271, 26850-26854), and here we show by immunofluorescence that IQGAP has a perinuclear localization, that it can be co-immunoprecipitated with Cdc42 from Golgi-enriched fractions, and that purified Golgi membranes are recognized by specific antibodies raised against IQGAP and Cdc42 in negative-stain immunogold electron microscopy experiments. Addition of activated, recombinant Cdc42 or solubilization of endogenous Cdc42 from Golgi membranes by the Rho-GDP dissociation inhibitor protein fails to solubilize IQGAP, suggesting that it associates with these membranes in a Cdc42-independent manner. Detergent solubilization of Golgi membranes leaves IQGAP and actin in an insoluble pellet but releases Cdc42 to the supernatant, whereas treatments that release actin from this detergent-insoluble pellet also release IQGAP. Addition of the COOH-terminal half of the IQGAP protein, which contains the Cdc42-binding domain, removes Cdc42 from Golgi membranes in a dose-dependent manner. These data suggest that IQGAP and Cdc42 are part of a cytoskeletal complex in Golgi membranes that may mediate Cdc42-regulated effects on the actin cytoskeleton in these membranes.     

Identification of the protein 4.1 binding interface on glycophorin C and p55, a homologue of the Drosophila discs-large tumor suppressor protein

Protein 4.1 is the prototype of a family of proteins that include ezrin, talin, brain tumor suppressor merlin, and tyrosine phosphatases. All members of the protein 4.1 superfamily share a highly conserved N-terminal 30-kDa domain whose biological function is poorly understood. It is believed that the attachment of the cytoskeleton to the membrane may be mediated via this 30-kDa domain, a function that requires formation of multiprotein complexes at the plasma membrane. In this investigation, synthetically tagged peptides and bacterially expressed proteins were used to map the protein 4.1 binding site on human erythroid glycophorin C, a transmembrane glycoprotein, and on human erythroid p55, a palmitoylated peripheral membrane phosphoprotein. The results show that the 30-kDa domain of protein 4.1 binds to a 12-amino acid segment within the cytoplasmic domain of glycophorin C and to a positively charged, 39-amino acid motif in p55. Sequences similar to this charged motif are conserved in other members of the p55 superfamily, including the Drosophila discs-large tumor suppressor protein. Our data provide new insights into how protein 4.1, glycophorin C, p55, and their non-erythroid homologues, interact with the cytoskeleton to exert their physiological effects.     

Identification of an actin cytoskeletal complex that includes IQGAP and the Cdc42 GTPase

The Rho subfamily of low molecular weight GTPases have been implicated in a variety of cellular functions that include reorganization of the actin cytoskeleton and stress-induced activation of the c-Jun kinase. The downstream targets that mediate the effects of Cdc42 on the actin cytoskeleton have yet to be fully identified. We have used the transient transfection of COS-7 cells with epitope-tagged Cdc42 to identify candidate signaling partners for this GTPase and identified the IQGAP protein as a major in vivo target for activated Cdc42. Epidermal growth factor stimulation of serum-starved COS-7 cells promoted the formation of a Cdc42-IQGAP complex, indicating that growth factors can increase the pool of activated Cdc42. Activated HA-Cdc42 co-localized with IQGAP or F-actin in vivo, whereas cells transfected with dominant-negative forms of Cdc42 (Cdc42(T17N)) showed predominantly dispersed distributions for both HA-Cdc42 and endogenous IQGAP. In detergent lysates from COS-7 cells transiently transfected with different forms of Cdc42, or from stably transfected CHO cells, the induction of actin polymerization by phalloidin resulted in the incorporation of both IQGAP and Cdc42 into actin-containing complexes. Taken together, these findings are consistent with a model whereby IQGAP serves as a target for GTP-bound Cdc42 providing a direct link between the activated GTPase and the actin cytoskeleton.