The activation dependent adhesion of macrophages to laminin involves cytoskeletal anchoring and phosphorylation of the alpha 6 beta 1 integrin

Macrophages require activation with either PMA (Mercurio, A. M., and L. M. Shaw. 1988. J. Cell Biol. 107:1873-1880) or interferon-gamma (Shaw, L. M., and A. M. Mercurio. 1989. J. Exp. Med. 169:303-308) to adhere to a laminin substratum. In the present study, we identified an integrin laminin receptor on macrophages and characterized cellular changes that occur in response to PMA activation that facilitate laminin adhesion. A monoclonal antibody (GoH3) that recognizes the integrin alpha 6 subunit (Sonnenberg, A., H. Janssen, F. Hogervorst, J. Calafat, and J. Hilgers. 1987. J. Biol. Chem. 262:10376-10383) specifically inhibited adhesion to laminin-coated surfaces. This antibody precipitated an alpha 6 beta 1 heterodimer (Mr 130/110 kD) from 125I surface-labeled macrophages. The amount of radiolabeled receptor on the cell surface did not increase after PMA activation. Thus, the induction of laminin adhesion cannot be attributed to de novo or increased surface expression of alpha 6 beta 1. By initially removing the Triton X-100-soluble fraction of macrophages and then disrupting the remaining cytoskeletal framework, we observed that 75% of the alpha 6 beta 1 heterodimer on the cell surface is anchored to the cytoskeleton in macrophages that had adhered to a laminin substratum in response to PMA. Significant cytoskeletal anchoring of this receptor was not observed in macrophages that had adhered to fibronectin or tissue culture plastic, nor was it seen in nonadherent cells. PMA also induced phosphorylation of the cytoplasmic domain of the alpha 6 subunit, but not the beta 1 subunit. Phosphorylated alpha 6 was localized to the cytoskeletal fraction only in macrophages plated on a laminin substratum. In summary, our results support a mechanism for the regulation of macrophage adhesion to laminin that involves specific and dynamic matrix integrin-cytoskeletal interactions that may be facilitated by integrin phosphorylation.     

Regulation of cellular interactions with laminin by integrin cytoplasmic domains: the A and B structural variants of the alpha 6 beta 1 integrin differentially modulate the adhesive strength, morphology, and migration of macrophages.

Several integrin alpha subunits have structural variants that are identical in their extracellular and transmembrane domains but that differ in their cytoplasmic domains. The functional significance of these variants, however, is unknown. In the present study, we examined the possibility that the A and B variants of the alpha 6 beta 1 integrin laminin receptor differ in function. For this purpose, we expressed the alpha 6A and alpha 6B cDNAs, as well as a truncated alpha 6 cDNA (alpha 6-delta CYT) in which the cytoplasmic domain sequence was deleted after the GFFKR pentapeptide, in P388D1 cells, an alpha 6 deficient macrophage cell line. Populations of stable alpha 6A, alpha 6B, and alpha 6-delta CYT transfectants that expressed equivalent levels of cell surface alpha 6 were obtained by fluorescence-activated cell sorter and shown to form heterodimers with endogenous beta 1 subunits. Upon attachment to laminin, the alpha 6A transfectants extended numerous pseudopodia. In contrast, the alpha 6B transfectants remained rounded and extended few processes. The transfectants were also examined for their ability to migrate toward a laminin substratum using Transwell chambers. The alpha 6A transfectants were three- to fourfold more migratory than the alpha 6B transfectants. The alpha 6-delta CYT transfectants did not attach to laminin in normal culture medium, but they did attach in the presence of Mn2+. The alpha 6-delta CYT transfectants migrated to a lesser extent than either the alpha 6A or alpha 6B transfectants in the presence of Mn2+. The alpha 6 transfectants differed significantly in the concentration of substratum bound laminin required for half-maximal adhesion in the presence of Mn2+:alpha 6A (2.1 micrograms/ml), alpha 6B (6.3 micrograms/ml), and alpha 6-delta CYT (8.8 micrograms/ml). Divalent cation titration studies revealed that these transfectants also differed significantly in both the [Ca2+] and [Mn2+] required to obtain half-maximal adhesion to laminin. These data demonstrate that the A and B variants of the alpha 6 cytoplasmic domain can differentially modulate the function of the alpha 6 beta 1 extracellular domain.     

The major laminin receptor of mouse embryonic stem cells is a novel isoform of the alpha 6 beta 1 integrin

Laminin is the first extracellular matrix protein expressed in the developing mouse embryo. It is known to influence morphogenesis and affect cell migration and polarization. Several laminin receptors are included in the integrin family of extracellular matrix receptors. Ligand binding by integrin heterodimers results in signal transduction events controlling cell motility. We report that the major laminin receptor on murine embryonic stem (ES) cells is the integrin heterodimer alpha 6 beta 1, an important receptor for laminin in neurons, lymphocytes, macrophages, fibroblasts, platelets and other cell types. However, the cytoplasmic domain of the ES cell alpha 6 (alpha 6 B) differs totally from the reported cytoplasmic domain amino acid sequence of alpha 6 (alpha 6 A). Comparisons of alpha 6 cDNAs from ES cells and other cells suggest that the alpha 6 A and alpha 6 B cytoplasmic domains derive from alternative mRNA splicing. Anti-peptide antibodies to alpha 6 A are unreactive with ES cells, but react with mouse melanoma cells and embryonic fibroblasts. When ES cells are cultured under conditions that permit their differentiation, they become positive for alpha 6 A, concurrent with the morphologic appearance of differentiated cell types. Thus, expression of the alpha 6 B beta 1 laminin receptor may be favored in undifferentiated, totipotent cells, while the expression of alpha 6 A beta 1 receptor occurs in committed lineages. While the functions of integrin alpha chain cytoplasmic domains are not understood, it is possible that they contribute to transferring signals to the cell interior, e.g., by delivering cytoskeleton organizing signals in response to integrin engagement with extracellular matrix ligands. It is therefore reasonable to propose that the cellular responses to laminin may vary, according to what alpha subunit isoform (alpha 6 A or alpha 6 B) is expressed as part of the alpha 6 beta 1 laminin receptor. The switch from alpha 6 B to alpha 6 A, if confirmed in early embryos, could then be of striking potential relevance to the developmental role of laminin.     

A synthetic peptide derived from the carboxy terminus of the laminin A chain represents a binding site for the alpha 3 beta 1 integrin

The purpose of this study was to identify the binding site(s) within laminin for the alpha 3 beta 1 integrin receptor. It has been previously shown, using proteolytic fragments and anti-laminin antibodies, that the region in laminin for alpha 3 beta 1 integrin binding is localized to the carboxy-terminal region at the end of the long arm (Gehlsen, K. R., E. Engvall, K. Dickerson, W. S. Argraves, and E. Ruoslahti. 1989. J. Biol. Chem. 264:19034-19038; Tomaselli, K. J., D. E. Hall, L. T. Reichardt, L. A. Flier, K. R. Gehlsen, D. C. Turner, and S. Carbonetto. 1990. Neuron. 5:651-662). Using synthetic peptides, we have identified an amino acid sequence within the carboxy-terminal region of the laminin A chain that is recognized by the alpha 3 beta 1 integrin. The amino acid sequence represented by the synthetic peptide GD-6 (KQNCLSSRASFRGCVRNLRLSR residues numbered 3011 to 3032) of the globular domain of the murine A chain supports cell attachment and inhibits cell adhesion to laminin-coated surfaces. By affinity chromatography, peptide GD-6-Sepharose specifically bound solubilized alpha 3 beta 1 from extracts of surface-iodinated cells in a cation-dependent manner, while it did not bind other integrins. In addition, exogenous peptide GD-6 specifically eluted bound alpha 3 beta 1 from laminin-Sepharose columns but did not elute the alpha 3 beta 1 integrin from a fibronectin-Sepharose column. Using integrin subunit-specific monoclonal antibodies, only those antibodies against the alpha 3 and beta 1 subunits inhibited cell adhesion to peptide GD-6-coated surfaces. Finally, a polyclonal antibody made against peptide GD-6 reacted specifically with both murine and human laminin and significantly inhibited cell adhesion to laminin-coated surfaces but not those coated with other matrix proteins. These results identify the laminin A chain amino acid sequence of peptide GD-6 as representing a binding site in laminin for the alpha 3 beta 1 integrin.     

Identification of a tetrapeptide recognition sequence for the alpha 2 beta 1 integrin in collagen

The alpha 2 beta 1 integrin serves as either a specific cell surface receptor for collagen or as both a collagen and laminin receptor depending upon the cell type. Recently we established that the alpha 2 beta 1 integrin binds to a site within the alpha 1 (I)-CB3 fragment of type I collagen (Staatz, W. D., Walsh, J. J., Pexton, T., and Santoro, S. A. (1990) J. Biol. Chem. 265, 4778-4781). To define the alpha 2 beta 1 recognition sequence further we have prepared an overlapping set of synthetic peptides which completely spans the 148-amino acid alpha 1(I)-CB3 fragment and tested the peptides for ability to inhibit cell adhesion to collagen and laminin substrates. The minimal active recognition sequence defined by these experiments is a tetrapeptide of the sequence Asp-Gly-Glu-Ala (DGEA) corresponding to residues 435-438 of the type I collagen sequence. The DGEA-containing peptides effectively inhibited alpha 2 beta 1-mediated Mg2(+)-dependent adhesion of platelets, which use the alpha 2 beta 1 integrin as a collagen-specific receptor, to collagen but had no effect on alpha 5 beta 1-mediated platelet adhesion to fibronectin or alpha 6 beta 1-mediated platelet adhesion to laminin. In contrast, with T47D breast adenocarcinoma cells, which use alpha 2 beta 1 as a collagen/lamin receptor, adhesion to both collagen and laminin was inhibited by DGEA-containing peptides. Deletion of the alanine residue or substitution of alanine for either the glutamic or aspartic acid residues in DGEA-containing peptides resulted in marked loss of inhibitory activity. These results indicate that the amino acid sequence DGEA serves as a recognition site for the alpha 2 beta 1 integrin complex on platelets and other cells.     

Activation of p53 function in carcinoma cells by the alpha6beta4 integrin.

The interaction of integrins with extracellular matrix is known to promote cell survival by inhibiting apoptotic signaling. In contrast, we demonstrate here that the alpha6beta4 integrin induces apoptosis in carcinoma cells by stimulating p53 function. Specifically, we show that expression of alpha6beta4 in carcinoma cells that lack this integrin stimulates an increase in the transactivating function of p53 as demonstrated by the ability of this integrin to up-regulate the expression of a p53-sensitive reporter gene as well as the endogenous p53 response gene, bax. In addition, we report that alpha6beta4 triggers apoptosis in carcinoma cells that express wild-type but not mutant p53 and that these alpha6beta4 functions are inhibited by a dominant negative p53 construct. Importantly, we provide a link between integrin signaling and p53 activation by demonstrating that the clustering of alpha6beta4 with a beta4 integrin-specific antibody promotes p53-dependent apoptosis in cells that express both alpha6beta4 and wild-type p53. These studies are the first to demonstrate that a specific integrin can promote apoptosis by activating p53. Moreover, given the ability of alpha6beta4 to stimulate invasion (Shaw, L. M., Rabinovitz, I., Wang, H. F., Toker, A., and Mercurio, A. M. (1997) Cell 91, 949-960), these studies suggest that the ability of alpha6beta4 to promote carcinoma progression will be enhanced in tumor cells that express mutant, inactive forms of p53.     

Interaction of the disintegrin and cysteine-rich domains of ADAM12 with integrin alpha7beta1

We describe a novel interaction between the disintegrin and cysteine-rich (DC) domains of ADAM12 and the integrin alpha7beta1. Integrin alpha7beta1 extracted from human embryonic kidney 293 cells transfected with alpha7 cDNA was retained on an affinity column containing immobilized DC domain of ADAM12. 293 cells stably transfected with alpha7 cDNA adhered to DC-coated wells, and this adhesion was partially inhibited by 6A11 integrin alpha7 function-blocking antibody. The X1 and the X2 extracellular splice variants of integrin alpha7 supported equally well adhesion to the DC protein. Integrin alpha7beta1-mediated cell adhesion to DC had different requirements for Mn2+ than adhesion to laminin. Furthermore, integrin alpha7beta1-mediated cell adhesion to laminin, but not to DC, resulted in efficient cell spreading and phosphorylation of focal adhesion kinase (FAK) at Tyr397. We also show that adhesion of L6 myoblasts to DC is mediated in part by the endogenous integrin alpha7beta1 expressed in these cells. Since integrin alpha7 plays an important role in muscle cell growth, stability, and survival, and since ADAM12 has been implicated in muscle development and regeneration, we postulate that the interaction between ADAM12 and integrin alpha7beta1 may be relevant to muscle development, function, and disease. We also conclude that laminin and the DC domain of ADAM12 represent two functional ligands for integrin alpha7beta1, and adhesion to each of these two ligands via integrin alpha7beta1 triggers different cellular responses.     

Ligand-dependent and -independent integrin focal contact localization: the role of the alpha chain cytoplasmic domain.

Many integrin receptors localize to focal contact sites upon binding their ligand. However, unoccupied integrin receptors do not localize to focal contact sites. Because the integrin beta 1 cytoplasmic domain appears to have a focal contact localization signal, there must be a mechanism by which this domain is kept inactive in the unoccupied state and becomes exposed or activated in the occupied receptor. We considered that this mechanism involves the alpha subunit cytoplasmic domain. To test this hypothesis, we have established two NIH 3T3 cell lines that express either the human alpha 1 wild-type subunit (HA1 cells) or the cytoplasmic domain deleted alpha 1 subunit (CYT cells). Both cell lines express similar levels of the human alpha 1 subunit, and there is no significant effect of the deletion on the dimerization and surface expression of the receptor. Furthermore, the deletion had no effect on the binding or adhesion via alpha 1 beta 1 to its ligand collagen IV. However, when these two cell lines are plated on fibronectin (FN), which is a ligand for alpha 5 beta 1 but not for alpha 1 beta 1, there is a striking difference in the cellular localization of alpha 1 beta 1. The HA1 cells show only alpha 5 in focal contacts, without alpha 1, demonstrating that all of the integrin localization is ligand dependent. In contrast, when the CYT cells are plated on FN, the mutant alpha 1 appears in focal contacts along with the alpha 5/beta 1. Thus, there is both ligand-dependent (alpha 5/beta 1) and ligand-independent (alpha 1/beta 1) focal contact localization in these cells. The truncated alpha 1 also localized to focal contacts in a ligand-independent manner on vitronectin. We conclude that the mutant alpha 1 no longer requires ligand occupancy for focal contact localization. These data strongly suggest that the alpha cytoplasmic domain plays a role in the normal ligand-dependent integrin focal contact localization.     

The beta1 cytoplasmic domain regulates the laminin-binding specificity of the alpha7X1 integrin

During muscle development, the laminin-specific alpha7 integrin is alternatively spliced in the putative ligand-binding domain to yield either the alpha7X1 or the alpha7X2 variant. The relative level of alpha7X1 and alpha7X2 is developmentally regulated. Similarly, the partner beta1 integrin cytoplasmic domain is converted from the beta1A to the beta1D splice variant. To determine whether beta1D modulates the activity of the alpha7 receptor, cells were transfected with alpha7X1 and beta1D cDNA. alpha7X1 coupled with beta1A failed to adhere to laminin-1, whereas cotransfectants expressing alpha7X1 and beta1D showed strong adhesion. Interestingly, alpha7X1 complexed with beta1A and beta1D displayed the same level of poor adhesion to laminin-2/4 or strong adhesion to laminin-10/11. These findings indicate that alpha7 function is regulated not only by X1/X2 in its extracellular domain but also by beta1 cytoplasmic splice variants. It is likely that expression of beta1D alters alpha7X1 binding to laminin isoforms by a process related to ligand affinity modulation. Functional regulation of alpha7beta1 by developmentally regulated splicing events may be important during myogenic differentiation and repair because the integrin mediates adhesion, motility, and cell survival.     

Molecular cloning and expression of the cDNA for alpha 3 subunit of human alpha 3 beta 1 (VLA-3), an integrin receptor for fibronectin, laminin, and collagen

alpha 3 beta 1 (VLA-3), a member of the integrin family of cell adhesion receptors, may function as a receptor for fibronectin, laminin, and collagen. A partial cDNA clone (2.4 kb) for the human alpha 3 subunit was selected from an endothelial cell lambda gt11 cDNA library by specific antibody screening. Several overlapping cDNA clones were subsequently obtained, of a total length of 4.6 kb from various cDNA libraries. The reconstructed alpha 3 cDNA was expressed on the surface of chinese hamster ovary cells as detected by an alpha 3- specific mAb after transfection, suggesting that the cDNA is authentic. Within this sequence was an open reading frame, encoding for 1,051 amino acids, including a signal peptide of 32 residues, a long extracellular domain (959 residues), a transmembrane domain (28 residues), and a short cytoplasmic segment (32 residues). Overall, the alpha 3 amino acid sequence was 25-37% similar to the other integrin alpha subunits that are cleaved, with most similarity to the alpha 6 sequence (37%), and less similarity to those alpha subunits that have I domains (15-20%, excluding the I domain sequence itself). Features most like those in other alpha subunits are (a) the positions of 18/19 cysteine residues, (b) three potential metal binding domains of the general structure DX(D/N)X(D/N)GXXD, and (c) the predicted transmembrane domain. The mass of alpha 3 calculated from its amino acid sequence is 113,505. The human alpha 3 sequence was 89% identical to hamster galactoprotein b3, and 70% similar to the chicken CSAT antigen band 2 protein partial sequence, suggesting that these two polypeptides are homologues of human alpha 3.     

Biochemical characterization and tissue distribution of the A and B variants of the integrin alpha 6 subunit

Two cytoplasmic variants of the alpha 6 integrin, alpha 6A and alpha 6B, have been identified previously (Hogervorst, F., I. Kuikman, A. G. van Kessel, and A. Sonnenberg. 1991. Eur. J. Biochem. 199:425-433; Cooper, H. M., R. N. Tamura, and V. Quaranta. 1991. J. Cell Biol. 115:843-850). Using synthetic peptides, containing sequences of their cytoplasmic domains, we have produced mAbs specific for either of the variants. These antibodies reacted with a variety of different epithelial tissues. In some tissues (e.g., salivary gland) both variants could be detected while in others only one of the variants was found (e.g., alpha 6A in epidermis and alpha 6B in kidney). Among nonepithelial cells and tissues, perineural fibroblasts and Schwann cells in peripheral nerves and platelets reacted with anti-alpha 6A, while microvascular endothelia reacted with both anti-alpha 6A and anti- alpha 6B. From our immunohistochemical results there is not evidence that combination with beta 1 or beta 4 is restricted to one of the two variants of alpha 6. This was confirmed by immunoprecipitation studies which showed that both beta 1 and beta 4 were coprecipitated by both anti-alpha 6A or anti-alpha 6B antibodies from cells. Also, the distribution of alpha 6A and alpha 6B subunits associated with beta 1 on cells attached to laminin was similar: both were found in focal contacts colocalizing with vinculin. In contrast, the alpha 6A subunit, associated with beta 4 in cultures of a squamous cell carcinoma cell line, was found to codistribute with bullous pemphigoid antigen 230 in hemidesmosomal-like structures. The alpha 6A and alpha 6B variants, immunoprecipitated from various cell lines, exhibited slightly different electrophoretic mobilities. Analysis of the antigens under reducing conditions showed that the mobility of the light chains, but not of the heavy chains, is different. In addition, in some cells the light chains of alpha 6A and alpha 6B, each are of two different sizes. Treatment with N-glycanase showed that these two light chain variants of alpha 6A and alpha 6B are not due to differences in N-linked glycosylation, and may therefore represent alternative proteolytic products of the alpha 6 precursor. We further demonstrate that alpha 6A, but not alpha 6B, is a major target for PMA-induced phosphorylation. Phosphorylated alpha 6A contained phosphoserine and a small amount of phosphotyrosine. There are also two variants of the integrin alpha 3 subunit with different cytoplasmic domains, but in the cell lines examined only alpha 3A could be demonstrated by RT- PCR.(ABSTRACT TRUNCATED AT 400 WORDS)     

Normal fertilization occurs with eggs lacking the integrin alpha6beta1 and is CD9-dependent

Previous results, based on inhibition of fertilization by an anti-alpha6 integrin mAb (GoH3), suggest that the alpha6beta1 integrin on mouse eggs functions as the receptor for sperm (Almeida, E.A., A.P. Huovila, A.E. Sutherland, L.E. Stephens, P.G. Calarco, L. M. Shaw, A.M. Mercurio, A. Sonnenberg, P. Primakoff, D.G. Myles, and J.M. White. 1995. Cell. 81:1095-1104). Because the egg surface tetraspanin CD9 is essential for gamete fusion (Kaji, K., S. Oda, T. Shikano, T. Ohnuki, Y. Uematsu, J. Sakagami, N. Tada, S. Miyazaki, and A. Kudo. 2000. Nat. Genet. 24:279-282; Le Naour, F., E. Rubinstein, C. Jasmin, M. Prenant, and C. Boucheix. 2000. Science. 287:319-321; Miyado, K., G. Yamada, S. Yamada, H. Hasuwa, Y. Nakamura, F. Ryu, K. Suzuki, K. Kosai, K. Inoue, A. Ogura, M. Okabe, and E. Mekada. 2000. Science. 287:321-324) and CD9 is known to associate with integrins, recent models of gamete fusion have posited that egg CD9 acts in association with alpha6beta1 in fusion (Chen, M.S., K.S. Tung, S.A. Coonrod, Y. Takahashi, D. Bigler, A. Chang, Y. Yamashita, P.W. Kincade, J.C. Herr, and J.M. White. 1999. Proc. Natl. Acad. Sci. USA. 96:11830-11835; Kaji, K., S. Oda, T. Shikano, T. Ohnuki, Y. Uematsu, J. Sakagami, N. Tada, S. Miyazaki, and A. Kudo. 2000. Nat. Genet. 24:279-282; Le Naour, F., E. Rubinstein, C. Jasmin, M. Prenant, and C. Boucheix. 2000. Science. 287:319-321; Miyado, K., G. Yamada, S. Yamada, H. Hasuwa, Y. Nakamura, F. Ryu, K. Su- zuki, K. Kosai, K. Inoue, A. Ogura, M. Okabe, and E. Mekada. 2000. Science. 287:321-324). Using eggs from cultured ovaries of mice lacking the alpha6 integrin subunit, we found that the fertilization rate, fertilization index, and sperm binding were not impaired compared with wild-type or heterozygous controls. Furthermore, a reexamination of antibody inhibition, using an assay that better simulates in vivo fertilization conditions, revealed no inhibition of fusion by the GoH3 mAb. We also found that an anti-CD9 mAb completely blocks sperm fusion with either wild-type eggs or eggs lacking alpha6beta1. Based on these results, we conclude that the alpha6beta1 integrin is not essential for sperm-egg fusion, and we suggest a new model in which CD9 acts by itself, or interacts with egg protein(s) other than alpha6beta1, to function in sperm-egg fusion.     

The integrin alpha 6 beta 4 is a laminin receptor

In this study, the putative laminin receptor function of the alpha 6 beta 4 integrin was assessed. For this purpose, we used a human cell line, referred to as clone A, that was derived from a highly invasive, colon adenocarcinoma. This cell line, which expresses the alpha 6 beta 4 integrin, adheres to the E8 and not to the P1 fragment of laminin. The adhesion of clone A cells to laminin is extremely rapid with half-maximal adhesion observed at 5 min after plating. Adhesion to laminin is blocked by GoH3, and alpha 6 specific antibody (60% inhibition), as well as by A9, a beta 4 specific antibody (30% inhibition). Most importantly, we demonstrate that alpha 6 beta 4 binds specifically to laminin-Sepharose columns in the presence of either Mg2+ or Mn2+ and it is eluted from these columns with EDTA but not with NaCl. The alpha 6 beta 4 integrin does not bind to collagen-Sepharose, but the alpha 2 beta 1 integrin does bind. Clone A cells do not express alpha 6 beta 1 as evidenced by the following observations: (a) no beta 1 integrin is detected in beta 1 immunoblots of GoH3 immunoprecipitates; and (b) no alpha 6 beta 1 integrin is seen in GoH3 immunoprecipitates of clone A extracts that had been immunodepleted of all beta 4 containing integrin using the A9 antibody. These data establish that laminin is a ligand for the alpha 6 beta 4 integrin and that this integrin can function as a laminin receptor independently of alpha 6 beta 1.     

p53 inhibits alpha 6 beta 4 integrin survival signaling by promoting the caspase 3-dependent cleavage of AKT/PKB

Although the interaction of matrix proteins with integrins is known to initiate signaling pathways that are essential for cell survival, a role for tumor suppressors in the regulation of these pathways has not been established. We demonstrate here that p53 can inhibit the survival function of integrins by inducing the caspase-dependent cleavage and inactivation of the serine/threonine kinase AKT/PKB. Specifically, we show that the alpha6beta4 integrin promotes the survival of p53-deficient carcinoma cells by activating AKT/PKB. In contrast, this integrin does not activate AKT/PKB in carcinoma cells that express wild-type p53 and it actually stimulates their apoptosis, in agreement with our previous findings (Bachelder, R.E., A. Marchetti, R. Falcioni, S. Soddu, and A.M. Mercurio. 1999. J. Biol. Chem. 274:20733-20737). Interestingly, we observed reduced levels of AKT/PKB protein after antibody clustering of alpha6beta4 in carcinoma cells that express wild-type p53. In contrast, alpha6beta4 clustering did not reduce the level of AKT/PKB in carcinoma cells that lack functional p53. The involvement of caspase 3 in AKT/PKB regulation was indicated by the ability of Z-DEVD-FMK, a caspase 3 inhibitor, to block the alpha6beta4-associated reduction in AKT/PKB levels in vivo, and by the ability of recombinant caspase 3 to promote the cleavage of AKT/PKB in vitro. In addition, the ability of alpha6beta4 to activate AKT/PKB could be restored in p53 wild-type carcinoma cells by inhibiting caspase 3 activity. These studies demonstrate that the p53 tumor suppressor can inhibit integrin-associated survival signaling pathways.     

Identification of novel interaction partners for the conserved membrane proximal region of alpha-integrin cytoplasmic domains

The alpha3Abeta1 integrin is a laminin receptor with a broad specificity for different laminin isoforms. Furthermore, it regulates the function of other integrins, like alpha2beta1, alpha5beta1 and alpha6Abeta1. In a yeast two hybrid screen of a human placenta cDNA library, we identified cDNAs coding for four different proteins that strongly interact with the conserved region of the cytoplasmic domain of the alpha3A integrin subunit. In addition to the cDNA for nucleotide exchange factor Mss4 and the putative tumour suppressor protein BIN1, two novel cDNAs were identified. Association analysis with different integrin subunits revealed them as cDNAs that encode binding proteins which react with a broad spectrum of alpha subunits. The conserved membrane proximal region of the alpha3A chain was identified as the binding site for all four proteins. They, therefore, may be involved in the regulation of general functions of integrins.     

Association of the membrane proximal regions of the alpha and beta subunit cytoplasmic domains constrains an integrin in the inactive state

The adhesiveness of integrins is regulated through a process termed "inside-out" signaling. To understand the molecular mechanism of integrin inside-out signaling, we generated K562 stable cell lines that expressed LFA-1 (alpha(L)beta(2)) or Mac-1 (alpha(M)beta(2)) with mutations in the cytoplasmic domain. Complete truncation of the beta(2) cytoplasmic domain, but not a truncation that retained the membrane proximal eight residues, resulted in constitutive activation of alpha(L)beta(2) and alpha(M)beta(2), demonstrating the importance of this membrane proximal region in the regulation of integrin adhesive function. Furthermore, replacement of the alpha(L) and beta(2) cytoplasmic domains with acidic and basic peptides that form an alpha-helical coiled coil caused inactivation of alpha(L)beta(2). Association of these artificial cytoplasmic domains was directly demonstrated. By contrast, replacement of the alpha(L) and beta(2) cytoplasmic domains with two basic peptides that do not form an alpha-helical coiled coil activated alpha(L)beta(2). Induction of ligand binding by the activating cytoplasmic domain mutations correlated with the induction of activation epitopes in the extracellular domain. Our data demonstrate that cytoplasmic, membrane proximal association between integrin alpha and beta subunits, constrains an integrin in the inactive conformation.     

Distinct and overlapping ligand specificities of the alpha 3A beta 1 and alpha 6A beta 1 integrins: recognition of laminin isoforms.

The ligand specificity of the alpha 3A beta 1 integrin was analyzed using K562 cells transfected with full-length alpha 3A cDNA and was compared with that of alpha 6A beta 1 in similarly transfected K562 cells. Clones were obtained that showed comparable surface expression of either alpha 3A beta 1 or alpha 6A beta 1 integrins. Those expressing alpha 3A beta 1 attached to and spread on immunopurified human kalinin and cellular matrices containing human kalinin, which is a particular isoform of laminin. In addition, alpha 3A transfectants adhered to bovine kidney laminins possessing a novel A chain variant. Binding to kalinin was blocked by a monoclonal antibody against the A chain constituent of kalinin and adhesion to both kalinin and kidney laminins by anti-alpha 3 and beta 1 monoclonal antibodies. The alpha 3A transfected cells bound more strongly to kalinin and bovine kidney laminins after treatment with the beta 1 stimulatory antibody TS2/16. A distinctly weaker and activation-dependent adhesion of alpha 3A transfectants was observed on human placental laminins possessing the Am chain variant (merosin), and no adhesion occurred on bovine heart laminins and murine EHS tumor laminin. Further inactive substrates were fibronectin, nidogen, and collagen types IV and VI, indicating that the alpha 3A beta 1 integrin is a much less promiscuous receptor than thought before. By contrast, alpha 6A transfected cells adhered to all laminin isoforms when stimulated with TS2/16. Adhesion also occurred only on bovine kidney laminins in the absence of TS2/16. These results demonstrate that both alpha 3A beta 1 and alpha 6A beta 1 integrins are typical laminin receptors but that their affinity and activation dependence for binding to various laminin isoforms differ considerably.     

The PDZ domain of TIP-2/GIPC interacts with the C-terminus of the integrin alpha5 and alpha6 subunits.

Different cDNA libraries were screened by the yeast two-hybrid system using as a bait the cytoplasmic sequence of integrin alpha6A or alpha6B subunits. Surprisingly, the same PDZ domain-containing protein, TIP-2/GIPC, was isolated with either of the variants, although their sequences are different. Direct interaction assays with the cytoplasmic domain of the integrin alpha1--7 subunits revealed that in addition to alpha6A and alpha6B, TIP-2/GIPC reacted also with alpha5, but not other alpha integrin subunits. The specificity of the interaction was confirmed by in vitro protein binding assays with purified peptides corresponding to integrin cytoplasmic domains. Further analysis with either truncation fragments of TIP-2/GIPC or mutated integrin cytoplasmic domains indicated that the interaction occurs between the PDZ domain of TIP-2/GIPC and a consensus PDZ domain-binding sequence, SDA, present at the C-terminus of the integrin alpha5 and alpha6A subunits. The integrin alpha6B subunit terminates with a different sequence, SYS, which may represent a new PDZ domain-binding motif.