The integrin family of cell adheson receptors mediates many of the interactions between cells and the extracellular matrix. Because the extracellular matrix has profound Influences on cell behavior, it seems llkely that t S transduce biochemical signals across the cell membrane. The nature ofthese putative signals has, thus far, rem elusive.Antibody-mediated clustering of integrin receptors was used to mimic the integrin clustering process that occurs during formation of adhesive contacts. Human epidermal carcinoma (KB) cells were incubated with an anti-lI integrin monocional antibody for 30 min on ice followed by incubation at 37C with anti-rat IgG. This treatment, which induced integrin clustering, stimulated the phosphorylation on tyrosine residues of a 115-to 130-kDa complex of proteins termed ppl3O. When integrins were clustered in the presence of the phosphatase inhibitor sodium orthovanadate, ppl3O showed a substantial increase in phosphorylation compared to the case in which integrins were clustered in the absence of vanadate. Maximal ppl3O phosphorylation was observed 10-20 min after initiation of integrin clustering in the absence of vanadate or after 5-10 min in its presence. These time courses roughly parallel the formation of integrin clusters on the cell surface as observed by fluorescence microscopy. ppl30 phosphorylation depended on the amount of anti-inte antibody present. Additionally, the tyrosine phosphorylation of ppl30 showed specificity since it was stmulated by antibodies to the integrin a3 and 3 subunits but not by antibodies to other integrin a subunits or to nonintgrin cell surface proteins. Immunoprecipitation experiments clearly demonstrated that ppl30 is not itself a PI integrin. It is postulated, therefore, that the integrinstimulated tyrosine phosphorylation of ppl30 may reflect part of an important signal transduction process between the extracellular matrix and the cell interior.Cell interactions with the extracellular matrix are important determinants of cellular morphology, growth, and differentiation (1-4). Contacts between cells and the extracellular matrix are mediated in part by members of the integrin superfamily of adhesive receptors (5-10). Integrins are heterodimeric cell surface glycoproteins consisting of noncovalently linked a and ( chains. The large extracellular amino-terminal domains of both chains associate to form an extracellular binding site for matrix proteins; each chain has a single a-helical transmembrane region and a short cytoplasmic domain (11,12). The abbreviated intracellular domains of integrins interact with a-actinin, talin, and probably other as yet to be discovered proteins to link integrins to the actin-containing cytoskeleton (9,13,14). Interference reflection microscopy in concert with fluorescence microscopy has shown that integrins can be clustered on the ventral surfaces of adherent cells in structures known as focal contacts (14-17). These structures provide a link (mediated through integrins) between extracellular matrix proteins...

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Background Integrins are cell surface receptors which, in part, mediate the adhesion of cells to the extracelluar matrix. In addition to providing a molecular “glue” essential for tissue organization and survival, integrins are dynamic signaling molecules. Integrins allow normal, nontransformed cells to sense that they are adhered to the extracellular matrix, thus providing a cell survival signal. This signal allows cells to proliferate in the presence of growth factors and in some instances prevents apoptosis. Integrins also mediate cell migration as it occurs in normal processes such as angiogenesis, wound healing, immune system function, and development. Aberrances in the expression and function of integrins contribute to many disease states including cancer. Results Focal adhesion kinase (FAK) becomes phosphorylated and activated during integrin‐mediated cell adhesion. Focal adhesion kinase is a signal transducer of integrins (and certain soluble growth factors). Cells derived from FAK −/− mouse embryos exhibit reduced migration relative to wild‐type cells. Cells which overexpress FAK show increased migration relative to wild‐type cells. Focal adhesion kinase promotes cell survival under certain in vitro conditions. Focal adhesion kinase is overexpressed in invasive and metastatic colon, breast, thyroid, and prostate cancers. Enhanced FAK immunostaining is detected in small populations of preinvasive (carcinoma in situ) oral cancers and in large populations of cells in invasive oral cancers. Conclusions Focal adhesion kinase is probably not a classical oncogene but may be involved in the progression of cancer to invasion and metastasis. It is hypothesized that overexpression of FAK in subpopulations of tumor cells leads to populations of cells with a high propensity toward invasion and metastasis. Focal adhesion kinase would have a dual role in this regard: Overexpression of FAK leads to (1) increased cell migration and (2) increased cell survival under anchorage‐independent conditions. Further work is needed to test this model and to determine whether FAK represents a viable target for anticancer therapy. © 1998 John Wiley & Sons, Inc. Head Neck 20: 745–752, 1998.

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Abstract. Integrin-mediated cell adhesion, or crosslinking of integrins using antibodies, often results in the enhanced tyrosine phosphorylation of certain intracellular proteins, suggesting that integrins may play a role in signal transduction processes. In fibroblasts, platelets, and carcinoma cells, a novel tyrosine kinase termed pp125 F^K has been implicated in integrinmediated tyrosine phosphorylation. In some cell types, integrin ligation or cell adhesion has also been shown to result in the increased expression of certain genes. Although it seems reasonable to hypothesize that integrin-mediated tyrosine phosphorylation and integrin-mediated gene induction are related, until now, there has been no direct evidence supporting this hypothesis. In the current report, we explore the relationship between integrin-mediated tyrosine phosphorylation and gene induction in human monocytes. We demonstrate that monocyte adherence to tissue culture dishes or to extraceUular matrix proteins is followed by a rapid and profound increase in tyrosine phosphorylation, with the predominant phosphorylated component being a protein of 76 kD (pp76). Tyrosine phosphorylation of pp76 and other monocyte proteins can also be triggered by incubation of monocytes with antibodies to the integrin/31 subunit, or by F(ab')2 fragments of such antibodies, but not by F(ab) fragments. The ligation of/31 integrins with antibodies or F(ab')2 fragments also induces the expression of immediate-early (IE) genes such as IL-1/L When adhering monocytes are treated with the tyrosine kinase inhibitors genistein or herbimycin, both phosphorylation of pp76 and induction of IL-1/~ message are blocked in a dose-dependent fashion. Similarly, treatment with genistein or herbimycin can block tyrosine phosphorylation of pp76 and IL-1/~ message induction mediated by ligation of/~ 1 integrin with antibodies. These observations suggest that protein tyrosine phosphorylation is an important aspect of integrin-mediated IE gene induction in monocytes. The cytoplasmic tyrosine kinase pp125 FAK, although important in integrin signaling in other cell types, seems not to play a role in monocytes because this protein could not be detected in these cells.PECIFIC cell-cell and cell-extraceUular matrix (ECM) ~ adhesion events are fundamental to many ditferent biological processes. In inflammatory reactions, monocytes are directed to sites of immunologic challenge by chemotactic factors. However, the migration of monocytes into tissues also involves adhesive interactions with the vascular endothelium and subsequently with ECM components and connective tissue cells (Zimmerman et al., 1992;Butcher, 1991;Osborn, 1990). The proteins that mediate many of the adhesive reactions of monocytes belong to the integrin family of cell surface receptors. Integrins are nonAddress all correspondence to R. L. Juliano,

S U M M A R YGastrin-releasing peptide (GRP) and its receptor (GRP-R) are not normally expressed by epithelial cells lining the colon but are aberrantly expressed in cancer, where they act as morphogens and regulate tumor cell differentiation. Studies of colon cancer formation in mice genetically incapable of synthesizing GRP-R suggested that this receptor's morphogenic properties were mediated via focal adhesion kinase (FAK). We therefore set out to determine the presence of both total and phosphorylated forms of FAK in human colon cancer specimens as a function of tumor cell differentiation and GRP/GRP-R co-expression. Ten colon cancers containing 25 regions of distinct differentiation were randomly selected from our GI Cancer Tumor Bank. All specimens were immunohistochemically probed using antibodies recognizing GRP, GRP-R, total FAK, and FAK specifically phosphorylated at tyrosine (Y) 397, 407, 576, 577, 861, and 925. Antibody-specific chromogen was determined by quantitative immunohistochemistry (IHC) for each region of defined differentiation. Here we confirm that GRP/GRP-R co-expression is a function of differentiation, with highest levels observed in well-differentiated tumor cells. We also show that the amount of total FAK and of FAK phosphorylated at Y397 and Y407 tightly correlates with differentiation and with the amount of GRP/GRP-R co-expression. These findings are consistent with GRP/GRP-R acting as a morphogen by activating FAK, and suggest that this occurs via phosphorylation of this enzyme at two specific tyrosine residues.

Abstract. The purpose of this study was to explore the functional role of the cytoplasmic domain of the ct subunit of the o~5//31 integrin, a fibronectin receptor. Mutant CHO cells that express very low levels of endogenous hamster a5 subunit (CHO clone B2) were transfected with an expression vector containing fulllength or truncated human a5 cDNAs to form chimeric human o~5/hamster/~1 integrins. Three transfectants were examined: B2a27 expresses a full-length human c~5 subunit with 27 amino acids in the cytoplasmic domain; B2al0 expresses an or5 with a 17-amino acid cytoplasmic truncation; B2al expresses an or5 with a 26-amino acid truncation. Levels of ot5//~l surface expression in B2a27 and B2al0 cells were similar to that in wild type CHO cells. The expression of ct5//31 in B2al cells was less, amounting to 15-20% of WT levels, despite message levels that were three to five times greater than those of B2a27. The transfectants were used to examine the role of the or5 cytoplasmic domain in cell adhesion, cell motility, cytoskeletal organization, and integrin-mediated tyrosine phosphorylation.The adhesion characteristics of B2a27 and B2al0 cells on fibronectin substrata were similar to each other and to wild type CHO cells. B2al ceils displayed slight reductions in the strength and rate of adhesion to fibronectin. Cell motility in the presence of fibronectin was similar for B2a27, B2al0, and wild type CHO cells, while the B2al cells were substantially less motile. Comparable degrees of cell spreading and extensive organization of actin filaments were observed for B2a27, B2al0, and wild type CHO cells on fibronectin substrata. The B2al cells spread to a lesser degree, and some organization of actin was observed; the untransfected B2 cells remained round on fibronectin substrata and showed no actin reorganization. Since the reduced motility and cell spreading observed in the B2al cells might be due either to reduced surface expression of ot5//31 or to the truncation in the or5 cytoplasmic domain, we used flow cytometric cell sorting to select populations of B2al and B2a27 cells expressing similar levels of cell surface a5. The deficits in spreading and motility were present in B2al cells expressing high levels of or5. Thus the region of the ix5 cytoplasmic domain adjacent to the membrane seems to play an important role in cytoskeletal organization and cell motility. We also examined whether ot subunit truncation would affect integrin-mediated tyrosine phosphorylation. When B2a27 cells interacted with fibronectin substrata, increased tyrosine phosphorylation was observed in proteins of ~125 kD. A similar pattern of phosphorylation was observed in wild type CHO, B2al0, and B2al cells, but not in B2 cells. Thus, the c~5 cytoplasmic domain does not seem to be essential for integrinmediated tyrosine phosphorylation of intracellular proteins.

We have proposed that gastrin-releasing peptide (GRP) and its receptor (GRP-R) are morphogens that when aberrantly re-expressed in colon cancer promote tumor cell differentiation and retard metastasis. Because circumstantial evidence suggested that these properties were mediated via focal adhesion kinase (FAK), the purpose of this study was to elucidate the role of GRP-induced activation of this enzyme on properties fundamental to metastasis including cell attachment, motility, and deformability. To do this, we studied 293 cells, a non-malignant epithelial cell line that we show expresses GRP and GRPR. To dissect out the role of FAK, 293 cells were modified to inducibly express the dominant negative enzyme FAK-related non-kinase (FRNK) under control of a Tet-On (i.e., doxycycline-sensitive) promoter. Under serum-free conditions, GRP acting in an autocrine manner caused FAK to be phosphorylated at Y397; and this could be completely inhibited either by incubating with the specific GRP-R antagonist D-Phe(6)(bombesin) methyl ester, or by upregulating FRNK using doxycycline. To measure cell attachment, we designed a cone-plate viscometer that recorded the shear stress required to detach cells from their underlying matrix. To assess motility, confluent cells were wounded and behavior assessed by time-lapse photography. To measure deformability, we recorded the ability of cells to be completely drawn into a micropipette <50% the size of the non-deformed cell. Control 293 cells adhered more avidly to their underlying matrix, rapidly remodeled wounded tissues without any increase in overall proliferation, and were less distensible than cells treated with antagonist or doxycycline. Thus, these findings suggest that expression of GRP/GRPR in cancer inhibits metastasis by enhancing cell attachment to the matrix, regulating motility in the context of remodeling, and decreasing deformability.