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Integrins are heterodimeric cell-adhesion receptors that comprise a family of over 20 different combinations of αand βsubunits that function in diverse cellular processes, including development, growth, motility, metastasis, apoptosis, inflammation, hemostasis, and bone resorption (for review, seerefs. 1,2). αvβ3 is a member of a subclass of integrins that show binding specificities to peptide sequences encoding an RGD (-Arg-Gly-Asp-) structural motif. Recent interest has focused on αvβ3, which has been shown to have important roles in tumor angiogenesis and metastasis (3,4), hyperplasia leading to restenosis (5), osteoclast-mediated bone resorption (6,7), and retinal neovascularization (8). Both in vitro and in vivo studies, employing specific antibodies to αvβ3, as well as short RGD-containing peptide sequences, have been used to demonstrate therapeutic utility in blocking the function of this receptor in animal models. [ABSTRACT FROM AUTHOR]

Upon binding to extracellular matrix (ECM) ligands, integrins aggregate on the plane of the plasma membrane and interact on the cytoplasmic side with elements of the cytoskeleton as well as signaling molecules. These events result in the organization of adhesive junctions, such as focal adhesions and hemidesmosomes, and the activation of signaling pathways that regulate gene expression (1,2). [ABSTRACT FROM AUTHOR]

When they are cultured on a reconstituted basement membrane gel, mammary epithelial cells round-up, aggregate, form cell-cell junctions, and undergo an apical/basal polarization that ultimately produces a central lumen. The resulting three-dimensional ‘mammospheres' closely resemble lactational alveoli in vivo and they fully differentiate when they are maintained in an appropriate mileau of lactogenic hormones. Specifically, the cells express milk protein genes, package the products in secretory vesicles, transport the vesicles to the apical cell surface, and release the products into the central lumen. Therefore, dynamic and reciprocal interactions between epithelial cells and the surrounding basement membrane are key regulators of phenotypic change in the developing mammary gland (1-4). [ABSTRACT FROM AUTHOR]

The development of new vessels plays a critical role in a number of physiological and pathological events such as embryonic development, wound healing, arthritis, and tumor growth and metastasis (1-4). One area that has received considerable attention is determining the role that integrins play in angiogenesis. Recently, we demonstrated that integrin αvβ3 was highly expressed on angiogenic blood vessels associated with a variety of solid tumors and during granulation tissue formation (5,6). Importantly, little if any αvβ3 was detected on quiescent vessels (5,6). In addition, antagonists of αvβ3 were shown to specifically inhibit angiogenesis in vivo. These findings suggest an important role for integrin αvβ3 during neovascularization. Thus, a great deal of attention has been focused on understanding the cellular and molecular mechanisms by which integrins regulate angiogenesis. Angiogenesis can be defined as the process by which new blood vessels form from pre-existing vessels. This process is similar to, but not identical with vasculogenesis, in which new blood vessels arise from blood islands and precursor cells called angioblasts (1-3). Thus, an in-depth understanding of the molecules that regulate this complex biological process is of fundamental importance in both basic and clinical research. In fact, recent advances in our understanding of the molecular and biochemical events that facilitate angiogenesis have provided insight into cellular invasion in general as well as the development of novel strategies for the treatment of neovascular diseases (5,7-11). [ABSTRACT FROM AUTHOR]

Cell-migration events play a key role in embryonic development and during physiological and pathological tissue reorganization, for example in wound healing, angiogenesis, inflammation, and tumor metastasis. In embryonic development cells proliferate from germ layers and early organ anlagen such as neural folds or somites and migrate to distinct targets along defined tracks to invade other organs and to build up new tissues. In the adult organism, cells migrate to repair tissue defects, to provide new microvasculature, to support immune defense in inflammatory regions, and to metastasize as a result of malign transformation (1,2). The precision of cell migration, the mechanism of locomotion, the control of time, speed, direction, and target of the migrating cells has been receiving increasing interest in cell biology and molecular medicine (3,4). [ABSTRACT FROM AUTHOR]

We have found that integrins control apoptosis in epithelial cells, and have named this general phenomenon "anoikis," the ancient Greek word meaning "homelessness" (1). Epithelial cells that have lost interaction with their extracellular matrix—or that interact with matrix through inappropriate integrin types (2) or other molecules—cannot colonize elsewhere, because of anoikis. This process is developmentally critical, and its loss can contribute significantly to tumor malignancy (reviewed in ref. 3). [ABSTRACT FROM AUTHOR]

Invasion of extracellular matrices is crucial to a number of physiological and pathophysiological states, including tumor cell metastasis, arthritis, embryo implantation, wound healing, and early development. To isolate invasion from the additional complexities of these scenarios a number of in vitro invasion assays have been developed over the years. Early studies employed intact tissues, like denuded amniotic membrane (1) or embryonic chick heart fragments (2), however recently, purified matrix components or complex matrix extracts have been used to provide more uniform and often more rapid analyses (for examples, see the following integrin studies). Of course, the more holistic view of invasion offered in the earlier assays is valuable and cannot be fully reproduced in these more rapid assays, but advantages of reproducibility among replicates, ease of preparation and analysis, and overall high throughput favor the newer assays. In this chapter, we will focus on providing detailed protocols for Matrigel-based assays (Matrigel=reconstituted basement membrane; reviewed in ref. (3)). Matrigel is an extract from the transplantable Engelbreth-Holm-Swarm murine sarcoma that deposits a multilammelar basement membrane. Matrigel is available commercially (Becton Dickinson, Bedford, MA), and can be manipulated as a liquid at 4°C into a variety of different formats. Alternatively, cell culture inserts precoated with Matrigel can be purchased for even greater simplicity. [ABSTRACT FROM AUTHOR]

Transcription is the first step in the process of gene expression. Controlling where and when a gene is transcribed requires the interaction of cis-acting DNA control elements located near the gene, with trans-acting regulators, such as transcription factors and RNA polymerase. Identification of these cis regulating sequences called promoters and enhancers is a major key to understanding transcriptional regulation at the molecular level. [ABSTRACT FROM AUTHOR]

Gene ablation, also known as knockout, is a powerful method to analyze gene function in vivo. Many integrin genes have been disrupted already, confirming but also contradicting previous results (1). In addition, new functions have been revealed, significantly increasing our understanding of the biological roles of integrins. Knockout of the remaining integrins will similarly elucidate their function during development. In addition, knockins of subtle mutations (2,3) and conditional knockouts (4,5), resulting in tissue-specific or temporally restricted gene ablation, will allow us to assess structure-function relationships of integrins in vivo and to investigate the function of integrins in a specific cell type at a specific time point in development. [ABSTRACT FROM AUTHOR]

Integrins are an important family of cell-surface receptors that mediate adhesive interactions. These transmembrane glycoproteins are composed of noncovalently associated α- and β-subunit heterodimers. Twelve different α-chains and eight different β-subunits combine to give more than 20 distinct integrins and alternative splicing of the α- and β-chains increases the complexity of their interactions (1). Integrins have been shown to mediate cell adhesion to at least 12 separate matrix components and are also involved in cell-cell contacts via interactions with members of the immunoglobulin and cadherin families (2). Whereas cellular adhesion to specific components of the extracellular matrix (ECM) is an important function of integrin receptors, signals initiated by ligation of these receptors also modulate cell spreading, migration, proliferation, differentiation, and survival (reviewed in refs. 1 and 3). Specific function-blocking anti-integrin antibodies are invaluable tools with which to study the role of these receptors in mediating the effects of ECM on cellular processes. [ABSTRACT FROM AUTHOR]

Cell-extracellular matrix interactions are mediated predominantly by integrins, cell-surface receptors that exist as heterodimers of noncovalently associated α and β subunits (1). Integrins have been implicated in organ and tissue development, normal and aberrant cellular growth, and modulation of intracellular signal-transduction mechanisms (2-7). The expression of individual integrin subunits in mammalian systems has been instrumental in studies aimed at examining the structure and function of integrins and in analyzing the ability of integrins to modulate cellular functions and intracellular signaling (for review, seeref. 8). [ABSTRACT FROM AUTHOR]

The interaction of integrins with their ligands is an essential step in regulating many cellular functions (1). The β2 integrins, which are exclusively expressed on leukocytes, are of critical importance for leukocyte functions (2-5). The discovery of an inherited defect in leukocyte adhesion (called leukocyte adhesion deficiency, LAD), caused by the lack of expression of β2 integrins on the cell surface, underscored the important biological role of these receptors in the inflammatory and immune responses (6). [ABSTRACT FROM AUTHOR]

Integrins are a well-studied class of cell-surface receptors involved in cell-matrix interactions. All integrins are heterodimeric glycoproteins composed of an α and a (β chain. Integrins belong to a large family of proteins. Currently over 16 α chains and 9 β chains have been reported. The particular combination of α and β chains largely determines ligand specificity (1,2). [ABSTRACT FROM AUTHOR]

Flow cytometry is the rapid analysis of populations of cells on a cell-by-cell basis by passing them individually through a beam of laser light, and the measurement of light emitted from the cells by scatter or fluorescence (1,2). Integrins have been studied by flow cytometry since the mid-1980s when immunologists started examining CD11 (integrins αM, αX, and αL) as markers of T-cell differentiation in mixed cell populations (3,4). These analyses highlight many of the advantages and utility of flow cytometry over other forms of integrin analysis. With the appropriate antibody, flow cytometry can identify individual cells in a mixed cell population. Cell-surface expression of integrins can be differentiated from intracellular pools by flow cytometry analysis, unlike Western blotting or immunoprecipitation. Multicolor analysis can simultaneously measure the levels of two or three different integrins, enabling the researcher to determine that both an α and β subunits are present on the plasma membrane. Lastly, flow cytometry can sterilely sort viable cells on the basis of their cell-surface integrin expression and retain their viability for further growth and analysis. [ABSTRACT FROM AUTHOR]

SDS PAGE/immunoblotting is the method of choice for the qualitative and quantitative detection of specific integrins in a given sample. In the simplest case, the cells under investigation are lysed, the lysates separated by SDS PAGE, transferred onto a membrane, and the integrins are then identified by using specific anti-integrin antibodies. If the aim of an experiment is limited to the identification or quantification of a specific integrin subunit in a given sample SDS PAGE/immunoblotting of a cell lysate using specific antibodies yields sufficient information. The advantage of separating and blotting the protein extract onto a membrane compared to a simple detection of a protein of interest, e.g., in an ELISA assay is the additional information about the size of the protein that is detected. This is particularly important when it is not clear if the antibodies used for detection of the protein cross-react with other proteins in the cell lysate. [ABSTRACT FROM AUTHOR]

Cell adhesion to the extracellular matrix causes a number of intracellular changes resembling those induced by receptor protein tyrosine kinases, including increased tyrosine phosphorylation of a number of cellular proteins, elevation of phosphoinositide 3-OH kinase (PI3-K) products, and increase in intracellular calcium concentrations (1). Although integrins do not possess any intrinsic enzymatic activity, they influence the activity of cytoplasmic kinases either directly or indirectly. All β1- and αv-containing integrins can activate the tyrosine kinase focal adhesion kinase (FAK) by an incompletely understood mechanism involving the cytoplasmic domain of the β subunit. Upon activation FAK undergoes autophosphorylation at Tyr 397 and combines with the Src Homology 2 (SH2) domain of a src-family kinase, such as c-src or c-fyn. The FAK-bound src family kinase in turn phosphorylates the C-terminal domain of FAK promoting the recruitment of additional signaling molecules, such as Grb2 (2). Autophosphorylated FAK has also been shown to be able to associate with PI3-K (3). [ABSTRACT FROM AUTHOR]

Monoclonal antibodies (MAbs) have proved to be an important tool for identifying novel cell-adhesion molecules and for determining their ligand binding specificity, function, and structure. Consequently, they have been instrumental in defining three families of adhesion receptors: the cadherin, immunoglobulin, and integrin families. Integrins include over 20 adhesion receptors that react with the extracellular matrix or cell-surface molecules. Integrins are composed of two distinct transmembrane glycoprotein subunits, α and β, which are noncovalently linked to each other. At least 15 α chains and 8 β chains have been observed in several possible combinations that determine the ligand specificity and function of the complex (1). [ABSTRACT FROM AUTHOR]

Integrins are a family of cell-surface receptors that recognize extracellular matrix proteins and mediate cell adhesion. Certain integrins bind to proteins containing the sequence Arg-Gly-Asp (RGD; ref. 1). Such RGD-binding integrins include α5β1, αvβ3, αvβ5 and αIIbβ3 (2,3). Peptide libraries displayed on filamentous phage have provided a straightforward method to characterize the peptide-binding specificity of these integrins. In phage display-based strategies, libraries of random peptides are expressed on the surface of bacteriophage. Each individual phage carries a single peptide. The peptide is fused to the phage capsid protein pIII by insertion of its encoding DNA sequence in the pIII gene of the phage genome. Libraries displaying peptides fused to the major capsid protein pVIII have also been used. Libraries using the pIII protein display 3-5 copies of each individual peptide whereas libraries using the pVIII protein display approx 3000 copies (4,5). Analysis of the phage-displayed peptides found during screenings on purified integrins revealed that the majority of the integrin-binding peptides contain an RGD motif (6-11). Other interesting ligand motifs were also found in the remaining population of integrin-binding peptides. Conversely, panning on immobilized fibronectin fragments containing the RGD sequence revealed peptide motifs present within integrins (12). We describe here strategies for the use of phage display libraries in cell-adhesion research, with emphasis on the peptide-binding specificity of different members of the integrin family. [ABSTRACT FROM AUTHOR]