Adhesive interactions between cells and ECM (Extracellular Matrix) proteins play a vital role in biological processes, including cell survival, growth, differentiation, migration, inflammatory responses, platelet aggregation, tissue repair and tumor invasion (Ref.4) and perturbing this coordination can lead to events such as malignant transformation. The major groups of proteins mediating these interactions are a family of cell surface receptors known as Integrins, named for their role in integrating the intracellular cytoskeleton with the ECM. The signals from these adhesion receptors are integrated with those originating from Growth Factor Receptors in order to organize the cytoskeleton, stimulate cell proliferation and rescue cells from matrix detachment and induced programmed cell death (Ref.1). These functions are critical in the regulation of gene expression, tissue development, inflammation, angiogenesis, tumor cell growth and metastasis. Integrin receptors are composed of Alpha and Beta subunit molecules in close noncovalent association that form structural and functional bridges between the ECM and cytoskeletal linker proteins within a cell. These subunits form 22 different heterodimeric receptor complexes and despite this high degree of redundancy, most Integrins have specific biological functions (Ref.2).
Three signaling pathways activated by Integrin receptors are cytoskeletal organization, cell proliferation and cell survival pathways (Ref.2). Integrins do not themselves possess a kinase domain or enzymatic activity but rely on specific ECM ligands (such as fibronectin, laminins, various collagens, tenascin, vitronectin and thrombospondin) which interact with the actin cytoskeleton at focal adhesions on the cell surface containing localized concentrations of Integrins, signaling molecules, and cytoskeletal elements. Binding of proteins such as Actinin-Alpha and Talin to Integrin cytoplasmic tails, and the subsequent recruitment of the actin-binding protein Vinculin are important steps in linking adhesion complexes to the actin cytoskeleton (Ref.3). Integrin aggregation by ligand binding, results in the oligomerization of FAK (Focal Adhesion Kinase), which is mediated by Talin. Autophosphorylation of FAK at residue Tyr397 results in the binding of SH2 domain of Src and Fyn which phosphorylates a number of FAK-associated proteins including Paxillin, tensin and the docking protein p130CAS (Crk-Associated Substrate). Phosphorylation of Tyr397 also leads to the recruitment of other SH2-containing proteins, including the PI3K (Phosphotidyl Inositol-3Kinase), PLC-Gamma (Phospholipase-C-Gamma) and the adapter protein GRB7 (Ref.5).
Src can also phosphorylate FAK at tyrosine residue 925, creating a binding site for the growth-factor-receptor-bound protein complex, the GRB2-SOS complex (Ref.2) and activate another small G-Protein, Ras. Once activated by FAK or SHC, Ras activates PI3K and Raf to the cytoplasmic membrane. Activated Src also phosphorylates CAS, enabling it to bind Crk and DOCK180 (Dedicator of Cytokinesis 180), leading to an increase in the affinity of the membranes for Rac. Activated Rac, in conjunction with activated CDC42, regulate numerous biochemical pathways, including activation of MEKKs (MAPK/ ERK Kinase Kinase), PAK (p21-Activated Kinase), MEKs (MAPK/ERK Kinases), Vav, and JNK (c-Jun NH2-terminal kinase), also called SAPK (Stress-Activated Protein Kinase), the key regulators of gene expression and cell cycle. ERK in turn activates transcription factors such as SRF (Serum Response Factor) and c-Myc that are involved in regulating growth and differentiation. Engagement of Integrins linked to SHC activates transcription from the SRE (Serum Response Element) and promotes progression through the G1-phase of the cell cycle in response to growth factors. Since growth factors also stimulate the Ras-MAPK pathway, the Integrins and Growth Factor Receptors synergize to enhance Ras-MAPK activation and also promote cell migration on the ECM in a transcription-independent manner (Ref.2).
The interaction of Integrins with ECM is not sufficient to induce its clustering and focal complex formation, but requires the activity of a small G-protein RhoA, a member of the Ras superfamily. RhoA is a molecular switch that shuttles between a GTP-bound ‘active’ state and a GDP-bound ‘inactive’ state and is activated by a variety of mitotic stimuli including those for GPCR (G-Protein Coupled Receptor) and Cytokine Receptors. CD47 associates with the Integrin heterodimer to form a protein complex with seven transmembrane segments that mimics the action of GPCR. In the case when Integrins are mechanically stressed, the complex stimulates Gs-mediated up-regulation of the cAMP cascade through AC (Adenyl Cyclase), resulting in nuclear translocation of the catalytic subunit of PKA
(Protein Kinase-A). RhoA is important for the organization of stress fibers and also in the regulation of acto-myosin contractility through Myosin PPtase (Myosin Phosphatase), MLCP (Myosin Light Chain Phosphatase) phosphorylation and through the recently identified serine/threonine kinase ROCK (Rho-Associated Coiled-Coil Containing Protein Kinase) (Ref.3). Integrins stimulate the production of PIP2 (Phosphatidyl Inositol Biphosphate) and this effect is mediated by RhoA through its interaction with a Type I isoform of PIP4, 5K (Phosphatidyl Inositol-4-Phosphate-5-Kinase). The increase in PIP2 synthesis by RhoA is potentially relevant to focal adhesion assembly because the actin binding activity of several cytoskeletal proteins such as Profilin, gelsolin and Vinculin is modulated by PIP2 enriched in focal adhesion plaques. In addition, Rho-GTP also regulates Integrin-clustering by disrupting the Integrin clustering and focal plaque formation. PI3K is also associated with Integrin-associated focal adhesion complexes (Ref.2) and provides protective signal acting through Akt/PKB (Protein Kinase-B) which blocks entry into apoptosis.
Integrins function as nodes within webs of signaling, adhesive and cytoskeletal pathways and is also central to a number of specialized pathways in the hematopoietic systems, allowing attachment of platelets to soluble ligands, lymphocytes to Antigen Presenting Cells and the phagocytosis of complement opsonized targets by granulocytes and macrophages (Ref.2). But like all systems, greater complexity also affords greater opportunities for subversion. Aberrations in Integrin signaling contribute to many different disease states from cancer-cell metastasis, angiogenesis and inflammatory disease to arthritis (Ref.3). Recent studies on Integrins have greatly improved the understanding of the central biological phenomenon, such as anchorage-dependence and have also generated a number of therapeutic applications and targets for tumor therapy. Several Integrin-based chemotherapeutic drugs that specifically block individual Integrins are under development, which target thrombosis, osteoporosis and tumor-induced angiogenesis in neovascular endothelial cells.