Integrins are cell surface receptors that interact with the extracellular matrix and mediate intracellular signals in response to the extracellular matrix including cellular shape, mobility, and progression through the cell cycle1. The integrin family of proteins are the major cell surface receptors involved in mediating cellular response to ECM binding. Composed of Alpha and Beta subunits, Integrin receptors form structural and functional linkages between the ECM and intracellular cytoskeletal linker proteins2. Signaling mediated from Intergrin/ECM interactions are also integrated with cellular responses to growth factor signaling to regulate cellular proliferation, cytoskeletal reorganization, and other responses necessary for cellular survival.
While not possessing kinase domains, Integrin receptors can activate a number of intracellular signaling pathways following ECM adhesive interactions. Within the ECM, Integrins have the ability to bind fibronectin, laminins, collagens, tenascin, vitronectin, and thrombospondin. Clusters of integrin/ECM interactions form focal adhesions, concentrating cytoskeletal components and signaling molecules within the cell. The cytoplasmic tail of integrins serve as a binding site for α-actinin and Talin which then recruit vinculin, a protein involved in anchoring F-actin to the membrane3. In addition to actin polymerization/ depolymerization, ligand binding to integrin receptors results in the Talin-mediated oligomerization of FAK (Focal Adhesion Kinase). The Tyr397 autophosphoryated FAK binds and activates Src and Fyn which in turn phosphorylate the FAK-associated proteins paxillin, tensin, and p130CAS. In addition phosphorylated FAK has been shown to phosphorylate PI3K, PLCγ, and GRB7 leading to their activation4. Activation of PI3K links integrin activation with the Akt signaling pathway for activation of cell survival mechanisms.
Phosphorylation of FAK at Tyr925 occurs by Src, thereby forming a complex with GRB2 and SOS, leading to the activation of Ras. Ras can function to activate numerous kinases including MEKKs, PAKs, MEKs, JNK, and SAPK. These kinases are key regulators of gene expression via the phosphorylation of multiple transcription factors including c-Myc, Elk1, Jun, and SRF (Serum Response Factor). Activated Src also phosphorylates p180CAS promoting the formation of a protein complex with Crk and DOCK180. This protein complex increases the membrane affinity for Rac, leading to further activation of the kinase pathways mentioned above2. Another signaling pathway utilized by integrin for MAPK activation is via integrin association with Caveolins. Caveolins are small membrane proteins (22 kD) that can associate to form high molecular mass proteins. Caveolin proteins contain a hydrophobic central region which allows embedding into the plasma membrane with cytoplasmic N- and C- terminal domains. Caveolin-1 (Cav-1) associates with integrins and Fyn (Src-related kinase), where Cav-1 mediates the phosphorylation of Shc by Fyn. Phosphorylated Shc serves as a binding site for Grb2 and SOS and further activation of Ras and the MAPK pathway5.
Collagen is a major component of the extracellular matrix and, in vertebrates, constitutes approximately 25% of total cellular protein. Fluorescent collagen conjugates (G13187, G13186, C13185) allow the visualization of the interactions between collagen and proteins involved in cell–cell and cell–surface adhesion.7 Recent research has demonstrated that Src family kinase activation occurs in lipid rafts8. The Vybrant® Lipid Raft Labeling Kits (V34403, V34404, V34405) are designed to provide convenient, reliable and extremely bright fluorescent labeling of lipid rafts in live cells.
Proteases have been used as a tool to investigate the role of surface molecules in fibronectin-mediated cell adhesion.9 Two EnzChek® Protease Assay Kits (E6638, E6639) measure the increase in fluorescence intensity that results from protease hydrolysis of a heavily labeled casein derivative. The EnzChek® Gelatinase/Collagenase Assay Kit (E12055) and EnzChek® Elastase Assay Kit (E12056), which use DQ® gelatin (D12060, D12052) or DQ® elastin as substrates, provide the speed, high sensitivity and convenience required for measuring gelatinase (collagenase) or elastase activity
The fungal metabolite brefeldin A (BFA) has proven valuable for dissecting the cellular processes, including vesicle formation10 and kinesin distribution,11 involved in exporting newly synthesized proteins.12 The green-fluorescent BODIPY® FL and red-orange–fluorescent BODIPY® 558/568 BFA derivatives (B7447, B7449) are selectively localized in the ER and Golgi apparatus in four different cell lines.13 BODIPY® 558/568 BFA may be used in conjunction with NBD C6-ceramide (N1154) to investigate the ER and Golgi apparatus simultaneously.
1. Ojaniemi, M. et. al. (1997) J. Biol. Chem. 272: 25993-8.
2. Kumar, C.C. (1998) Oncogene 17: 1365-73.
3. Martin, K.H. (2002) Science 296: 1652-3.
4. Parsons, J.T. (2000) Oncogene 19: 5606-13.
5. Salanueva, I.J. (2007) J. Cell. Mol. Med. 5: 969-980
6. Braut-Boucher, F. et. al. (1995) J Immunol Methods 178: 41-51
7. Lu, ML, et al. (1992) J Cell Sci 101: 873-83
8. Hitosugi, T. (2007) Cancer Research 67: 8139-8148
9. Tarone, et. al. (1982) J. Cell Biol. 94:179-186
10. Veeit, B., et al. (1993) J Cell Biol. 122: 1197-1206
11. Lippincott-Schwartz, J. et al. (1995) J Cell Biol 128: 293-306
12. Misumi, Y. et al. (1986) J Biol Chem 261: 11398-11403
13. Deng, Y. et al. (1995) J Histochem Cytochem 43: 907-915.
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