Integrins are cell surface receptors that interact with the extracellular matrix. They mediate intracellular signals in response to the extracellular matrix including cellular shape, mobility, and progression through the cell cycle . The integrin family of proteins is 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 proteins . 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. Thermo Scientific™ has a wide range of products to help with integrin research.
While not possessing kinase domains, integrin receptors can activate a number of intracellular signaling pathways and targets 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 membrane .
In addition to actin polymerization/depolymerization, ligand binding to integrin receptors results in the Talin-mediated oligomerization of FAK (Focal Adhesion Kinase). The Tyr397 auto phosphorylated FAK binds and activates Src and Fyn which in turn phosphorylate the FAK-associated proteins paxillin, tensin and p130CAS. Furthermore, phosphorylated FAK has been shown to phosphorylate PI3L, PLCγ and GRB7 leading to their activation . 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, and forms 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 SFD (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 above .
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 pathway .
The fundamental role of cell–cell and cell–matrix adhesion in the morphology and development of organisms, organs and tissues has made identification of molecular mediators of cell adhesion an important research focus in cell biology and immunology. A number of different assays are available to detect cellular adhesion and the cellular processes influenced by integrin signaling mechanisms.
We offer antibodies, ELISAs, Luminex multiplex assays and growth factors for key targets in the integrin signaling pathway.
Featured below is western blot, immunofluorescence, and flow cytometry data using Thermo Scientific products.
Western blot analysis of Integrin β1 [pTpT788/789] (Product # 44-872G) was performed on extracts of serum-starved mitotic HeLa cells generated by treatment with 100 ng/mL taxol for 16 hours. The samples were run on a 10% tris-glycine gel. Then, proteins were transferred to a PVDF membrane.
Flow cytometry analysis of CD18 in PBMC cells (green) compared to an isotype control (blue). Human blood was collected, combined with a hydrophilic polysaccharide, centrifuged, transferred to a conical tube and washed with PBS. 50 µL of cell solution was added to each tube at a dilution of 2x10^7 cells/mL, followed by the addition of 50 µL of isotype control and primary antibody (Cat. No. MA1810) at a dilution of 0.5 µg/test. Cells were incubated for 30 min at 4°C and washed with a cell buffer, followed by incubation with a DyLight 488-conjugated goat anti-mouse IgG (H+L) secondary for 30 min at 4°C in the dark. FACS analysis was performed using 400 µL of cell buffer.
Immunofluorescent analysis of CD18 (green) showing staining in the cytoplasm of Hela cells (right) compared to a negative control without primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a integrin beta-2/CD18 monoclonal antibody (Cat. No. MA1810) in 3% BSA-PBS at a dilution of 1:20 and incubated overnight at 4 °C in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a flourescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.
|Product Name||Catalog Number|
|Phospho-FAK pTyr397 Antibody||44624G|
|Phospho-SRC pTyr418 Antibody||44660G|
|Phospho-JNK (SAPK) pThr183/pTyr185 Antibody||44682G|
|Caveolin 1 Antibody||PA1064|
|Phospho-Paxillin pTyr118 Antibody||44722G|
|Phospho-PAK1/2/3 pSer141 Antibody||44940G|
|Phospho-PI3K p85 pTyr458+p55 pTyr199 Antibody||PA517387|
|Collagen I Antibody||PA126204|
|alpha Actinin 4 Antibody||MA1800|
|Phospho-CrkL pTyr207 Antibody||PA517741|