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Unformatted text preview: 7 © 1999 by CRC Press LLC Chapter Specificity of Integrin Signaling Kishore K. Wary, Agnese Mariotti, and Filippo G. Giancotti Contents I. Introduction II. Experimental Overview III. Coimmunoprecipitation of Integrins with Caveolin-1 and Caveolin-1 with Fyn A. Protocol B. Reagents IV. Integrin Ligation A. Crosslinking of Integrins with Soluble Antibodies B. Ligation of Integrins with Antibody-Coated Polystyrene Beads C. Adhesion to Anti-Integrin Antibody- or ECM-Coated Dishes D. Reagents V. Coimmunoprecipitation of Integrins and Caveolin-1 with Shc A. Protocol B. Reagents VI. Caveolin-1 Associated c-Fyn Tyrosine Kinase Assay A. Protocol B. Reagents VII. Coimmunoprecipitation of Fyn with Shc Following Integrin Ligation A. Protocol B. Reagents VIII. Tyrosine Phosphorylation of Shc and Recruitment of Grb-2 A. Protocol B. Reagents © 1999 by CRC Press LLC IX. Association of Shc GST-Fusion Proteins with Tyrosine Phosphorylated β 4 A. Protocol B. Reagents Acknowledgments References I. Introduction The binding of integrins to extracellular matrix (ECM) proteins promotes their aggregation on the plane of the plasma membrane and their interaction with cytoskeletal elements as well as signaling molecules. These events lead to the assembly of focal adhesions and the activation of signaling pathways which regulate gene expression. 1,2 Ligation of all β 1 and α v-containing integrins results in the recruitment and activation of Focal Adhesion Kinase (FAK). This process appears to involve the interaction of FAK with talin, since it requires the portion of β1 cytoplasmic domain which binds talin. Upon activation and autophosphorylation, FAK combines with a Src-family kinase, c-Src or c-Fyn, which phosphorylates its C-terminal domain promoting the recruitment of other signaling molecules. The signaling pathways activated by the FAK-Src family kinase complex appear to play a role in regulating the assembly/disassembly of focal adhesions during cell migra- tion, regulating cell proliferation, and protecting cells from programmed cell death. 3 We have recently provided evidence that ligation of α 1 β 1, α 5 β 1, α v β 3, and α 6 β 4, but not α 2 β 1, α 3 β 1, and α 6 β 1, causes recruitment and tyrosine phosphory- lation of Shc. 4,5 Shc is an adaptor protein containing a Src Homology-2 (SH2), a phosphotyrosine binding (PTB), and a collagen homology domain. Shc is known to link various tyrosine kinases to Ras . Upon recruitment to activated tyrosine phos- phorylated signal transducers via the SH2 and/or PTB domain, Shc is phosphorylated on tyrosine and binds the Grb2/mSOS complex . This process results in the recruit- ment of mSOS to the plasma membrane and consequent activation of Ras. We have observed that the α 6 β 4 integrin is associated with a cytoplasmic tyrosine kinase that phosphorylates the cytoplasmic domain of β 4. Since the SH2 and the PTB domain of Shc can interact directly in vitro with distinct tyrosine phosphorylated residues on the...
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This note was uploaded on 05/06/2010 for the course MECH. 28197 taught by Professor Dr.shafii during the Spring '10 term at Sharif University of Technology.
- Spring '10