3385_Ch16 - Chapter 16 Expression Cloning of Proteins That...

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16 © 1999 by CRC Press LLC Chapter Expression Cloning of Proteins That Modify Integrin Activation Csilla A. Fenczik, Joe W. Ramos, and Mark H. Ginsberg Contents I. Introduction II. General Considerations III. Specific Protocols A. Cell Sort B. Hirt Supernatants C. Analysis of Clones D. Materials and Equipment Acknowledgments References I. Introduction Integrins transmit information in both directions across the plasma membrane. Inte- grin affinity for ligands (“activation”) can be modulated by intracellular signals (inside-out signaling). 1 Conversely, integrin ligand-binding can modify cell shape, growth, survival, and gene expression (outside-in signaling). 2 The expression cloning strategy we describe uses inside-out signaling as a selective marker. The dynamic regulation of integrin adhesion by inside-out signaling is a crucial aspect of integrin function that is important in hemostasis, leukocyte extravasation, cell migration, and fibronectin matrix assembly. Inside-out signaling is energy dependent, cell-type
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© 1999 by CRC Press LLC specific, and requires the cytoplasmic domains of both the α and β subunits. 3 The proteins that mediate these cytoplasmic signals have not been fully defined. One approach to identifying the proteins involved in inside-out signaling is to find proteins that directly interact with the integrin cytoplasmic domains. This has been done using both biochemical approaches and yeast two-hybrid screens. Several proteins have been shown to interact with integrin cytoplasmic domains by one or both of these methods including talin, α -actinin, filamin, β 3 -endonexin, and Rack1. 4-9 However, the functional relevance of these proteins in regulating inside-out integrin signaling remains unclear. Genetic analysis has been a successful method for mapping intracellular signal transduction pathways in vivo. 10 In general, these approaches have involved the isolation and characterization of mutants that perturb functions by disrupting sig- naling cascades. Recently, methods have been developed to isolate proteins that complement overexpressed signaling mutants in cultured cells. 11-13 We have used concepts from these techniques to develop expression cloning strategies to identify potential integrin regulatory proteins in a cell culture system. The advantage of this approach is that it does not rely on a physical interaction between integrins and possible regulatory molecules, but rather on a functional relationship. A second important advantage is that all proteins expressed by the library are tested without inherent bias. The major disadvantage is that there are certain functional interactions that are not likely to be uncovered by such an approach. For instance, if a multi- protein complex is required for the rescue of integrin suppression, it may be impos- sible to identify it by this method. We have developed two screens to isolate proteins that regulate integrin acti-
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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.

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3385_Ch16 - Chapter 16 Expression Cloning of Proteins That...

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