3385_Ch01 - © 1999 by CRC Press LLC Section I...

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Unformatted text preview: © 1999 by CRC Press LLC Section I Protein-Protein Interactions and Early Events in Integrin Signaling 1 © 1999 by CRC Press LLC Chapter The Use of Chimeric Receptors in the Study of Integrin Signaling Anthony M. Mastrangelo, Amy L. Bodeau, Suzanne M. Homan, Allison L. Berrier, and Susan E. LaFlamme Contents I. Introduction II. Activation of Tyrosine Phosphorylation A. Overview B. Protocols C. Materials D. Buffers III. Inhibition of Cell Attachment A. Overview B. Protocols C. Materials IV. Inhibition of Cell Spreading A. Overview B. Protocols C. Materials D. Buffers . V. Concluding Remarks. Acknowledgments References © 1999 by CRC Press LLC I. Introduction Integrins mediate the bidirectional transfer of signals across the plasma membrane. These signals regulate cell adhesion as well as adhesion-dependent aspects of cell behavior including cell proliferation, survival, and differentiation. 1-3 Integrin β cyto- plasmic domains function in all steps of the adhesion process, including cell attach- ment, cell spreading, the formation of focal adhesions, and cell migration. 4,5 They are thought to function in these processes by interacting with specific cytoplasmic proteins, thereby connecting integrins with the cell’s cytoskeletal and signal trans- duction systems. Although several cytoplasmic proteins have been demonstrated to bind to β cytoplasmic domains, their roles in regulating integrin function have not yet been clearly defined. 2,5 To study the function of β cytoplasmic tails and the molecular mechanisms involved, we constructed chimeric receptors containing wild-type and mutant inte- grin β subunit cytoplasmic tails connected to the extracellular and transmembrane domain of the human interleukin-2 (IL-2) receptor which functions merely as a reporter domain ( Figure1 ). Clustering these chimeric receptors on the cell surface can activate signaling pathways by mechanisms similar to endogenous integrins. 6 Therefore, these chimeric receptors can be used to identify and analyze signaling events triggered by integrin β cytoplasmic domains. Additionally, these chimeric receptors can function as dominant inhibitors of endogenous integrin function in a variety of processes including cell attachment and spreading, fibronectin matrix assembly, fibronectin-mediated phagocytosis, and high-affinity ligand binding. 7-11 Identifying the mechanisms and protein interactions involved in these dominant negative effects will provide important insights into the role of β cytoplasmic domains in regulating integrin function. FIGURE1 Chimeric receptors. Chimeric receptors consist of the extracellular and transmembrane (TM) domains of the tac subunit of the IL-2 receptor 32 connected to various integrin β cytoplasmic domains. The amino acid sequences of the wild-type β 1 and β 3 cytoplasmic domains are shown. The control receptor (C) consists of the extracellular and transmembrane domains of the IL-2 receptor connected to an intracellular lysine (K) residue. © 1999 by CRC Press LLC...
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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_Ch01 - © 1999 by CRC Press LLC Section I...

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