3385_Ch02 - Chapter 2 Using Synthetic Peptides to Mimic...

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2 © 1999 by CRC Press LLC Chapter Using Synthetic Peptides to Mimic Integrins: Probing Cytoskeletal Interactions and Signaling Pathways Carol A. Otey Contents I. Introduction II. Peptides in Affinity Chromatography A. Overview B. Protocol III. Small-Scale Peptide Resin Pull-Down Experiments A. Overview B. Protocol IV. Peptides in Microtiter-Well Binding Assays A. Overview B. Protocol V. Peptides in Single-Cell Microinjection A. Overview B. Protocol VI. Peptides on Pins and on Paper VII. Materials and Instruments Acknowledgments References
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© 1999 by CRC Press LLC I. Introduction Integrins are transmembrane proteins, with the largest portion of the molecule oriented to the extracellular side of the membrane. This creates a challenge for researchers whose interests are focused on the cytoplasmic domains, as this portion of the integrin molecule is relatively quite small. Numerous approaches have been used to probe the function of integrin cytoplasmic domains in different experimental systems, and several of these methods are discussed elsewhere in this volume. Fusion proteins of various types have been used to mimic integrin tails, but the fusion partners can create problems of non-specific binding in certain types of assays. To make a more “pure” integrin cytoplasmic domain, synthetic peptides can be utilized. Synthetic peptides have been used to identify novel cytoskeletal binding partners for integrin cytoplasmic domains, to map the binding sites for integrin-associated proteins, to estimate the affinity of these binding interactions, and even to probe the functions of integrin binding partners in living cells. The following sections describe methods for using synthetic peptides in a variety of solid-phase binding assays, and in single-cell microinjection experiments. II. Peptides in Affinity Chromatography A. Overview Many important functions of integrins are mediated by proteins that bind to the short cytoplasmic domains of the two integrin subunits. These cytoplasmic domains serve to physically connect integrins to the actin cytoskeleton, forming an important mechanical linkage between the outside of the cell and the inside. In addition, integrin cytoplasmic domains interact with catalytic binding partners to initiate outside-in signaling pathways. It is likely that cytoplasmic binding partners are also involved in regulating the affinity of integrins through the mechanism known as inside-out signaling. One way of identifying novel cytoplasmic binding partners for a specific integrin subunit is to use synthetic peptides in an affinity chromatography column, to “fish out” proteins that bind specifically and with high affinity to a particular integrin tail. The integrin-derived peptides are immobilized on a column matrix, and lysates of cultured cells or tissues are passed over the column. After the column is washed extensively, any bound proteins are eluted and further character- ized. This technique is a good choice for making a preliminary investigation into
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3385_Ch02 - Chapter 2 Using Synthetic Peptides to Mimic...

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