Cell interactions with extracellular matrix components play important roles in a number of biological processes such as embryonic development, wound healing, and malignant transformation. Our long-term goal is to how the associations of cells with extracellular matrix molecules can stimulate changes in cell morphology, locomotion, growth properties, and differentiation state. The mechanism by which receptor engagement at an adhesive membrane triggers behavioral responses by cells is not understood. However, it is likely that at least some of the proteins involved in transmission of signals to the interior of the cells are concentrated at the cytoplasmic face of the adhesive membrane. Two interactive proteins that have emerged as candidates for participation in signal transduction events at sites of membrane-substratum contact are zyxin and the cysteine rich protein, CRP. Both zyxin and CRP exhibit LIM domains, zinc-binding sequences that are also found in a number of transcription factors and a protooncogene product. We postulate that the LIM domain serves as a protein-binding interface and that the cytoskeletal LIM proteins mediate the assembly of a signal transduction machine at adhesive membranes. Biophysical, biochemical, molecular and cell biological strategies will be employed to probe the structure and function of zyxin and CRP. Spectroscopic approaches and site-directed mutagenesis will be utilized to examine the structural features of the LIM domain. The amino acid residues that participate in zinc coordination will be defined and the importance of the metal for the structure and function of the LIM domain will be evaluated. The results from these experiments will have general impact on our understanding of the properties of the LIM domain, a sequence motif found in a number of proteins involved in signal transduction. Mapping studies will be performed to characterize the associations between zyxin and its two known protein partners, alpha-actinin and CRP and to define the sequences in zyxin and CRP that target them for association with the adhesion plaque and the actin cytoskeleton. An interaction cloning approach will be employed in an effort to define the full complement of zyxin-binding partners. To probe the functions of zyxin and CRP in living cells, the effects of elimination or over-expression of zyxin or CRP sequences on cell behavior will be examined. Finally, an evolutionary comparison of avian, murine and Drosophila zyxin and CRP sequences will highlight the highly conserved and, by implication, functionally important regions of the two proteins. These studies will also lay the foundation for future molecular and genetic analyses of zyxin and CRP function. The broad-based approach proposed in this application will provide insight into the structure and function of the LIM domain as well as the two cytoskeletal proteins that display this zinc-binding motif.
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