This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Directed cell migration is essential for spatial ordering of cells during embryogenesis and development. The goal of this research is to determine what role motion and flexibility play in the ability of proteins to recognize and bind specifically to other proteins in cell migration signaling. The chosen model system for this study is the Mig10 protein of the nematode C. elegans. C. elegans is a well characterized system in which the goal of understanding cell migration at the molecular level is attainable. Through genetic analysis the gene Mig10 has been shown to be required for the long range anterior to posterior migration of embryonic neurons and the proper development of the excretory canals. A Mig10 mutation causing severe truncation of the Mig10 protein results in several abnormal phenotypes. The phenotypes include Wit (withered-tail), Egl (egg-laying-defective), Unc (uncoordinated) and protrusion of the vulva. The protein coded by the gene (a.k.a. Mig10) shares a central multidomain region of sequence, termed GM (Grbs and Mig), with the Grb7 protein family. Through homology analysis a Ras-Associating (RA) domain and a Pleckstrin Homology (PH) domain have been identified in the central GM region of the Mig10 and Grb7 family proteins. Adjacent RA and PH domains are also found in other proteins, for example lamellipodin and RIAM (both involved in cytoskeletal rearrangement) and beta-amyloid precursor protein-binding protein, RARP1 (function is unknown). Detailed information describing the function of the Mig10 GM region is not yet existent although some information describing the role of these domains in the Grb7 protein is available. In the Grb7 protein both the PH domain and the RA domain are required for cell migration related to Grb7 signaling. Both the RA and PH domains have eluded easy functional classification, in that the RA domain has not been shown to bind G-proteins, and the PH domain may be a member of a growing group of PH domains that have dual function, binding both phospholipids and proteins. It is possible that the RA and PH domains work in concert, or that prior phospholipid binding is necessary for their activation. Most biochemical methods would be unable to identify function of these domains if that is the case. A combination of structural and dynamic information may the only approach that can elucidate the function of these mysterious domains. The goals of this proposal are: 1) Expression and purification of the Mig10 Pleckstrin Homology and Ras-Associating-like protein domains in quantities sufficient for structure determination, 2) Obtaining the solution structures of both the Mig10-PH and Mig10-RA domains, and 3) Measure the nuclear relaxation behavior of both the PH and RA domains in an effort to relate flexibility and dynamic movement to the location of potential binding sites and ability to bind specific ligands. The accuracy of binding site prediction will then be tested through mutagenesis analysis in C. elegans.
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