Actin-membrane interactions are involved in neuronal outgrowth, immune cell function, wound healing, and the spread of metastatic cancer. However, little is known about the integral membrane proteins that mediate this interaction in ambeboid cells, e.g., Neutrophils and metastatic cancer cells. Ponticulin, an integral membrane protein that binds directly to F-actin and nucleates assembly is the major high affinity link between the plasma membrane and the actin cytoskeleton in the model system D. discoideum. The specific goals of the proposed research are: (1) To construct and express a genetically engineered synthetic ponticulin gene, ponS; (2) To identify the F-actin binding site in ponticulin by site- directed mutagenesis of ponsS, coupled with analysis of mutant ponticulin proteins expressed in D. discoideum; (3) To identify disulfide bridges that stabilize the multipartite F-actin binding site by MALDI TOF mass spectroscopy and site directed mutagenesis (4) To investigate the topography of ponticulin by insertional epitope scanning mutagenesis and alanine insertional mutagenesis. The ultimate goals of this proposed research are two fold. First, to understand at the molecular level the relationship between the structure and function of an F-actin binding integral membrane protein. This information will increase our understanding of the mechanism by which the actin-based cortical cytoskeletons interacts with the plasma membrane in normal motile processes, e.g., chemotaxis and phagocytosis. Second, to begin to understand the structure of a beta-strand/barrel transmembrane protein. Although it has been estimated that 40% if gene products are membrane associated, only a few types of membrane protein structure have been determined. Information gained by these studies will increase our understanding of an entire class of membrane protein. Information gained about the structure-function relationship of ponticulin also may have implications for immune cell disorders and cancer metastasis.
| Hitt, Anne L; Iijima-Shimizu, Miho; DuBay, Mary J et al. (2003) Identification of a second member of the ponticulin gene family and its differential expression pattern. Biochim Biophys Acta 1628:79-87 |
| Hitt, Anne L; Laing, Suzette D; Olson, Stacey (2002) Development of a fluorescent F-actin blot overlay assay for detection of F-actin binding proteins. Anal Biochem 310:67-71 |
