GTP binding proteins (GTPases, G proteins) mediate a large number of important cellular processes. This family of proteins can serve as biological switches, turning signaling processes on or off in response to incoming extracellular signals. Their switching capability arises from conformational changes in the molecule in response to binding GTP or GDP. Exchange of bound GDP for GTP, in response for example to growth factor binding, G proteins become activated, triggering a cascade of events leading to cell shape changes or cell division. Spontaneously or catalyzed by the interaction with regulatory proteins, the G protein hydrolyzes bound GDP to GTP, thus resetting the system to the nonsignaling state. A wide variety of cancers are associated with mutations in G proteins (the best known examples are the mutations of the H-Ras protein) or proteins which regulate G proteins (e.g., the Dbl oncoprotein). The focus of this project is the three dimensional solution structure of Arf6, a GTP binding protein related to H-Ras. Arf signaling has been implicated in cell shape alterations associated with cell migration, differentiation and metastasis. Also, the abnormalities in lipid metabolism seen in breast and ovarian cancer are associated with Phospholipase D, which is activated by the Arf proteins. The Arf proteins have an additional signaling mechanism which involves the myristylation of the C-terminus. In the GDP-bound form, the myristate is presumably associated with the protein; whereas, associated with activation by exchange of GTP for GDP, the lipid group is inserted into the membrane. We intend to use NMR spectroscopy to determine the solution structure and backbone dynamics of Arf6 in its GDP-bound and GMPPCP-bound (GMPPCP is a stable analog of GTP) forms as well as in its myristylated and unmyristylated forms. The second part of the project will attempt to determine the orientation of the protein with respect to the membrane using lipid vesicles. The key to this project is the production of myristylated Arf6 in bacteria by coexpressing Arf6 and N-myristyl transferase. This has led to the production of high levels of myristylated protein that provides a unique opportunity to study the structure of a Ras-like G protein in its native, lipid-modified state. Because of the importance of the myristyl switch in Arf function, the orientation of the lipid and the structure of the myristylated forms of the protein will be of considerable interest to the design of therapeutic agents directed toward these important signaling molecules.
| Afroze, Syeda H; Uddin, M Nasir; Cao, Xiaobo et al. (2011) Internalization of exogenous ADP-ribosylation factor 6 (Arf6) proteins into cells. Mol Cell Biochem 354:291-9 |
| Gizachew, Dawit; Oswald, Robert (2006) NMR structural studies of the myristoylated N-terminus of ADP ribosylation factor 6 (Arf6). FEBS Lett 580:4296-301 |
