Two-photon laser scanning microscopy, fluorescence spectroscopy, molecular biology, and biochemical approaches will be used to elucidate how 3-OH phosphoinositide regulate the intracellular behavior of Cytohesin-1, whose physiological role has been suggested to be the regulation of adhesion through its interaction with the beta2 subunit of integrin receptors in leukocytes. Integrins arbitrate communications between the cell and its external environment in regulation of many cellular functions, including embryonic development, tumor cell growth and metastasis, programmed cell death, and leukocyte homing. Altered regulation of cellular adhesion can also contribute to a number of disorders, among them rheumatoid arthritis, cardiovascular disease, and cancer. The physiological importance of leukocyte integrins is underscored by the finding that individuals lacking the beta2 subunit suffer from a severe syndrome, leukocyte adhesion deficiency, characterized by an inability to clear pathogens, recurrent infections, and frequently, death at an early age. The proposal consists of three specific aims: i) To determine if the Pleckstrin Homology (PH) domain of Cytohesin-1 is required to recruit Cytohesin-1 to a cellular membrane surface. ii) To determine if binding of the Cytohesin-I PH domain to a specific 3-OH phosphoinositide is sufficient to recruit Cytohesin-1 to a cellular membrane. iii) To determine whether binding of Cytohesin-1 to the plasma membrane is sufficient for it to co-localize with integrin beta2 and/or with ADP Ribosylation Factor. This project is centered on understanding how DE-phosphoinositide lipid second messengers may regulate the spatial and temporal localization of Cytohesin-1 under receptor control.
