Harry Hines Division of Toxinology, USAMRIID, Fort Detrick, MD Office: 301-619-4241 FAX: E-mail: harry.hines@det.amedd.army.mil Job Title: Principal Investigator Ph.D. in Biophysical Sciences from the University of Houston

Speaker: Harry Hines, Division of Toxinology, United States Army Medical Research Institute for Infectious Diseases (USAMRIID), Fort Detrick, Frederick, MD

Topic: Proteomics of PC12 Lipid Rafts

Place: Building 549, Executive Board Room, NCI at Frederick, Frederick, MD

Time: Tuesday, February 15, 2005, at 2:00 PM

Abstract: Detergent-resistant microdomains/membranes (DRMs), also known as lipid rafts or detergent-insoluble glycolipid-rich membranes (DIGs), are biochemically defined regions of the cell membrane enriched in cholesterol, certain lipids, glycosylphosphatidylinositol (GPI)-linked proteins, and some integral, membrane-spanning proteins that are resistant to solublization by non-ionic detergents such as Triton X-100 at 4oC. The in vivo nature and exact function of DRMs remains controversial, but it is known that concentrating certain lipids in these microdomains also seems to concentrate certain proteins, especially those involved in cell signaling processes. In addition, lipid rafts may also act as a cellular portal, linked to the endocytotic pathway, that provides a mechanism to control the entry of beneficial macromolecules and particles into the cell. This portal can also be exploited by macromolecules such as protein toxins and particles such as bacteria and viruses that apparently capitalize upon the concentration of signaling molecules in DRMs to facilitate cell entry.

PC-12 (rat pheochromocytoma) cells derive from the neural crest and are commonly used as a model system to study neuronal cell differentiation and certain neuronal cell functions such as neurotransmitter release. NGF-induced differentiation transforms PC-12 cells into cells that possess sympathetic neuron characteristics. NGF stimulation of PC-12 cells arrests growth in the G1 phase of the cell cycle, promotes neurite formation, promotes the formation of synaptic-like vesicles, and induces electrical excitability. With these changes, DRM protein composition may also change with differentiation, which may affect its function as a cellular portal for particle and macromolecule entry. For example, the binding of clostridial toxins to PC12 cells is enhanced after NGF differentiation. Therefore, identifying changes in DRM protein composition may provide insights into changes in function that accompanies PC-12 cell differentiation and its influence on endocytosis.

In this study, we used one-dimensional SDS-PAGE for protein separation and nLC-nESI/MS/MS to identify proteins co-isolated with PC-12 cell DRMs before and after treatment with NGF. Proteins were assigned to three categories: proteins present in DRMs from treated and untreated cells (NGF+/-), proteins present in DRMs from untreated cells (NGF-), and proteins present in DRMs from NGF-treated cells (NGF+). Generally, 35 proteins were assigned to NGF+, 40 proteins were assigned to NGF-, and 75 proteins were assigned to NGF+/-, giving a total of 150 identified proteins. In addition to proteins typically described as plasmalemma proteins, proteins assigned to mitochondria, endoplasmic reticulum, and vesicles were detected also.