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. This year one PhD student, one masters student and a full time technician joined the lab as well as a honors undergraduate student. They have each successfully competed for INBRE funding and my PhD student is applying for external fellowships. This and purchase of an additional thermocycler increased lab productivity considerably allowing us to gather enough data to clarify and focus the CAV1/AhR research. Analyses of approximately 30 Hepa1c1c7 clones by RTPCR allowed us to identify a series of clones with increasing levels of CAV1 expression and determine that AhR-dependent gene expression (i.e., CYP1A1 induction) was enhanced by low level CAV1 expression. This effect reached a maximum in clones expressing moderate levels of CAV1 and then AhR-dependent gene expression began to decrease with additional CAV1 expression creating a bell shaped AhR-dependent gene expression curve with increasing CAV1. Immunofluorescence microscopy revealed CAV1 localization to the cytoplasm at low expression levels which switched to predominantly plasma membrane localization in high CAV1 expressing clones. This reconciles all our preliminary data and is consistent with recently published data indicating that increasing CAV1 protein results in formation of high molecular weight oligomers. As CAV1 oligomerizes in vivo, it is transported from cytosolic pools to the plasma membrane. We have evaluated endogenous CAV1 expression and localization in three additional cell lines. These results are now part of a R15 proposal being submitted May 05 and should be ready for publication late 2005. The additional lab personnel have also allowed for significant development on two additional projects that should be ready for funding/publication in 2006. As the preliminary data allows for more clarified hypotheses, I am working with NIH program staff to identify and target my proposals to appropriate funding mechanisms.
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