Research Summary

My laboratory utilizes biochemical, biophysical, molecular, and proteomic approaches to study cell signaling pathways. We use human fibroblasts grown in cell culture as one model cell system to investigate early signaling events that are initiated when cells are stimulated with mitogens. We also use HEK-293 cells for studies where we introduce exogenous genes or siRNA constructs against endogenous genes, or, in some cases, fibroblasts derived from transgenic mice to evaluate the role of selected proteins in cell growth control. We also work in a cultured hippocampal cell system (H19-7 cells) when investigating questions pertaining to excitable cells. There are basically two areas of interest in our laboratory: 1) the mechanism for regulation of intracellular Ca²⁺ in response to mitogen stimulation and the downstream events regulated by Ca²⁺ entry and 2) the use of proteomics to elucidate tyrosine phosphorylation pathways downstream of the bradykinin (BK) receptor. 

For the project on Ca²⁺ regulation, we are utilizing Ca²⁺-sensitive fluorescence indicators combined with sophisticated image analysis techniques to monitor mitogen-induced Ca²⁺ changes in cultured cells. We are particularly interested in the mechanism for stimulation of Ca²⁺ entry via either receptor-operated channels or store-operated channels. The latter are plasma membrane channels whose activity are stimulated in response to depletion of intracellular Ca²⁺ stores, a process that seems to utilize unique signaling pathways. We have recently provided evidence for the involvement of tyrosine kinase activity in the regulation of Ca²⁺ entry via store-operated channels. We have demonstrated a role for c-src in the regulation of Ca²⁺ entry based on studies utilizing transfection techniques to overexpress c-src, as well as fibroblasts derived from c-src knockout transgenic mice. We are utilizing biochemical and molecular approaches to identify which targets of c-src are important in the regulation of store-operated channels. 

On another front, we are trying to identify the proteins responsible for forming the store-operated  and receptor-operated Ca²⁺ channels.  We used RT-PCR methods to screen for expression of mammalian homologs of Drosophila Trp (the gene hypothesized to code for store-operated Ca²⁺ channels in Drosophila) in HEK-293 or H19-7 cell. These cells express up to six of the 7 TRPC (a subfamily of Trp genes) proteins identified. To date, we have made constructs which express hairpin siRNA specific for individual TRPC homologs and we stably express these constructs in HEK-293 or H19-7 cells to selectively suppress one or more TRPC homologs to evaluate the role of these proteins in both store-operated and carbachol-stimulated Ca²⁺ entry. We have demonstrated that TRPC1 and TRPC3 are involved in mediating store-operated Ca²⁺ entry in both cell types, and TRPC7 is involved in HEK-293 cells but not in H19-7 cells. On the other hand, TRPC4 plays no role in store-operated Ca²⁺ entry in either cell type, but plays a major role in mediating carbachol-stimulated Ca²⁺ entry in HEK-293 cells. Of particular interest is our observation that in cells where expression of TRPC4 is selectively suppressed, low doses of carbachol can no longer generate repetitive Ca²⁺ oscillations. This indicates that TRPC4 mediates the Ca²⁺ entry required to maintain continuous Ca²⁺ oscillations in response to carbachol. We are continuing to analyze the contribution of TRPC5 and TRPC6, and combinations of various TRPC homologs, to Ca²⁺ channel activity initiated by a variety of stimuli (EGF, UV radiation, apoptosis stimuli, and cell cycle variations). We also will investigate the role of Ca²⁺ entry, via various TRPC channels, on downstream events such as transcription, cell growth, and apoptosis. We have recently identified a peptide toxin from scorpion venom that selectively inhibits store-operated Ca²⁺ channels and this toxin will be useful in identifying events regulated downstream of store-operated Ca²⁺ channels. 

For the project on BK-induced tyrosine phosphorylation, we are stimulating HEK-293 cells expressing the B2 BK receptor with bradykinin, extracting cell proteins, purifying tyrosine phosphorylated proteins on an immunoaffinity column, and using the fraction specifically eluted from the column to identify the tyrosine phosphorylated proteins using a proteomics approach.  We run the purified proteins on 2D gels, cut out protein spots that are differentially regulated by BK, and do peptide mass fingerprinting by mass spectroscopy to identify the proteins of interest. These studies are done in collaboration with Argonne National Laboratory. 

top