Research

There is great interest in therapeutic efforts to modulate G protein-coupled receptor (GPCR) signaling in a variety of cellular systems.  Our lab is focused on gaining a better understanding of GPCR signaling components, where they localize in the cell, and how these pathways are altered in various disease states.  One area of interest involves GPCR signaling pathways that elevate or reduce cAMP levels (i.e. prostaglandin, adrenergic, and opioid receptors).  We use molecular cloning, expression of cloned receptors, and a variety of cell biology and biochemical approaches to examine mechanisms for activation and deactivation of cAMP generation through adenylyl cyclase, an effector enzyme.   In addition to investigating the functional aspect of this signaling pathway, our lab is also investigating the morphological distribution of these components, i.e. compartmentation to caveolar microdomains.  Caveolae, or "little caves," are cholesterol and sphingolipid enriched invaginations of the plasma membrane (~50-100 nm in size).  Our lab has identified localization of certain GPCRs, G-proteins, adenylyl cyclase isoforms, and cyclic nucleotide phosphodiesterase isoforms to these discrete regions of the plasma membrane.  Our objective is to understand how compartmentation to these lipid rafts affects signal transduction and how this specific organization is altered during disease states such as pulmonary hypertension, cardiac ischemia, and congestive heart failure.  One goal of these studies is to develop novel gene therapy strategies to modulate cellular responses through changes in expression of limiting components in the signaling pathways. Currently we study adult cardiac myocytes and fibroblasts, vascular smooth muscle cells, and vascular endothelial cells. People involved in these projects include: Rik Bundey, Fiona Murray, Chih-Min Tang, Lingzhi Zhang, Steve Fuhs, Utako Yokoyama.

Nucleotide modulation of neurotrophin-stimulated differentiation. Pictured on the right is an immunolabeled PC12 cell.  The Hoescht-stained nucleus (blue) and TrkA receptors (red, NGF receptor) are visible.

Currently, we are investigating the role of nucleotides in the nerve growth factor (NGF)-stimulated differentiation and catecholamine release by an immortalized pheochromocytoma cell line, the PC12 cell.  We are currently focused on the effects of nucleotides, as transduced through P2 receptors, on PC12 cell neurite extension and norepiniephrine release in the PC12 cell by investigating G-protein receptor expression, and downstream regulators of GPCR signaling.  In other studies, we assess release of nucleotides and activation by and regulation of expression of P2Y receptors in a well-differentiated renal epithelial cell line (MDCK) and in PC-12 adrenal chromaffin cells. We are studying aspects of nucleotide responses of Jurkat T cells. This work was conducted by a recent graduate student in the lab, David Arthur.

The second messenger cyclic AMP promotes cell death (apoptosis) of certain cells but prevents apoptosis of other cell types. The precise mechanisms for these contrasting effects are not well understood.

We are using S49 lymphoma cells - a cell line in which mutants have been isolated along the pathway for cyclic AMP generation and action - as the system to explore pro-apoptosis and T84 and other intestinal epithelial cells to assess anti-apoptosis and are using a combined cellular and molecular strategy, including use of DNA microarrays and real-time PCR, to define components involved in pro- and anti-apoptotic responses to cyclic AMP. In other studies we are seeking to define how the cyclic AMP-regulated effector enzyme, protein kinase A, regulates such components. Scientists involved in this project include Lingzhi Zhang and Fiona Murray. 

We are also studying the role of cyclic nucleotide phosphodiesterases in chronic lymphocytic leukemia using peripheral blood mononuclear cells (PBMC) isolated from normal and CLL patients. The goal of this project is to test the hypothesis that agents that inhibit specific PDE isoforms, by raising the level of cyclic nucleotides, may selectively kill CLL cells or enhance the killing induced by other anti-leukemic agents.