Lipids are important regulators of the activity of many proteins including those involved in cardiac, vascular, pulmonary, and neural regulation, yet little is known about the molecular mechanisms mediating these effects. My laboratory is engaged in a program of basic research to elucidate the molecular mechanisms by which lipids act as specific ligands to regulate cellular responses, and to investigate in select areas of cardiovascular disease now aberrations of signaling pathways may play a vital role in the pathogenesis of human disease.

We have used the family of phosphoinositide-specific phospholipase C isoforms as a model to study protein-lipid interactions. Upon stimulation by various hormones these membrane associated enzymes hydrolyze polyphosphoinositides to yield second messengers such diacylglycerol and inositol 1,4,5-trisphosphate. The determinants of enzyme function are complex and can be broken down into several primary components: substrate binding and catalysis, enzyme translocation, and regulation. During the past several years we have identified the structural motifs in the PLC delta 1 isoform that mediate each of the three primary functions. All three motifs require the binding of a specific lipid for function. The structural motif mediating translocation also modulates the rate catalysis, and is encoded by a unique domain termed the pleckstrin homology or PH domain. This newly discovered protein module of 100 amino acids exists in many molecules (including PLC d1) which participate in signal transduction. Our work is among the first to demonstrate a clear function in signal transduction for the PH domain, and serves a paradigm for the activation and regulation of many signaling molecules.

Work in progress includes characterization of a new family of PLC isoforms, and the elucidation of the physiological roles of various PLC isoforms using primary cell culture and transgenic mice.

TECHNIQUES

My laboratory is one whose research is solely undertaken to answer questions which we feel are important. Therefore, since the questions are of utmost importance, we are not a technique driven laboratory. We utilize many different techniques in our work including those from cell biology, protein biochemistry, molecular biology, pharmacology and genetics. The following methods are employed currently in the laboratory: radionuclide ligand binding and analysis, high performance stearic-exclusion liquid chromatography, cell culture, affinity chromatography, photoaffinity labeling, SDS-gel electrophoresis, protein assay, Western blotting, DNA/RNA isolation and characterization by Northern and Southern blotting, isolation of novel genes and construction of DNA molecules by PCR, DNA sequencing, site directed mutagenesis, mammalian cell transfection and establishment of stably expressing cell lines, assay of signal transduction pathways via adenylyl cyclase, phospholipase C, D and A2; isolation of alleles by single stranded conformation polymorphism analysis (SSCP), measurement of intracellular calcium fluxes via fluorescent calcium sensitive dyes, and the generation and assessment of transgenic mice.

Figure Legends

• Figure 1: Molecular model of PH domain from PLC delta 1. Indicated amino acid residues at positions 30,32,36, and 54 are critical to interaction with lipid ligand IP3 (shown in red).
• Figure 2: Schematic of PLC delta 1 showing functional domains identified by the Lomasney and King laboratories largely as a result of characterization of site directed point mutants.
• Figure 3: Proposed model for regulation of PLC delta 1. PLC delta 1 is primarily a cytoplasmic enzyme. During activation it translocates to the plasma membrane, where lipid ligands and substrate reside. PIP2 binds to the PH domain of PLC delta 1 and activates hydrolysis of substrate. Once PLC delta 1 is attached to the interface, hormones which activate PIP5K can indirectly activate PLC delta 1 by increasing concentration of the agonist PIP2. The products of hydrolysis DAG and IP3 are second messengers, which in turn regulate various effector molecules. PLC delta 1 is inhibited by interaction with tissue transglutaminase (TG), a cytoplasmic enzyme. While bound to TG, PLC delta 1 is unable to translocate to the plasma membrane and is therfore inhibited. The GTP bound form of TG interacts very weakly with PLC delta 1. Therefore, hormones which increase GTP concentrations could potentially release PLC delta 1 from TG and hence release it from inhibition.