University of Virginia Health System

Dr. James C. Garrison

Mechanisms by which Hormones and Growth Factors Regulate Cell Function
The overall goal of our research is to elucidate the mechanisms by which hormones and growth factors regulate cellular function. Presently, our major effort is directed toward understanding how the family of seven transmembrane domain receptors interact with G proteins to generateintra cellular signals. We are currently pursuing two major projects.. spIn the first project, we are attempting to understand how the diversity of G protein \(*a and \(*b\(*g subunits leads to specificity intrans membrane signaling. By using cDNA clones coding for the multiple subunits of G proteins to over express the proteins in thebaculovirus/Sf9 insect cell system, we can obtain large amounts of Gprotein \(*a subunits or \(*b\(*g dimers of defined composition. Using these techniques, we have demonstrated that recombinant\(*b\(*g dimers of defined composition have differential abilities to support the interaction between \(*a subunits and receptors or to directly activate effectors. These exciting results provide a potential explanation for signaling selectivity in the intact cell. Thus, we are expanding our study of the functional differences of recombinant \(*b\(*g dimers to better understand the specificity of cell signaling.

One important issue is to define a functional role for \(*b\(*g dimers containing divergent \(*b subunits such as \(*b\s-4\d5\u\s+4. Our initial results with dimers composed of the \(*b\s-4\d5\u\s+4\(*g\s-4\d2\u\s+4 subunits indicate selective coupling to the Gq \(*a subunit and activation of a restricted set of effectors. We are now studying a panel of dimers containing the \(*b\s-4\d5\u\s+4 subunit combined with various \(*g subunits for their ability to couple receptors to \(*a subunits an to activate effectors. We are also studying the function of the variant \ (*b\s-4\d3S\u\s+4 dimer, identified in hypertensive patients, which is missing one WD-40 repeat.

In another aspect of this study, we are determining which domains of the \(*g subunit interact with receptors and effectors. By using a series of chimeric \(*g subunits containing switched N- and C-terminal domains from a pair of active and inactive \(*g subunits, we are examining the role of these domains using assays of receptor interaction and effector function. The final area of this study examines the possibility that the prenyl group of the \(*g subunit participates in activating the \(*b\(*g dimer. Dimers containing \(*gsub units modified with the C\s-215\s+2 farnesyl group are usually less active than dimers containing \(*g subunits modified with the C\s-220\s+2 geranylgeranyl group. We are testing the recent observation that the prenyl group interacts directly with the \ (*b subunit to determine if the two lipids cause different conformational changes in the \(*b subunit. The approach is to make mutant \(*b subunits in which the prenyl group is not able to fold back into the \(*b subunit and test their activity in assays of receptorin teraction and effector function.

In a second project, we are studying the cross-talk between the pathways activated by G proteins and those activated by oncogenes and mitogens. Historically, these pathways were considered very separate; however, there now appears to be significant crosstalk between them. Thus, occupation of G protein coupled receptors can stimulate the activity of tyrosine kinases, guanine nucleotide exchange factors and growth regulating pathways. G proteins markedly stimulate the p110-\(*gisoform of phosphatidylinositol 3-kinase (PtdIns 3-kinase) leading to production of phosphatidylinositol (3,4,5) tris phosphate. This lipid is an important signal that activates the phosphatidylinositol dependent protein kinase, PDK-1, leading to activation of protein kinase B and a host of signaling events. The initial focus of our work is to understand the mechanisms by which G protein \(*a and \(*b\(*g subunits activate the PtdIns 3-kinase. We are determining the ability of pure, recombinant, G protein \(*a subunits and \(*b\(*g dimers of defined composition to activate the purified p110-\(*g isoform of PtdIns3-kinase.

In another aspect of this project, we are determining the domains in the \(*b and \(*g subunits that activate PtdIns 3-kinase. We are using \(*b\(*g dimers with selected point mutations in the \(*b subunit in combination with mutated and/or chimeric \(*g subunits to evaluate the domains in the \(*b\(*g dimer which activate the PtdIns3-kinase. In the final aspect of this project, we are attempting to understand how known regulatory mechanisms affect the activity of \(*b\(*g dimers on PtdIns 3-kinase. We are examining the role of protein phosphorylation in regulating PI 3-kinase both in terms of phosphorylation of the kinase itself and phosphorylation of the \(*b\(*g dimers which activate this effector.