Our goal is to determine the function of the individual members of the inhibitory family of G-proteins. We have taken a genetic approach to produce mutants by homologous recombination which lack each of these specific gene products in order to determine their role in normal cell physiology. Analysis of these mutants, each lacking a specific alpha(i) G-protein, should enable us to determine the function of each of these G-proteins, just as the cyc-cells revealed the functions of alpha/s containing G-proteins. We wish to address the following basis questions and hypotheses: 1) What is the specificity of receptor-alpha(i) interactions? and 2) What is the specificity of alpha(i)-effector interactions? To accomplish these goals, we will take two genetic approaches, the production of homozygous mutant cell lines lacking each of the alpha(i) subunits, and the production of a transgenic mouse line lacking an alpha(i) subunit by blastocyst mediated transgenesis. We have developed a novel method for the production of homozygous mutant cell lines. This method has been readily adaptable for a number of genes and has allowed us to produce cell lines with more than one gene inactivated. These cell lines should prove to be invaluable tools to test our hypotheses and determine the specific roles of the alpha(i) G- proteins. Using these cells lines we have expressed heterologous receptors. We will test coupling of alpha(i)1, alpha(i)2 and alpha(i)3, to a number of receptors including D2-dopamine receptor, alpha2 adrenergic receptor and the 5HT1A serotonin receptor. We have identified a specific requirement for alpha(i)2 in the signal transduction from the alpha2-adrenergic receptor to intracellular Ca++ responses but no effect of the knockout on inhibition of cAMP accumulation. We will extend these studies to include testing the role of alpha(i) G-proteins on mitogenesis induced by bombesin. Embryonic stem cells are cultured cell line derived from the inner cell mass of normal mouse blastocyst. These cells are capable of forming all tissues of the mouse if reintroduced into a normal blastocyst by microinjection. They are also capable of in vitro differentiation to number of cell types including beating cardiocyte, skeletal muscle, neurons, glia, and hematopoietic cells. We will take advantage of this pluripotential to produce ES cells lacking the alpha(i) proteins, differentiate them in vitro, and then test coupling to muscarinic receptors to cardiac K+ channels by patch clamp. Although many questions about the function of alpha(i) subunits are best answered in cultured cell lines, some questions require the production of a mutant mouse line by blastocyst mediated transgenesis. In particular, we will study the role of alpha(i)2 on the growth and differentiation of adrenal cortical and ovarian stromal cells, where alpha(i)2 has been implicated as a proto-oncogene.
