G protein signaling constitutes a fundamental mechanism of intercellular communication used by all eukaryotes. The signaling pathways have several components, each encoded by distinct multigene families, including the seven transmembrane domain receptors, the heterotrimeric G protein alpha, beta and gamma subunits and various intracellular effector proteins. G proteins couple extracellular signals received by receptors to the regulation of effector proteins that generate intracellular second messengers. In lower eukaryotes, G proteins are required to generate cellular responses to environmental cues during development. In mammals, G proteins are essential components of sensory transduction systems and regulate a variety of other specialized functions in terminally differentiated cells. G proteins are also expressed early in mammalian development and in pluripotent progenitor cells of adults, but almost nothing is known about their biological function. The long term goals of this proposal are to understand G protein function in early mammalian development and hematopoiesis. The G protein alpha subunit is a crucial regulator of signaling specificity and is an excellent target for functional studies. We have identified a sub-family of alpha subunits that activate phospholipase Cbeta. One of these alpha subunit genes, Gna15, is expressed specifically in hematopoietic cells. With the exception of those G proteins that are expressed only in specialized sensory transduction cells, Gna15 has the most restricted expression pattern of any G protein subunit. We will use mouse embryonic stem cells and transgenic mouse technology to express mutations in Gna15 and study their effects on hematopoiesis. One of the problems in studying mammalian signal transduction is the complexity of these systems and potential functional redundancy inherent in multigene families. Gna15 is co-expressed in myeloid and lymphoid lineages with Gna11, a distantly related gene in the same sub-family. Gna11 is ubiquitously expressed and may partially complement a deficiency in Gna15. We will study the overlapping and unique functions of Gna11 and Gna15 by creating mutations in either gene separately and together in mice. Gna11 and Gna15 are adjacent on mouse chromosome 10. To create the double mutant, the proximity of Gna11 and Gna15 demands that either we delete both genes sequentially from the same chromosome in ES cells or simultaneously in a single recombination event. We propose to obtain simultaneous deletion of Gna11 and Gna15 by two independent protocols: either a novel method of deletion in the male germline or a more convential approach in ES cells. Disrupting Gna15 and/or Gna11 signaling may arrest hematopoietic differentiation. We will identify deficiencies in hematopoiesis in homozygous mutants and chimeric mice. Hematopoietic lineages will be traced using labeled antibody markers to identify the affected cells or their immediate progenitors by flow cytometry and fluorescence activated cell sorting (FACS). We will then ask fundamental questions regarding the components of the disrupted signal transduction pathway in these cells; what are the ligands, receptors and effectors that are coupled by Gna11 and Gna15?
