This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Signaling through heterotrimeric G proteins is essential for a variety of cellular responses including neurotransmission, hormonal response, olfactory transduction, phototransduction, cell migration and apoptosis. In response to the multitude of G protein coupled receptors (GPCR) that control these diverse pathways, vertebrates have evolved multiple isoforms of the alpha, beta and gamma subunits that selectively associate to form heterotrimeric G proteins. The zebrafish genome has 26alpha, 9beta and 17gamma subunits, which would make a total of 3978 potential different heterotrimers, if all combinations were made. Although the signaling partners and binding affinities of individual G protein isoforms have been investigated in vitro, the endogenous function of many heterotrimers and the extent of their functional redundancy remain uncharacterized. To address the functional diversity of heterotrimers containing specific Ggamma subunits in vivo, we expressed dominant negative versions of the Ggamma subunits to disrupt the signaling necessary for a known GPCR-mediated event, primordial germ cell (PGC) migration. We show that the vast majority of prenylation-deficient Ggamma subunits can disrupt PGC migration by altering the subcellular localization of signaling components. This disruption manifests in an inability of PGCs to migrate directionally. We identified a distinct subset of wild type Ggamma subunits that have the ability to reverse this semi-dominant negative effect, suggesting that Ggamma subunits have distinct and overlapping signaling capacities in vivo. To understand the roles Ggamma protein domains have in contributing to differences in Ggamma signaling capacity, we constructed Ggamma chimeras. Analysis of these chimeras demonstrated that multiple regions and motifs within the central and c-terminal regions influence the ability of Ggamma to mediate the signaling pathways necessary for directional PGC migration. Our results also indicate that prenylation-deficient Ggamma subunits can be used to disrupt GPCR-mediated signaling events in vivo.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
2P41RR011823-16
Application #
8365902
Study Section
Special Emphasis Panel (ZRG1-CB-L (40))
Project Start
2011-09-01
Project End
2012-06-30
Budget Start
2011-09-01
Budget End
2012-06-30
Support Year
16
Fiscal Year
2011
Total Cost
$12,768
Indirect Cost
Name
University of Washington
Department
Biochemistry
Type
Schools of Medicine
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
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