Influence of cytosolic AGS3 on receptor--G protein coupling.
Activator of G protein signaling 3 (AGS3) activates the Gbetagamma mating pathway in yeast in a manner that is independent of heptahelical receptors. It competes with Gbetagamma subunits to bind GDP-bound Gi/o(alpha) subunits via four repeated G protein regulatory (GPR) domains in the carboxyl-terminal half of the molecule. However, little is known about the functional role of AGS3 in cellular signaling. Here the effect of AGS3 on receptor-G protein coupling was examined in an Sf9 cell membrane-based reconstitution system. A GST-AGS3-GPR fusion protein containing the four individual AGS3-GPR domains inhibits receptor coupling to Galpha subunits as effectively as native AGS3 and more effectively than GST fusion proteins containing the individual AGS3-GPR domains. While none of the GPR domains distinguished among the three G(i)alpha subunits, both individual and full-length GPR domains interacted more weakly with G(o)alpha than with G(i)alpha. Cytosolic AGS3, but not membrane-associated AGS3, can interact with G(i)alpha subunits and disrupt their receptor coupling. Immunoblotting studies reveal that cytosolic AGS3 can remove G(i)alpha subunits from the membrane and sequester G(i)alpha subunits in the cytosol. These findings suggest that AGS3 may downregulate heterotrimeric G protein signaling by interfering with receptor coupling.
Ma
Hongzheng
H
Department of Biochemistry and Molecular Pharmacology, West Virginia University School of Medicine, Morgantown, West Virginia 26506-9142, USA.
Peterson
Yuri K
YK
Bernard
Michael L
ML
Lanier
Stephen M
SM
Graber
Stephen G
SG
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001921:Brain / Q000378:metabolism; D002352:Carrier Proteins / Q000494:pharmacology*; D002462:Cell Membrane / Q000737:chemistry; D003600:Cytosol / Q000378:metabolism; D016166:Free Radical Scavengers / Q000378:metabolism; D020555:GTP-Binding Protein Regulators / Q000502:physiology*; D005982:Glutathione Transferase; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism*; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism*; D015151:Immunoblotting; D021122:Protein Subunits; D051381:Rats; D011985:Receptors, Serotonin / Q000378:metabolism*; D044263:Receptors, Serotonin, 5-HT1; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012701:Serotonin / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013347:Subcellular Fractions
D002352:Carrier Proteins; D016166:Free Radical Scavengers; D020555:GTP-Binding Protein Regulators; C498913:Gpsm1 protein, rat; D021122:Protein Subunits; D011985:Receptors, Serotonin; D044263:Receptors, Serotonin, 5-HT1; D011993:Recombinant Fusion Proteins; D006153:Guanosine Diphosphate; D012701:Serotonin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D005982:Glutathione Transferase; D020962:Heterotrimeric GTP-Binding Proteins