Abstract
G gamma(13) is a divergent member of the G gamma subunit family considered to be a component of the gustducin G-protein heterotrimer involved in bitter and sweet taste reception in taste bud cells. G gamma(13) contains a C-terminal asparagine-proline-tryptophan (NPW) tripeptide, a hallmark of RGS protein G gamma-like (GGL) domains which dimerize exclusively with G beta(5) subunits. In this study, we investigated the functional range of G gamma(13) assembly with G beta subunits using multiple assays of G beta association and G beta gamma effector modulation. G gamma(13) was observed to associate with all five G beta subunits (G beta(1-5)) upon co-translation in vitro, as well as function with all five G beta subunits in the modulation of Kir3.1/3.4 (GIRK1/4) potassium and N-type (alpha(1B)) calcium channels. Multiple G beta/G gamma(13) pairings were also functional in cellular assays of phospholipase C (PLC) beta 2 activation and inhibition of G alpha(q)-stimulated PLC beta 1 activity. However, upon cellular co-expression of G gamma(13) with different G beta subunits, only G beta(1)/G gamma(13), G beta(3)/G gamma(13), and G beta(4)/G gamma(13) pairings were found to form stable dimers detectable by co-immunoprecipitation under high-detergent cell lysis conditions. Collectively, these data indicate that G gamma(13) forms functional G beta gamma dimers with a range of G beta subunits. Coupled with our detection of G gamma(13) mRNA in mouse and human brain and retina, these results imply that this divergent G gamma subunit can act in signal transduction pathways other than that dedicated to taste reception in sensory lingual tissue.
Highlights
One major class of cellular signal transduction pathways is controlled by heterotrimeric guanine nucleotide-binding proteins (“G proteins”)
Guanine nucleotide exchange activity of agonist-occupied G protein-coupled receptors facilitates dissociation of G␣1⁄7GTP and G␥ subunits and allows both moieties to modulate a variety of downstream effectors; for the free G␥ dimer, these effectors include the second messenger generators adenylyl cyclase and phospholipase C- (PLC)1 as well as ion channels such as G protein-coupled inward-rectifying potassium (GIRK) channels and N-type calcium channels [3, 4]
G␥13 is unique in possessing an asparagine-proline-tryptophan (NPW) tripeptide prior to the C-terminal isoprenylation signal sequence; this conserved tripeptide ends in phenylalanine (i.e. NPF) in all other mammalian G␥ polypeptides
Summary
Expression Constructs—The entire open reading frame of mouse G␥13 was amplified by PCR (sense primer 5Ј-GGGATCCGACGCCATGGAGGAGTGGGATG-3Ј, antisense primer 5Ј-GTCTAGAGTGTGGGTCAGGCTCATAGG-3Ј) from a MarathonTM mouse brain cDNA library (CLONTECH), digested with EcoRI and XbaI, and subcloned in-frame with an N-terminal tandem hemagglutinin (HA)-epitope tag into pcDNA3.1 (Invitrogen) as previously described [20]. Following removal of beads by centrifugation at 10,000 ϫ g for 1 min, the supernatants were transferred to fresh tubes containing anti-HA monoclonal antibody 12CA5 (Roche Molecular Biochemicals) and incubated with constant rotation for 1 h at 4 °C. Proteins were separated on 12% SDS-PAGE gels and electroblotted onto polyvinylidene difluoride membranes (Millipore) for detection of G subunits with anti-Myc mouse monoclonal antibody conjugated to horseradish peroxidase (9E10-HRP; Roche Molecular Biochemicals), followed by enhanced chemiluminescence (Amersham Bioscience, Inc.). G␥ Subunit Inhibition of G␣q-stimulated PLC1 Activity—G␥ subunit-mediated inhibition of G␣q-stimulated [3H]inositol phosphate accumulation was measured as described above, except that COS-7 cells were transiently transfected with 20 ng of G␣q vector in addition to 300 ng of PLC1 vector, 200 ng each of the indicated G and/or G␥ subunit expression vectors, and pcDNA3.1 vector DNA to a total of 720 ng/well
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