Gβ Association and Effector Interaction Selectivities of the Divergent Gγ Subunit Gγ13

Abstract

G␥ 13 is a divergent member of the G␥ 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␥ 13 contains a C-terminal asparagine-proline-tryptophan (NPW) tripeptide, a hallmark of RGS protein G␥-like (GGL) domains which dimerize exclusively with G␤ 5 subunits. In this study, we investigated the functional range of G␥ 13 assembly with G␤ subunits using multiple assays of G␤ association and G␤␥ effector modulation. G␥ 13 was observed to associate with all five G␤ subunits (G␤ 1-5 ) upon co-translation in vitro, as well as function with all five G␤ subunits in the modulation of Kir3.1/3.4 (GIRK1/4) potassium and N-type (␣ 1B ) calcium channels. Multiple G␤/ G␥ 13 pairings were also functional in cellular assays of phospholipase C (PLC) ␤2 activation and inhibition of G␣ q -stimulated PLC␤1 activity. However, upon cellular co-expression of G␥ 13 with different G␤ subunits, only G␤ 1 /G␥ 13 , G␤ 3 /G␥ 13 , and G␤ 4 /G␥ 13 pairings were found to form stable dimers detectable by co-immunoprecipitation under high-detergent cell lysis conditions. Collectively, these data indicate that G␥ 13 forms functional G␤␥ dimers with a range of G␤ subunits. Coupled with our detection of G␥ 13 mRNA in mouse and human brain and retina, these results imply that this divergent G␥ subunit can act in signal transduction pathways other than that dedicated to taste reception in sensory lingual tissue.One major class of cellular signal transduction pathways is controlled by heterotrimeric guanine nucleotide-binding proteins ("G proteins"). The conventional model of heterotrimeric G-protein signaling involves serpentine cell-surface receptors (G protein-coupled receptors) coupled to a membrane-associated heterotrimer composed of a GTP-hydrolyzing G␣ subunit and a G␤␥ dimeric partner (1, 2). The WD-repeat ␤-propeller protein G␤ and the ␣-helical isoprenylated polypeptide G␥ form an obligate heterodimer that binds tightly to GDP-bound G␣, enhancing G␣ coupling to receptor and inhibiting its release of GDP. Guanine nucleotide exchange activity of agonist-occupied G protein-coupled receptors facilitates dissociation of G␣⅐GTP 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).Considerable functional diversity is possible within the large combinatorial range of potential G␤␥ dimers (5, 6), given the existence of at least six G␤ subunits (G␤ 1-4 , the outlier G␤ 5 , and its retinal-specific isoform G␤ 5L ; Refs. 7 and 8) and 11 G␥ subunits (three farnesylated and eight geranylgeranylated species; Fig. 1). However, outside the unique nature of G␥ 1 (the farnesylated G␥ of the retinal phototransduction cascade; Refs. 9 -12), specific roles for particular G␥ subunit...

Comprehensive analysis of heterotrimeric G-protein complex diversity and their interactions with GPCRs in solution

“…6. Similarly, the β 2 γ 13 -dimer, which could not be identified when mixing subunits that had been separately translated in vitro (32), could be identified when both subunits were cotranslated in vitro (37). This finding suggests the need for cotranslational assembly.…”

“…Formation of a heterotrimeric Gαβγ complex may stabilize intrinsically unstable βγ-dimer combinations. Indeed, the dogma of the stable βγ-dimer under nondenaturing conditions (35) has been challenged, because certain βγ-dimers have been found to show a tendency to dissociate into β and γ when taken out of the cellular context (36)(37)(38)(39). Moreover, there is evidence that dimers like β 2 γ 1, β 2 γ 13 , and β 3 γ 1 play functional roles in the cell (37, 40, 41), although they have not been identified in previous dimerization assays.…”

Differential Sensitivity of Phosphatidylinositol 3-Kinase p110γ to Isoforms of G Protein βγ Dimers

“…The ␥ subunits separate into 5 subfamilies, one with ␥ 1,8,11 , one containing ␥ 2,3,4,9, one comprised of ␥ 7,12 , and another containing ␥ 5,10 (22,46). G␥ 13 is quite divergent and forms its own subfamily (22, 46) but seems to have functional activities comparable with ␥ 2 (35). To minimize the complexity of exploring all 84 possible combinations of the existing ␤ and ␥ subunits, we chose subunits known to be effective activators of PtdIns 3-kinase such as ␤ 1 and ␥ 2 and selected ␤ or ␥ isoforms from each of the subfamilies to compare with these benchmarks.…”

“…The second subfamily is comprised of ␥ subunits 2,3,4,9, whereas the third and fourth subfamilies only consist of two members each, ␥ subunits 7 and 12, and ␥ subunits 5 and 10, respectively. G␥ 13 is the most divergent ␥ and forms its own subfamily (22,46) but has signaling properties similar to ␥ 2 (35).…”

“…The baculovirus for ␥ 13 was the kind gift of Dr. David Siderovski, University of North Carolina (35). The baculoviruses encoding the "conformational change" mutants (H311A, R314A, W332A) and the "prenyl pocket" mutants (T329K, S331A, W339A, V315A, F335A, K337A) in the ␤ 1 subunit were prepared as described (36).…”