The proline-rich N-terminal domain of G18 exhibits a novel G protein regulatory function

Abstract

The protein G18 (also known as AGS4 or GPSM3) contains three conserved GoLoco/GPR domains in its central and C-terminal regions that bind to inactive Galpha(i), whereas the N-terminal region has not been previously characterized. We investigated whether this domain might itself regulate G protein activity by assessing the abilities of G18 and mutants thereof to modulate the nucleotide binding and hydrolytic properties of Galpha(i1) and Galpha(o). Surprisingly, in the presence of fluoroaluminate (AlF(4)(-)) both G proteins bound strongly to full-length G18 (G18wt) and to its isolated N-terminal domain (G18DeltaC) but not to its GoLoco region (DeltaNG18). Thus, it appears that its N-terminal domain promotes G18 binding to fluoroaluminate-activated Galpha(i/o). Neither G18wt nor any G18 mutant affected the GTPase activity of Galpha(i1) or Galpha(o). In contrast, complex effects were noted with respect to nucleotide binding. As inferred by the binding of [(35)S]GTPgammaS (guanosine 5'-O-[gamma-thio]triphosphate) to Galpha(i1), the isolated GoLoco region as expected acted as a guanine nucleotide dissociation inhibitor, whereas the N-terminal region exhibited a previously unknown guanine nucleotide exchange factor effect on this G protein. On the other hand, the N terminus inhibited [(35)S]GTPgammaS binding to Galpha(o), albeit to a lesser extent than the effect of the GoLoco region on Galpha(i1). Taken together, our results identify the N-terminal region of G18 as a novel G protein-interacting domain that may have distinct regulatory effects within the G(i/o) subfamily, and thus, it could potentially play a role in differentiating signals between these related G proteins.

FIGURE 1.

A, shown is the amino acid sequence of G18. The three GoLoco motifs are underlined. The proline residues are shown in white on a black background, and arginines that could potentially contribute to the N-terminal effects are indicated in bold type. B, tissue distribution of G18 is shown. Various tissues from 3-month-old C57BL/6 mice were isolated, total RNA was extracted, and reverse transcriptase-PCR followed by PCR was performed using primers specific for the open reading frame of G18. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. The control lane indicates the reference length of G18 using the PCR product from the plasmid.

FIGURE 2.

Constructs and purified proteins. A, shown is the domain architecture of different constructs used in the study. B, His-tagged proteins were purified from E. coli strain BL21 (DE3) using Ni-NTA affinity purification followed by fast protein liquid chromatography. Protein purity was estimated by Coomassie staining. The correct molecular size of G18ΔC (which may have run anomalously due to its high proline content) was verified by mass spectrometry.

FIGURE 3.

Protein-protein interaction between G proteins and G18. Purified His₆-Gα_i1 or Gα_o was incubated with excess GDP±AlF₄⁻ for 30 min at 4 °C, purified GST-tagged G18 or one of its mutants was added to the solution, and the incubation was continued for another 2 h before adding glutathione-Sepharose 4B beads. After overnight incubation at 4 °C on a rotating platform, the mixture was centrifuged and washed, and the resulting pellet was retained for immunoblotting (IB) analysis. Membranes were probed with anti-His antibody. Input represents 10% of the protein used in the pulldown assay. A representative blot of three independent experiments is shown.

FIGURE 4.

Protein-protein interaction between G proteins and the N terminus of G18. Purified His₆-Gα_i1 or Gα_o was incubated with excess GDP+AlF₄⁻ for 30 min at 4 °C, purified GST-tagged G18 or its isolated N terminus (G18ΔC) was added to the solution, and the incubation was continued for another 2 h before adding Ni-NTA-agarose beads. The protein mix was further incubated overnight at 4 °C on a rotating platform, samples were centrifuged, and the resulting pellet was retained for immunoblotting (IB) analysis. Membranes were probed with anti-GST antibody. Input represents 10% of the protein used in the pulldown assay. A representative blot of three independent experiments is shown.

FIGURE 5.

The effects of G18 on G protein GTPase activity under presteady state conditions. Purified His₆-Gα_i1 (A) or His₆-Gα_o (B) was incubated with [γ-³²P]GTP (1 × 10⁶cpm/assay) for 15 min at 30 °C (Gα_i1) or 20 °C (Gα_o). A single round of GTP hydrolysis was measured at 0 °C in the presence of 10 mm Mg²⁺ and RGS4, G18, or one of its mutants as indicated. Data points shown are the means ± S.E. from three independent experiments.

FIGURE 6.

The effects of G18 on G protein nucleotide exchange. Purified His₆-Gα_i1 (A) or His₆-Gα_o (B) was preincubated with G18 at 4 °C. Binding assays were initiated by adding 0.5 μm [³⁵S]GTPγS (1.25 × 10⁵ cpm/pmol) at 30 °C (Gα_i1) or 20 °C (Gα_o). The binding of GTPγS to Gα proteins was measured after 30 min (Gα_i1) or 60 min (Gα_o) of incubation. Nonspecific binding was estimated in the presence of excess unlabeled GTPγS, and these values were subtracted from the results. The data are presented as the mean ± S.E. of three to five independent experiments performed in duplicate. *, p < 0.05, compared with G protein alone (one-way analysis of variance with Tukey's multiple comparison test).

FIGURE 7.

The effects of G18 on Gα protein GTPase activity under steady state conditions. Purified His₆-Gα_i1 (A) or His₆-Gα_o (B) was mixed with G18 at 4 °C. The protein mixture was incubated with [γ-³²P]GTP (1 × 10⁶ cpm/assay) in the presence of 6 mm Mg²⁺ at 30 °C (Gα_i1) or 20 °C (Gα_o). The free ³²P_i level was measured after 60 min of incubation. The data are presented as the mean ± S.E. of three independent experiments performed in duplicate. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with G protein alone (one-way analysis of variance with Tukey's multiple comparison test).

FIGURE 8.

The effects of G18 on receptor- and agonist-stimulated G protein GTPase activity. A and B, Sf9 cell membranes overexpressing M2 muscarinic acetylcholine receptor and heterotrimeric Gα_i1 or Gα_o were prepared as indicated under “Experimental Procedures.” Carbachol was used to activate M2 receptor. Steady state GTPase activities of G proteins were measured in the presence (solid line) or absence (dashed line) of RGS4 and the indicated concentrations of G18wt. Nonspecific signal was determined in the absence of added purified proteins and in the presence of tropicamide. The data are presented as the mean ± S.E. of three-four independent experiments. C and D, purified His₆-Gα_i1 or His₆-Gα_o was incubated with [γ-³²P]GTP (1 × 10⁶ cpm/assay) for 15 min at 30 °C (Gα_i1) or 20 °C (Gα_o). A single round of GTP hydrolysis was measured at 0 °C in the presence of 10 mm Mg²⁺ (□) and RGS4 (▴) or RGS4+G18 (▾). Data points shown are the means ± S.E. from three independent experiments.

FIGURE 9.

The effects of G18 mutants on receptor- and agonist-stimulated G protein GTPase activity. M2-G_i1 and M2-G_o membranes from sf9 cells were assayed for agonist-stimulated steady state GTPase activity in the presence of RGS4 and the indicated concentrations of G18 mutants, as described in Fig. 7. G18wt activity (Fig. 7) is shown as a dashed line for comparison in each panel. The data points shown are means ± S.E. from three to four independent experiments.