Sequence alignment between G_γ subunits and the GGL domains of RGS6, RGS7, RGS9, RGS11, and EGL-10. Residues conserved among RGS proteins are in black boxes; residues in common between RGS proteins and one or both G_γ consensus lines (Cons-A, Cons-B) are shown in shaded boxes. Asterisk denotes first residue of the RGS domain. b, bovine; c, C. elegans; h, human; m, mouse; r, rat.

Purification of G_β5/RGS11 heterodimers after expression in Sf9 cells. Cells were infected with recombinant baculoviruses encoding either (A) hexahistidine-tagged G_β5 and full-length RGS11 or (B) hexahistidine-tagged G_β5 and RGS11ΔD (aa 219–467). Fractions were subjected to electrophoresis through polyacrylamide gels containing sodium dodecylsulfate and stained with Coomassie blue. Lanes: 1, 15 μg of soluble lysate; 2, 1 μg of Ni-NTA eluate; 3, 1 μg of Mono Q eluate. (C) The peak from the Mono Q column shown in B (150 μg of protein) was chromatographed over a Pharmacia 16/60 Superdex 200 gel filtration column, and fractions (0.2 ml) were monitored for absorbance at 280 nm or (D) were analyzed electrophoretically and stained with Coomassie blue.

Northern blot analyses of RGS11 and Gβ5 expression patterns. Blots of (A) 20 μg total RNA or (B and C) 2 μg poly(A+) RNA from various human tissues were serially hybridized with a human RGS11 cDNA probe, a mouse G_β5 cDNA probe, and, as a control for RNA loading and quality, a human glyceraldehyde-3-phosphate dehydrogenase probe.

G_β binding specificity of the GGL domain. HA-tagged G_γ or RGS proteins were either cotranslated in vitro (A and B) or cotransfected into COS-7 cells (C) with individual G_β subunits, immunoprecipitated (IP) with anti-HA mAb, and visualized by SDS/PAGE and autoradiography (A and B) or immunoblotting (C) with indicated antisera (Blot). (A) G_β subunit association in vitro with G_γ1, G_γ2, and full-length RGS11 proteins. (B) G_β subunit association in vitro with truncated RGS7 protein (ΔDΔC, aa 202–395 of SwissProt accession no.P49802), truncated RGS11 proteins (ΔDΔC, aa 219–423; ΔDΔG, aa 283–467), and a chimeric protein (Fusion) composed of the RGS11 GGL domain (aa 219–283) fused to the rat RGS12 PDZ domain (aa 1–91 of SwissProt accession no. O08774). (C) G_β subunit association with G_γ2, full-length RGS11, and truncated RGS11 proteins (ΔD, aa 219–467; ΔDΔG, aa 283–467) in COS-7 cells.

Molecular modeling of the G_β5/GGL domain interface. The model of G_β5 (yellow) associated with the GGL domain of RGS11 (blue) is shown with the N-terminal DEP and C-terminal RGS domains of RGS11 drawn as blue spheres. (Inset) Specific residues at the G_β5–GGL interface. Residues Val-274, Leu-313, and Ala-353 from G_β5, which are thought to be important for specificity, are green. Analogous residues Leu-248, Tyr-251, and Trp-274 from RGS11 are white.

The G_β5/RGS11ΔD heterodimer is a GAP for G_oα. (A) Myristoylated G_oα bound with [γ-³²P]GTP (90 nM) served as the substrate for G_β5/RGS11ΔD in single turnover GAP assays conducted in solution at 4°C. Production of ³²P_i was monitored after the addition of Mg²⁺ to initiate the reaction and either 0, 500 nM, or 1 μM G_β5/RGS11ΔD. Reactions containing 150 nM RGS4 served as positive controls. Data shown are representative of more than three separate experiments. (B) Inhibition of the G_β5/RGS11ΔD-stimulated GTPase activity of G_oα by transition-state complexes of various G_α subunits. Transition-state (GDP-AlF₄⁻) complexes of myristoylated G_oα, myristoylated G_iα1, G_sα, G_qα, and G_tα were incubated with G_β5/RGS11ΔD for 30 min on ice in a buffer containing 10 mM NaF, 5 mM MgCl₂, and 20 μM AlCl₃. This mixture was then diluted 10-fold by addition of [γ-³²P]GTP-G_oα in buffer containing 40 μM GTP, 5.5 mM 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate, 50 mM NaHepes (pH 8.0), 1 mM DTT, 1 mM EDTA, 0.1 mg/ml of BSA, and 4% glycerol. The final concentrations of myristoylated G_oα-GTP substrate and G_β5/RGS11ΔD were 200 nM; the final concentrations of the competing G_α-transition state complexes are indicated. Each point represents the initial rate of GTP hydrolysis, determined by fitting a nine-point time course to a linear regression. The initial rate of GTP hydrolysis by G_oα was 0.04/min in the absence of G_β5/RGS11ΔD and 0.2/min in its presence.