Ric-8A and Ric-8B bind different sets of G protein subunits. A, whole rat brain membrane detergent extracts (11.4 mg) were incubated with 100 μg of purified GST-TEV, GST-TEV-Ric-8A, GST-TEV-Ric-8BFL, or GST-TEV-Ric-8BΔ9 proteins and applied to glutathione-Sepharose 4B resin. The resins were washed, and bound proteins were released by TEV protease digestion. Detergent extract input material (Ext.), the proteins released by TEV digestion, and increasing concentrations of purified G protein subunit standards (Gα_i1, Gα_q, Gα₁₃, Gα_{s short}, Gβ₁γ₂) were resolved by SDS-PAGE. The gels were transferred to nitrocellulose and Western blotted with G protein subunit-specific antisera as indicated. Immunoblot (IB) signals were calibrated by densitometry analysis in supplemental Fig. S2. The total amount of G protein isolated with each GST-Ric-8 bait is reported in ng and as percentage recovery of input. B, TAP-tagged Ric-8A or Ric-8BFL were stably expressed in HeLa S3 cells and purified from soluble (detergent-free) cell lysates by tandem affinity chromatography (glutathione-Sepharose 4B and FLAG affinity resins). Eluates from the FLAG affinity column and the indicated amounts of purified G protein subunit standards were resolved by SDS-PAGE and Western blotted with G protein subunit-specific antisera. The amount of each G protein subunit (pg/μg of input) that co-purified with TAP-tagged Ric-8A or Ric-8BFL was measured by quantitative densitometry analysis of the immunoblots. C, purified GST-TEV-Ric-8 proteins (500 nm) were incubated with purified Gα_i1 or Gα_{s short}, with or without Gβ₁γ₂ (1 μm). The protein mixtures were adsorbed to glutathione-Sepharose 4B resin. The resins were washed, and proteins bound specifically were released by TEV protease digestion. The released proteins were processed in reducing SDS sample buffer, resolved by SDS-PAGE, and visualized by Coomassie Blue staining.

Ric-8B delayed GTPγS binding to nucleotide-free Gα_s after stimulating rapid GDP release. Purified Gα_{s short} or myristoylated Gα_i1 (100 nm each) was loaded to completion with 10 μm [α-³²P]GDP (SA 50,000 cpm/pmol) and then added to reactions with or without purified Ric-8 proteins as indicated. Gα GDP release was measured at 25 °C by quenching aliquots of each reaction in AlF₄⁻-containing buffer and filtering them onto nitrocellulose filters. The filters were washed, dried, and subjected to scintillation counting to quantify the amount of GDP that remained bound to Gα at each time point. The inverse of the percentage of maximal GDP release (■) was co-plotted with the percentage of maximal Ric-8-stimulated (●) or intrinsic (○) GTPγS binding (at 25 °C) over time for Gα_{s short} alone (A), Gα_i1 and Ric-8A (B), Gα_{s short} and Ric-8BFL (C), or Gα_{s short} and Ric-8BΔ9 (D). The data were fit to exponential one-phase association functions using GraphPad Prism version 5.0. All assay results are representative of at least three independent experiments that contained 2–3 replicates/assay.

Ric-8BFL and Ric-8BΔ9 bind differentially to GDP- and GTPγS-bound Gα_{s short} and Gα_q. Ric-8B proteins (5 μm) were mixed with Gα_{s short} (A and B) or Gα_q (10 μm each) (C and D) in the presence of 100 μm GDP or [³⁵S]GTPγS (SA 35,000 cpm/pmol) and incubated for 15 min at 22 °C. The protein/nucleotide mixtures were centrifuged to remove particulate and gel-filtered over Superdex 75 and Superdex 200 columns arranged in tandem. The column eluates were fractionated, and protein-containing fractions were analyzed by Coomassie-stained SDS-PAGE and scintillation counting to quantify the amount of GTPγS contained in each fraction (blue traces, [³⁵S]GTPγS experiments only). UV absorbance traces (AU₂₈₀) of the column eluates for the GDP (red traces) and GTPγS (black traces) experiments were co-plotted with the GTPγS measurements (blue traces) on double-labeled y axis plots. Left to right, species eluted in decreasing molecular weight from the columns.

Ric-8 proteins catalyze GTPγS/GTPγS futile nucleotide exchange. Gα_{s short} (A) and Gα_q (B) were loaded to completion with [³⁵S]GTPγS as described. GTPγS release from Gα (100 nm) was measured over the indicated time courses at 25 °C (Gα_{s short}) or 30 °C (Gα_q) after the addition of 100 μm non-radioactive GTPγS (○) and/or Ric-8BFL (■), Ric-8A (●), and/or Ric-8BΔ9 (▴) (for Gα_{s short} only) (500 nm each) using the GTPγS binding assay nitrocellulose filter binding method. The data were fit to one-phase exponential dissociation functions (Ric-8BFL/Gα_{s short} and Ric-8A/Gα_q) or otherwise plotted by linear regression using GraphPad Prism. Results are the mean ± S.E. of three independent experiments. Note that most error bars are smaller than the actual plotted symbols, and some points were hidden by other data.

Ric-8BFL is a Gα_q, Gα₁₃, and Gα_s/Gα_olf GEF, and Ric-8BΔ9 is a Gα_s GEF. The kinetics of GTPγS binding to Gα_{s short} (A and B), Gα_olf (C), Gα_q (D), Gα₁₃ (E), and Gα_i1 (F) were measured in the absence (○) or presence of Ric-8 proteins (closed symbols). Purified G proteins (100 nm each) were added to reactions containing 10 μm [³⁵S]GTPγS (SA 10,000 cpm/pmol) and the following purified Ric-8 proteins: 200 nm (●), 500 nm (■), and 2 μm (▴) Ric-8BFL or Ric-8BΔ9 or 200 nm Ric-8A (♦) (A and B); 200 nm Ric-8BFL (●), Ric-8BΔ9 (▴), or Ric-8A (♦) or 1 μm Ric-8BFL (■) or Ric-8BΔ9 (▾) (C); 200 nm Ric-8BFL (■), Ric-8BΔ9 (▴), or Ric-8A (●) (D–F). The reactions were incubated at 25 °C (30 °C for Gα_i1) for the indicated times. Triplicate aliquots were withdrawn from the reactions, quenched, and filtered through nitrocellulose filters. The filters were washed, dried, and subjected to scintillation counting to quantify the amount of G protein-bound GTPγS at each time point. The data were fit to exponential one-phase association functions or linear regression using GraphPad Prism version 5.0. Results are presented as the mean ± S.E. (error bars) of three experiments. A (inset), each data set was plotted as the percentage of maximal GTPγS bound to show that Ric-8BFL stimulated the rate of observed Gα_{s short} GTPγS binding with increasing Ric-8BFL concentration. Notes that most error bars are smaller than actual plotted symbols. Intrinsic Gα rates (○) were measured in each experiment, although these points were often hidden by other data.

Ric-8B regulation of Gα_{s short} and Gα_q steady state GTP hydrolytic activities are GTP-dependent. Gα_{s short} (A, C, and E) or Gα_q (B, D, and F) (50 nm each, total of 1 pmol/assay) was mixed in triplicate with Ric-8BFL (■), Ric-8BΔ9 (▴), or Ric-8A (●) (0–2.5 μm) and the indicated concentrations of [γ-³²P]GTP (SA 10,000–70,000 cpm/pmol) to initiate steady state GTPase reactions at 25 °C. The reactions were quenched after 5–7 min in acidic charcoal suspension and processed as described. C, the Ric-8BΔ9 (△) preparation did not contain a contaminating GTPase. In the GTP titration experiments (E and F), Gα alone (○) or Gα and the indicated Ric-8 proteins (500 nm each, closed symbols) were used. All assay results are representative of at least three independent experiments that contained 3 replicates/assay. Error bars, S.E.

Ric-8 proteins do not affect Gα_{s short} single turnover GTPase activity. Gα_{s short} was loaded with [γ-³²P]GTP at 25 °C in buffer lacking Mg⁺² and separated from free GTP by rapid gel filtration. Gα_{s short}-[γ-³²P]GTP (60 nm, actual concentration) single turnover GTPase reactions were initiated at 4 °C by the addition of buffer containing MgCl₂ (○), MgCl₂ and Ric-8BFL (■), Ric-8BΔ9 (▴), or Ric-8A (●) (500 nm each). Duplicate reactions were quenched in acidic charcoal suspension at the indicated times and processed as described. Data are representative of three or more independent experiments. The mol of phosphate (Pi) released/mol of Gα_{s short} over time were plotted using GraphPad Prism version 5.0 and one-phase association functions. Note that some points were hidden by other data.

Ric-8B regulation of Gα_s catalysis. Ric-8B is a Gα_s GRF with GTP-dependent GEF activity. At low GTP (<10 μm), Ric-8B-stimulated Gα_{s short} GDP release (step 1) was significantly faster than observed Ric-8B-stimulated GTP(γS) binding (step 1 plus step 2), whereas intrinsic Gα_{s short} GDP release and observed GTPγS binding rates were equivalent. Ric-8B-FL potently inhibited Gα_{s short} steady state GTPase activity (step 5) at low GTP, due to its capacity to dramatically increase the K_m of GTP for Gα_{s short} (∼385 nm to an estimated value of ∼42 μm) and to stimulate futile GTP for GTP exchange (step 3). Ric-8BΔ9 did not increase the K_m of GTP for Gα_{s short} nearly as much (∼2.6 μm) and did not stimulate futile GTP/GTP exchange. These results probably reflect a higher affinity that Ric-8BFL has over Ric-8BΔ9 for Gα_s. Neither Ric-8B isoform had any effect on Gα_s single turnover GTPase activity (step 4). At higher GTP (>10 μm), Ric-8BFL and Ric-8BΔ9 stimulated Gα_{s short} nucleotide exchange and steady state GTPase activities. At physiological GTP, Ric-8B-catalyzed exchange is predicted to proceed in the forward direction to produce activated Gα_s-(GTP).