Ric-8 Proteins Are Molecular Chaperones That Direct Nascent G Protein α Subunit Membrane Association

Ric-8A^−/− and Ric-8B^−/− mouse ES cell lines derived from blastocysts are viable

Transgenic Ric-8A– or Ric-8B–deleted C57BL/6J mice were created with VelociGene technology (44). The complete Ric-8A coding sequence or the first three exons and a portion of the third intron of Ric-8B were replaced with LacZ expression cassettes under the control of the endogenous Ric-8 promoters (fig. S1). Homozygous Ric-8A^−/− or Ric-8B^−/− mice were not born and exhibited lethality before E8.5 (n > 100 mice). Heterozygote mice of each strain were reproductive and had no obvious defects. Fifty-three and 49 preimplantation blastocysts (chemically delayed from E3.5 to E6.5) were harvested from Ric-8A^+/− or Ric-8B^+/− intercrosses and grown in culture. Viable inner cell masses were detached and subcultured to enable outgrowth of mouse ES (mES) cell lines. Those surviving derivation resulted in four Ric-8A^+/+, nine Ric-8A^+/−, three Ric-8A^−/−, one Ric-8B^+/+, three Ric-8B^+/−, and seven Ric-8B^−/− viable mES cell lines as determined by genomic polymerase chain reaction (PCR)–based genotyping (fig. S1). Ric-8A and Ric-8B were not required for embryonic development of viable blastocysts, placing the timing of embryonic death between E4 and E8.5 for both gene deletions. No obvious morphological differences were observed among mES cell lines cultured on mitotically inactivated mouse embryonic fibroblasts (MEFs) or in feeder-free culture (Fig. 1A). Ric-8A and Ric-8B promoters were active in mES cell colonies. Dose-dependent β-galactosidase activity was observed as a function of LacZ reporter gene copy number (Fig. 1A). mES cell colonies of each genotype uniformly expressed the pluripotency marker Oct3/4 (Fig. 1A) (45).

Relative amounts of Ric-8A and Ric-8B were measured by quantitative Western blotting of whole-cell lysates prepared from two independently derived wild-type, Ric-8A^−/−, and Ric-8B^−/− cell lines. Ric-8A and two Ric-8B isoforms (Ric-8BFL and Ric-8BΔ9) were not found in the Ric-8A^−/− or Ric-8B^−/− cell lines, respectively (Fig. 1B). The amounts of the Ric-8BFL and Ric-8BΔ9 isoforms were increased ~30 and ~80%, respectively, in the Ric-8A^−/− cells compared to those in wild-type cells, and the amount of actin was reduced by 40%. The amounts of Ric-8A and actin were unchanged in the Ric-8B–deleted cells.

Ric-8A and Ric-8B are required to maintain normal amounts of G protein subunits at steady state

We tested the effects of complete ablation of Ric-8A or Ric-8B on steady-state G protein amounts in crude membrane fractions (150,000g pellets, P150) and whole-cell lysates prepared from duplicate wild-type, Ric-8A^−/−, and Ric-8B^−/− mES cell lines. Equal amounts of total protein (20 μg of P150 fraction and 60 μg of whole-cell lysate) were analyzed by Western blotting with G protein subunit–specific antisera (Fig. 2A). In both Ric-8A^−/− cell lines, we observed a marked reduction in the amounts of Gα_i1/2, Gα_o, Gα_q, and Gα₁₃ in the membrane to <5% of those of wild-type cells. The amount of total Gβ (including Gβ₁ to Gβ₄) was reduced by 40% compared to that in wild-type cells, and the amount of membrane Gα_s was reduced modestly (~20%). The amount of Gα_s protein in the membrane of both Ric-8B^−/− cell lines was reduced by 85% compared to that of wild-type cells, but the amounts of all other G proteins were similar to those of wild-type cells. In whole-cell lysates from Ric-8A^−/−or Ric-8B^−/− cells, the same patterns of overall reduction in the amounts of G proteins were observed, with the exception of Gα_s in the Ric-8A^−/− cells. The magnitude of the reductions in the amounts of Gα_o, Gα₁₃, and Gα_s in whole-cell lysates of Ric-8A^−/− cells was not great as those in the membrane fraction, indicating that the overall defects in G protein abundance were likely a consequence of Ric-8–mediated association of G proteins with the membrane or stabilization of G proteins at the membrane after attainment of membrane residence. In the absence of Ric-8A, some G proteins were partially shifted to the soluble fraction. The amount of polymerized actin (F-actin) was also reduced in the Ric-8A^−/− cells compared to that in wild-type cells, but no such reduction occurred in the Ric-8B–deleted cell lines.

Gα and Gβγ subunit abundances are interdependent (33). We tested whether transient transfection of Ric-8A^−/− and Ric-8B^−/− mES cell lines with plasmids encoding FLAG-tagged Gβ₁ and Gγ₂ subunits would rescue the steady-state deficits in G protein α subunits in these cell lines (fig. S2). In Ric-8A^−/− cells, transient overexpression of Gβγ restored the amount of Gα_s in crude membranes to that of wild-type cells, but the amount of Gα_i1/2 was only rescued from 2.2 to 3.5% of that of wild-type cells. We observed similar percentages in the Gβγ-dependent increase in the amounts of membrane-associated Gα_s and Gα_i1/2 in wild-type cells. In Ric-8B^−/− cells, the amount of membrane Gα_i1/2 was increased in response to the increase in Gβγ abundance, but the amount of membrane Gα_s remained reduced at ~6% of that of wild-type cells.

We then performed genetic complementation tests of the Ric-8A– or Ric-8B–null cell defects. A Ric-8A^−/− mES cell line made to stably express human Ric-8A complementary DNA (cDNA) as a transgene (Tg) did not exhibit the defects in G protein and F-actin abundance that were observed in Ric-8A^−/− cells, and restored the amounts of Ric-8BFL and Ric-8BΔ9 to amounts similar to those in wild-type cells (Fig. 2B). We could not generate genetically complemented Ric-8B^−/− cell lines because stable expression of cDNA encoding Ric-8BFL or Ric-8BΔ9 in mES cells seemed to be repressed. The abundance of the mES cell pluripotency marker Oct3/4 in Ric-8A^−/− cells was 40% greater than that in wild-type cells but was reduced to ~10% above that of wild-type cells upon complementation with Ric-8A. The absence of Ric-8A may contribute to the increased pluripotent potential of mES cells, a role consistent with observations that the single ric-8 genes of C. elegans and Drosophila are required for asymmetric cell division and differentiation (10, 46).

We conducted quantitative PCR analyses comparing the relative abundances of G protein subunit mRNAs in wild-type mES cell lines with those in Ric-8A^−/− or Ric-8B^−/− cell lines to test the possibility that Ric-8 proteins regulated G protein abundance through a pretranslational mechanism (Fig. 2C). The relative amounts of Gα_i, Gα₁₃, Gα_q, and Gβ mRNAs remained largely unchanged in the Ric-8A–null and Ric-8B–null cell lines compared to those in wild-type cells. The abundance of Gα_s mRNA was increased twofold in the Ric-8A^−/− cell lines compared to that in wild-type cells, perhaps through a compensatory mechanism. A modest increase in the abundance of Ric-8B mRNA was observed in the Ric-8A^−/− cells, which coincided with the increased amounts of Ric-8BFL and Ric-8BΔ9 proteins.

G proteins are reduced in abundance but not absent from the plasma membrane in Ric-8A– and Ric-8B–null mES cells

To assess the residency of G protein α subunits and Gβγ dimers in the plasma membranes of wild-type, Ric-8B^−/−, and Ric-8A^−/− cells, we transiently transfected the mES cell lines with plasmids encoding green fluorescent protein (GFP)– or yellow fluorescent protein (YFP)–tagged G protein α subunits and a split YFP pair that reconstituted the Gβ₁γ₇ dimer (Fig. 3) (47). Analysis of confocal microscopy images revealed that GFP-Gα_i, GFP-Gα_q, YFP-Gα_sL, and split YFP-Gβ₁γ₇ were present in the plasma membranes of Ric-8A–deleted cells, but at substantially reduced intensities compared to those in the plasma membranes of transfected wild-type cells. In the Ric-8A^−/− cell line expressing the Ric-8A cDNA, the fluorescence intensities of each expressed G protein in the plasma membrane were restored. In the Ric-8B^−/− cells, only YFP-Gα_sL exhibited a reduced intensity in the plasma membrane. These results were consistent with the patterns of reduced abundances of endogenous G proteins at steady state that were observed earlier (Fig. 2). We conclude that Ric-8 proteins are not essential for the trafficking of overexpressed G protein heterotrimers to the plasma membrane but are indeed necessary to establish or maintain normal amounts of G proteins in the membrane.

The biosynthesis and initial association of G proteins with intracellular membranes are defective in Ric-8A^−/− mES cells

The reduced abundances of select G protein α subunits in Ric-8A– or Ric-8B–null cells might be accounted for if Ric-8 proteins functioned as chaperones that escorted nascent G protein α subunits to the membrane, aided the folding of nascent G α subunits, or both. We developed metabolic labeling and coimmunoprecipitation procedures to address the mechanism of synthesis of G protein α subunits and assembly of G protein heterotrimers in Ric-8A^−/− cells. Wild-type mES cells were first labeled with [³⁵S]methionine and [³⁵S] cysteine for 60 min. Labeled whole-cell lysates were subjected to immunoprecipitation with antiserum (B084) against Gα_i1/2 (48) in the presence of guanosine diphosphate (GDP) or Mg²⁺ and guanosine 5′-O-(3′-thiotriphosphate) (GTP-γ-S). Gβγ is dissociated from Gα when the latter becomes bound to GTP-γ-S. The immunoprecipitates were analyzed by Western blotting with antiserum against Gα_i1/2 and antiserum (U49) against Gβ₁ (48), or were subjected to autoradiography. When GTP-γ-S was included, Gβ₁ was not present in the Gα_i1/2 immunoprecipitates, and two prominent ~37-kD bands were also absent on the autoradiograph (Fig. 4A). We concluded that these bands represented two or more endogenous Gβ isoforms that bound to endogenous Gα_i1/2.

We next measured the time course of Gα_i1/2 synthesis and its association with nascent Gβγ subunits in the Ric-8A^−/− and genetically complemented mES cell lines. The mES cells were metabolically pulse-labeled for up to 1 hour and subjected to immunoprecipitation with antiserum against Gα_i1/2 at the indicated times (Fig. 4B). Gα_i1/2 was synthesized at equivalent rates in Ric-8A^−/− cells stably expressing pcDNA or the human Ric-8A cDNA (Tg), showing that the translation of G protein α subunits was not affected by the loss of Ric-8A; however, we uncovered other differences between these cell lines. In the Ric-8A^−/− cells, two Gα_i species of different apparent molecular masses were reproducibly recovered from the Gα_i1/2 immunoprecipitates over the course of the pulse labeling study, whereas only one species of lower apparent mobility was recovered from the Ric-8A–complemented Ric-8A^−/− cells (Fig. 4B and fig. S3). At 60 min of cell labeling, the ratio of the two Gα_i1/2 species approached 1:1 in the Ric-8A^−/− cells. Before this, most of the nascent Gα_i1/2 had a higher apparent mobility. Gα_i subunits are myristoylated cotranslationally, and the modified and unmodified forms can be resolved by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) (1, 34, 49). We tested whether these might be the two forms of nascent Gα_i observed in Ric-8A^−/− cells by examining the SDS-PAGE mobilities of transiently expressed Glu-Glu–tagged wild-type Gα_i1 or the myristoylation-defective mutant Gα_i1 G2A (fig. S3). Nascent, wild-type Glu-Glu–tagged Gα_i1 migrated distinctly faster than did Glu-Glu–tagged G2A Gα_i1, as was expected for the myristoylated protein; however, a wild-type Glu-Glu–tagged Gα_i1 doublet could not be resolved in samples from Ric-8A^−/− cells that were subjected to immunoprecipitation with antibody against Glu-Glu despite the clear resolution of Glu-Glu–tagged wild-type Gα_i1 and Glu-Glu–tagged G2A Gα_i1 in adjacent lanes. This suggested that the endogenous nascent Gα_i doublet observed in the Ric-8A^−/− cell lines might not correspond to the myristoylated and unmodified forms of the protein. Gα_i may be subject to a hitherto unknown modification during early biosynthesis that is revealed when Ric-8A is not present.

Next, we investigated the temporal association of newly synthesized Gα_i1/2 and Gα_q with cell membranes and the formation of G_i heterotrimers. Ric-8A^−/− cells expressing Ric-8A (Tg) or sham cDNA were pulse-labeled for 10 min and were fractionated into soluble and crude membranes by centrifugation at different chase times (0, 15, and 30 min). Gα_i1/2 or Gα_q was immunoprecipitated from the subcellular fractions and visualized with coimmunoprecipitating Gβγ by autoradiography (Fig. 4C). Gβγ was not efficiently coimmunoprecipitated with the antiserum against Gα_q. At each time of chase, ~45 to 50% of the newly made Gα_i1/2 and ~90% of the newly made Gα_q was defectively retained in the soluble fraction of Ric-8A^−/− cells. The reduced portion of nascent Gα_i1/2 that fractionated with membranes in the Ric-8A^−/− cells became bound to Gβγ with high stoichiometry (~1:1). In the Ric-8A–complemented Ric-8A^−/− cells, membrane-associated nascent Gα_i1/2 reached a stoichiometry of only ~0.4 to 0.6 mol of Gβγ per mole of Gα_i1/2. These results are consistent with the Gα_i:Gβγ stoichiometry measurements made in the whole-cell pulse-labeling experiment (Fig. 4B), but were unexpected given the defects in the steady-state amounts of Gα_i1/2 and total Gβγ in the Ric-8A^−/− cells. We speculate that cotranslationally myristoylated Gα_i is better able to complete initial endomembrane association in the absence of Ric-8A than are other classes of G protein α subunits because these other α subunits are not myristoylated. Although the overall abundance of nascent membrane-bound Gβγ may also be reduced in Ric-8A^−/− cells, the availability of free Gβγ sites to accept nascent, myristoylated Gα_i may actually be higher because of the bulk reduction in the amounts of all membrane Gα subunits.

The defect in the association of Gα_q with membranes was more marked than that of Gα_i1/2 in Ric-8A^−/− cells, and we reasoned that a lengthened kinetic delay might account for this defect. We immunoprecipitated Gα_q with two polyclonal Gα_q-specific antisera (Z808 and Z811) from subcellular fractions of cells pulse-labeled for 2 hours (which approximated the steady-state condition) (Fig. 4D). Nearly all of the Gα_q was membrane-associated in Ric-8A–complemented Ric-8A^−/− mES cells, but a reduced quantity was present predominantly in the cytosol of Ric-8A^−/− cells. Unlike Gα_i1, nascent Gα_q is not cotranslationally lipidated and it has a stringent requirement for Ric-8A for its initial membrane association.

Enhanced G protein turnover accounts for reduced steady-state amounts in Ric-8A–null mES cells

The large Ric-8A–dependent reductions in the steady-state amounts of Gα and total Gβ could be explained if the nascent Gα subunits that did not associate with the membrane were rapidly degraded. We measured the protein turnover rates of endogenous Gα_i1/2, Gα_q, and Gβ₁ in Ric-8A^−/− cells expressing Ric-8A (Tg) or sham cDNA. Cells were pulse-labeled for 60 min and chased over a 24-hour time course. Gα_i1/2, Gα_q, Gβ₁, Ric-8A, and γ-tubulin were individually immunoprecipitated and visualized by SDS-PAGE or autoradiography (Fig. 5). The turnover rates of Gα_i1/2 and Gα_q were enhanced markedly in the Ric-8A–deleted cells, with half-lives (t_1/2) of 2.6 ± 0.6 and 1.7 ± 0.2 hours compared to 16.5 ± 0.7 and 18 ± 0.9 hours, respectively, when Ric-8A was expressed. Gβ₁ was also degraded faster in Ric-8A–deleted cells with a t_1/2 of 3.8 ± 0.7 hours compared to 16.1 ± 2.7 hours in cells in which Ric-8A was expressed. An unrelated control protein, γ-tubulin was degraded with similar kinetics in both cell lines, and the t_1/2 of Ric-8A turnover was 16.2 ± 1.1 hours. In the Ric-8A^−/− cells, the substantial delay in Gβ₁ turnover in comparison to that of Gα (Δt_1/2 ~1.2 to 2.1 hours) may reflect the higher stability of Gβ₁ compared to that of Gα, or it could delineate the sequence of events in the turnover of these proteins in the absence of Ric-8A.

Basal and β-adrenergic–stimulated AC activities are attenuated in Ric-8A^−/− and Ric-8B^−/− mES cells

G protein allosteric modulators of AC enzymes include the stimulatory Gα_s protein and the inhibitory Gα_i protein, as well as Gβγ-specific regulation of AC isoforms (50). Ric-8A deletion resulted in reduced amounts of Gα_i and total Gβ proteins and modestly lowered amounts of membrane-bound Gα_s, whereas deletion of Ric-8B affected the abundance of Gα_s alone. We measured total AC activity in duplicate wild-type and Ric-8–null mES cell lines. Basal amounts of adenosine 3′,5′-monophosphate (cAMP) in Ric-8B^−/− and Ric-8A^−/− were lower than that in wild-type cells (Fig. 6A). Stimulation of wild-type, Ric-8A^−/−, and Ric-8B^−/− cells with a β-adrenergic receptor (β-AR) agonist produced a similar result. The maximal amounts of isoproterenol-induced cAMP were lower in Ric-8B–null cells than in wild-type cells, but were most reduced in Ric-8A–null cells. Isoproterenol was equipotent in each of the three cell types [median effective concentration (EC₅₀) range of ~410 to 515 nM], suggesting that the observed dysregulation of cAMP accumulation did not occur at the level of β-ARs in the absence of Ric-8A or Ric-8B (Fig. 6B). The defects were manifested in the initial phase of cAMP accumulation because a saturating concentration of isoproterenol (10 μM) in the presence of phosphodiesterase inhibitors stimulated wild-type cells to accumulate cAMP at the fastest rate (100 fmol/min) and Ric-8A^−/− cells to accumulate cAMP at the slowest rate (38 fmol/min), whereas Ric-8B^−/− cells produced cAMP at an intermediate rate (58 fmol/min) (Fig. 6C). Treatment of mES cells with forskolin, a direct activator of AC, recapitulated the results observed with isoproterenol. Forskolin potency was largely unaffected by Ric-8A or Ric-8B deletion, but the maximal response to forskolin was blunted the most in Ric-8A^−/− cells and to an intermediate extent in Ric-8B^−/− cells (Fig. 6D and fig. S4). We observed the known synergism between forskolin and isoproterenol in each cell type, but similar overall patterns of cAMP accumulation persisted.

The dose-dependent relationship between isoproterenol and cAMP accumulation in the Ric-8A^−/− mES cell line genetically complemented with Ric-8A (Tg) or expressing sham cDNA showed that stable Ric-8A expression rescued the defect in cAMP accumulation in Ric-8A–null cells (Fig. 6E). In summary, basal as well as hormone- and forskolin-stimulated cAMP amounts were most depressed in Ric-8A^−/− cell lines and to an intermediate extent in the Ric-8B^−/− cell lines. The simplest interpretation of these reductions in the amounts of cAMP in Ric-8B^−/− cells is that they are a result of the ~85% loss in the abundance of the AC activator, Gα_s. It is not intuitively clear why cAMP concentrations were further depressed in Ric-8A–null cells because these cells expressed substantially more Gα_s and less Gα_i than did the Ric-8B^−/− cells. The pronounced reduction in the amount of total G proteins, particularly steady-state Gβγ abundance in the Ric-8A^−/− cells, may have contributed to the reduced total AC activities. Alternatively, misregulation at the level of AC enzyme(s) or combinatorial effects thereof with the reduced G protein amounts seems likely.

Lysophosphatidic acid receptor–dependent and Rho-dependent modulation of F-actin amounts and structure are perturbed in Ric-8A–null mES cells

Lysophosphatidic acid (LPA) receptors activateG_12/13 family G proteins, whose Gα₁₂ and Gα₁₃ subunits in turn bind to p115 Rho GEFs to activate Rho family guanosine triphosphatases (GTPases) (51). Activated Rho signals to alter the actin cytoskeleton and the adhesive properties of cells. Ric-8A stimulates guanine nucleotide exchange on Gα₁₃ (1), and we found that it was required for optimal Gα₁₃ abundance (Fig. 2). Whole-cell actin and precipitable actin (F-actin) were also reduced 40 and 70%, respectively, in Ric-8A^−/− cells, but not in Ric-8B^−/− cells (Figs. 1B and 2A). To test whether the defect in F-actin was attributed to aberrant G_12/13-mediated regulation of Rho signaling, we measured basal (continuous serum only) and LPA-stimulated amounts of Rho-GTP by glutathione S-transferase (GST)–Rhotekin pull-down assays in Ric-8A^−/− mES cells that stably expressed Ric-8A (Tg) or sham cDNA (Fig. 7A). We found that the total amounts of Rho in these cells were equivalent, but that the basal amount of Rho-GTP was markedly reduced in the Ric-8A^−/− cells compared to that in the Ric-8A–complemented Ric-8A^−/− cells. The percentage increase in LPA-stimulated production of Rho-GTP over basal amounts was similar in both cell types, but LPA-induced Rho-GTP amounts were substantially higher in Ric-8A^−/− cells than in Ric-8A^−/− cells complemented with Ric-8A cDNA.

We then transiently transfected the cell lines with plasmids encoding dominant-negative or constitutively active RhoA proteins and incubated the cells with rhodamine-phalloidin to visualize actin microfilaments and filopodia by microscopy (Fig. 7B). Sham-transfected Ric-8A^−/− cells had reduced amounts of actin microfilaments and filopodia-like structures compared to those of Ric-8A^−/− cells stably expressing Ric-8A cDNA (Tg). Expression of dominant-negative RhoA reduced the numbers of phalloidin stained actin structures in the Ric-8A–expressing cells, whereas constitutively active RhoA increased the numbers of such structures in both cell types. Fractionation of total soluble G-actin from precipitable F-actin corroborated the microscopic analysis. The amounts of G-actin were unchanged, but substantially less F-actin was present in the Ric-8A–null cells than was in the Ric-8A–expressing cells (Fig. 7C). Constitutively active RhoA restored the deficit in F-actin observed in Ric-8A^−/− cells. Signaling downstream of Rho was not perturbed in Ric-8A^−/− cells, which implied that the misregulation of the actin cytoskeleton was at the level of Rho activation. Likely candidates of this misregulation include Gα_12/13 and Gα_q subunits because these G proteins activate Rho GEFs and were reduced in abundance >95% in Ric-8A^−/− cells relative to wild-type cells.

Reagents, antibodies, and plasmids

Rabbit polyclonal antiserum 2414 against Ric-8B was produced by Capralogics Inc., using purified full-length Ric-8B protein as an immunogen. Rabbit polyclonal antiserum against Ric-8A was used to detect Ric-8A protein (54). G protein subunit–specific antisera were used to detect Gα_i1/2 (BO84), Gβ_1-4 (B600) (55), Gα_o (S214), Gα_s (584), Gβ₁ (U49) (48), Gα_q (WO82, Z811, and Z808) (56, 57), and Gα₁₃ (B860) (58). Antibodies against actin, α-tubulin, γ-tubulin, and Oct3/4 were from Sigma-Aldrich Inc. Antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was from Santa Cruz Biotechnology Inc. Antibody against Rho (clone 55) was from Millipore. cDNA encoding human Ric-8A was subcloned by linker-based PCR from HeLa cell cDNA into the Nhe I and Not I sites of pcDNA3.1/Hygro or pcDNA3.1/Neo. Plasmids encoding Gα_i-GFP and Gα_q-GFP were gifts from A. Smrcka and S. Malik. Plasmids encoding Gα_sL-YFP, YFP–N-β₁, and YFP–C-γ₇ were gifts from C. Berlot (47, 59). cDNAs encoding human Glu-Glu–tagged Gα_i1, FLAG-Gβ₁, Gγ₂, RhoT19N, and RhoG12V in pcDNA3.1/Neo (Invitrogen) were obtained from the S&T cDNA Resource Center. Sequences encoding wild-type and Glu-Glu–tagged G2A Gα_i1 were amplified by PCR (using a mutant sense strand primer to create G2A) and subcloned into pcDNA3.1/Hygro (Invitrogen).

Engineering of mouse Ric-8A– and Ric-8B–null alleles

Targeted mES cells harboring null alleles of Ric-8A (VelociGene ID: VG #546) or Ric-8B (VelociGene ID: VG#547) were generated with VelociGene technology (44). Bacterial artificial chromosomes (BACs) containing mouse genomic DNA encompassing Ric-8A or Ric-8B were selected by a PCR-based screen from a 129/Svj mouse genomic DNA BAC library (Incyte). The BAC ID for Ric-8A was from BAC ES release 2: 109m11 and contained ~135 kb of genomic DNA. The BAC ID for Ric-8B was from BAC ES release 2: 292o14 and contained ~170 kb of genomic DNA. To generate the targeting vectors, we replaced a 4961–base pair (bp) region of the Ric-8A locus consisting of the entire coding sequence with a transmembrane domain–LacZ/Neomycin phosphotransferase (TM-LacZ/Neo)–encoding cassette by bacterial homologous recombination (BHR) (60-63). An ~30,689-bp region spanning the first three exons and a portion of the third intron of the Ric-8B locus was also replaced with this cassette. In preparation for targeting, the modified BACswere subjected to restriction digest with Not I to generate linear targeting vectors with BAC homology arms of about 40 and 95 kb, for Ric-8A, or 50 and 100 kb, for Ric-8B, flanking the TM-LacZ/Neo cassettes. The ES line F1H4, a C57BL/6-129SvJ hybrid, was electroporated with the linearized constructs. 288 Ric-8A and 288 Ric-8B ES cell clones were selected and genotyped by loss-of-allele (LOA) assays (44). Seventeen of 288 Ric-8A clones and 8 of 288 Ric-8B clones were targeted, indicating targeting frequencies of 5.9 and 2.8%, respectively. Mice were generated by microinjection of the targeted ES cells into C57BL/6 blastocysts and backcrossed to C57BL/6 mice.

Derivation and culture of mES cell lines

Three- to 5-week-old Ric-8A^+/− or Ric-8B^+/− (C57BL/6) mice were superovulated by intraperitoneal injection of 5 IU of pregnant mare serum gonadotropin (PMSG), whichwas followed 48 hours later by injection of 5 IU of human chorionic gonadotropin (hCG) (Sigma-Aldrich). Mice were mated and, 48 hours after fertilization, were injected intraperitoneally with 0.1 mg of tamoxifen and subcutaneously with 3 mg of medroxyprogesterone acetate (Sigma-Aldrich) to prevent blastocyst implantation. After 4 days (delayed ~E6.5), pregnant mice were killed, uteri and oviducts were dissected, and blastocysts were flushed out with Dulbecco’s modified Eagle’s medium (DMEM) buffered with 20 mM Hepes (pH 7.4) (Invitrogen). Blastocysts were cultured individually on 0.1% gelatin–coated plates in RESGRO medium (Millipore). After 2 days of growth, inner cell masses were mechanically detached and subcultured in RESGRO medium. Derived mES cell lines were maintained in ES cell medium [DMEM containing 15% fetal bovine serum (FBS), 1× nonessential amino acid mixture (Invitrogen), 0.1 mM β-mercaptoethanol, and recombinant leukemia inhibitory factor (LIF) (10 μg/ml; Millipore)]. MEFs were isolated from E14.5 wild-type C57BL/6 mouse embryos as described previously (64). To assess β-galactosidase activity in stem cells, we washed the cells with phosphate-buffered saline (PBS), fixed them with 0.2% glutaraldehyde, and stained them in 2 μM MgCl₂, 4 μM ferrous cyanide, 4 μM ferric cyanide, and X-galactosidase (0.5 mg/ml) in PBS at 37°C for up to 2 hours.

AC assays

In standard assays, 4 × 10⁴ ES cells were seeded per well in 96-well plates 24 hours before commencement of the assay. For the basal cAMP cell titration experiment, 1 × 10⁴ to 75 × 10⁴ cells were seeded per well. The medium was exchanged with DMEM containing 20 mM Hepes (pH 7.4), 0.1 mM isobutylmethylxanthine (IBMX), 0.1 mM rolipram, and 0.1% bovine serum albumin (BSA) for all experiments. For the isoproterenol time course experiment, cells were incubated with isoproterenol (10 μM) for 0 to 5 min at 22°C. For the isoproterenol dose-response experiments, cells were incubated with isoproterenol (0 to 10 μM) for 2 min at 22°C. To measure the combined effects of forskolin and isoproterenol, we incubated cells with isoproterenol (100 nM) with or without soluble forskolin (50 μM, Tocris Bioscience) for 5 min at 22°C. Reactions were quenched with the LANCE cAMP Detection Kit lysis reagent (PerkinElmer), and the accumulated cAMP was measured according to the manufacturer’s instructions with a Victor 3V plate reader (PerkinElmer). Signals were measured at 615 and 665 nm.

Measurements of Rho-GTP and F-actin abundance

Cells were either left untreated or treated with oleoyl-l-α-lysophosphatidic acid (LPA, 10 μM; Avanti Polar Lipids Inc.) for 3 min and lysed in the lysis/binding/wash buffer supplied in the EZ-Detect Rho Activation Kit (Pierce). Total protein was quantified by Amido Black Protein Assay (65). Equivalent samples were supplemented with GST-Rhotekin RBD, and Rho-GTPwas isolated according to the manufacturer’s instructions (Pierce). Total Rho and Rho-GTP were visualized by Western blotting analysis with an antibody against Rho that detected Rho protein (clone 55, Millipore). ES cells grown on gelatin-coated glass were rinsed in PBS, fixed with 4% paraformaldeyhyde (PFA), blocked with 5% BSA in PBS, and stained with rhodamine-phalloidin (0.25 U/ml; Invitrogen) and 4′,6-diamidino-2-phenylindole (DAPI) (0.1 μg/ml; Invitrogen). F-actin structures and nuclei were imaged by confocal microscopy with a 60× oil immersion objective [numerical aperture (NA) = 1.35]. For fractionation of F-actin and G-actin, cells were lysed by nitrogen cavitation (Parr Industries), and lysates were centrifuged at 150,000g for 60 min. Laemmli sample buffer was added to the soluble and precipitate fractions. Samples were resolved by SDS-PAGE and analyzed by Western blotting with an antibody against actin (Sigma-Aldrich).

Quantitative PCR analysis

Total RNA was isolated from feeder-free ES cells with the RNA queous RNA isolation kit (Ambion). RNA was converted to cDNA with a Transcriptor First Strand cDNA Synthesis Kit (Roche). PCR assays (each containing 20 μl of reaction volume) were performed in triplicate with an ABI Prism 7000 Real-Time PCR cycler (Invitrogen) and consisted of 1 μg of cDNA, uniquely designed combinations of TaqMan probes and primers (Roche Universal Probe Library Assay Design Center), and FastStart Universal Probe Master Mix with ROX (Roche). The relative amounts of mRNAs in the Ric-8 knockout cell line compared to those in wild-type cells were calculated with the ΔΔC_t method (66) and an actin reference gene TaqMan assay. All TaqMan assays were verified to be >85% efficient.

Metabolic labeling and immunoprecipitations

Feeder-free ES cells were grown in gelatin-coated dishes and starved in cysteine- and methionine-free DMEM (Mediatech Inc.) supplemented with 2 mM l-glutamine, 1× nonessential amino acid mixture (Invitrogen), 0.1 mM β-mercaptoethanol, and 15% dialyzed FBS (Invitrogen) for 20 to 45 min. Cells were metabolically pulse-labeled at 37°C in this medium supplemented with l-[³⁵S]cysteine and l-[³⁵S]methionine (200 to 300 μCi/ml; EXPRE³⁵S³⁵S Protein Labeling Mix, PerkinElmer) for the indicated times. Cells subjected to chase were washed once and incubated at 37°C in chase medium (ES cell medium without LIF, supplemented with 1 mM l-cysteine and 1 mM l-methionine). For biosynthesis and coimmunoprecipitation experiments, labeled cells (from ~90% confluent 60-mm dishes) were washed with ice-cold PBS and solubilized in PBS containing 2% NP-40, 1 μM GDP, and a mixture of protease inhibitors [phenylmethylsulfonyl fluoride (23 μg/ml), N_α-tosyl-l-lysine-chloromethyl ketone (21 μg/ml), l-1-p-tosylamino-2-phenylethyl-chloromethyl ketone (21 μg/ml), leupeptin (3.3 μg/ml), and lima bean trypsin inhibitor (3.3 μg/ml) (Sigma-Aldrich)]. Clarified detergent lysates were incubated with antisera for 18 hours at 4°C followed by incubation with Protein A–Agarose (Roche) for 4 hours at 4°C. Protein A–captured antibody-antigen complexes were solubilized in Laemmli sample buffer and resolved on 12% tris-glycine-SDS gels. Gels were dried and subjected to autoradiography. For subcellular fractionation pulse-chase labeling experiments, labeled cells (from two ~90% confluent 10-cm dishes) were lysed by nitrogen cavitation in fractionation lysis buffer (PBS containing 1 mM EDTA, 1 μM GDP, and protease inhibitor mixture). The postnuclear 500g supernatants were centrifuged at 150,000g for 30 min. Soluble fractions were supplemented with 1% sodium cholate and 1% NP-40, precleared with Protein A–Agarose, and subjected to immunoprecipitation as described earlier. Crude membrane fractions were dounce-homogenized in fractionation lysis buffer containing 1% sodium cholate and 1% NP-40 and rotated for 10 min at 22°C to extract proteins. The membrane extracts were clarified by centrifugation and subjected to immunoprecipitation. For protein turnover measurements, metabolically labeled cells (from ~90% confluent 60-mm dishes) were lysed in cholate lysis buffer [40 mM tris (pH 7.7), 1 mM EGTA, 2 mM MgCl₂, 1% sodium cholate, BSA (100 μg/ml), and protease inhibitor mixture] and clarified by centrifugation at 21,000g. To immunoprecipitate partially denatured individual antigens (Gα_i1/2, Gα_q, Gβ₁, Ric-8A, and γ-tubulin), we added SDS (0.5%) and Triton X-100 (1%) and precleared the supernatants with Protein A–Agarose before immunoprecipitation.

Cell imaging

For Oct3/4 immunofluorescence experiments, cells seeded on coverslips were rinsed twice with PBS and fixed with 4% PFA in PBS for 20 min at 22°C. Fixed cells were treated with PBS containing 0.1% Triton X-100, blocked in 5% BSA in PBS, and incubated with antibody against Oct3/4. Cells were washed and incubated with Alexa Fluor 568–conjugated antibody against mouse antibody (Invitrogen). Washed cells were stained with DAPI, mounted, and imaged with a Nikon TE200 inverted microscope with 10× or 20× objective lenses coupled to a monochrometer-based epifluorescence illumination system (TILL Photonics). Excitation was performed at 405 and 568 nm, and images were captured with a high-speed, digital charge-coupled device (CCD) camera (TILL Photonics). To image live cells that were transfected with plasmids encoding fluorescently tagged G protein subunits, we exchanged the cell medium with Hank’s balanced salt solution (HBSS). Cells were imaged on an Olympus BX61WI upright microscope coupled to an Olympus Fluoview 1000 multiphoton/confocal (FV1000MP) microscope equipped with a suite of diode and gas lasers. Imaging was performed with a 60× water immersion objective (NA = 0.9).

Statistical analysis

Error bars represent the SEM for at least three independent experiments as indicated throughout. Two-tailed unpaired t tests with confidence intervals of 95% were conducted as indicated.