Mammalian Ric-8A (Synembryn) Is a Heterotrimeric Galpha Protein Guanine Nucleotide Exchange Factor*

Gregory G. Tall, Andrejs M. Krumins, and Alfred G. GilmanDagger

From the Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9041

Received for publication, November 21, 2002, and in revised form, December 26, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The activation of heterotrimeric G proteins is accomplished primarily by the guanine nucleotide exchange activity of ligand-bound G protein-coupled receptors. The existence of nonreceptor guanine nucleotide exchange factors for G proteins has also been postulated. Yeast two-hybrid screens with Galpha o and Galpha s as baits were performed to identify binding partners of these proteins. Two mammalian homologs of the Caenorhabditis elegans protein Ric-8 were identified in these screens: Ric-8A (Ric-8/synembryn) and Ric-8B. Purification and biochemical characterization of recombinant Ric-8A revealed that it is a potent guanine nucleotide exchange factor for a subset of Galpha proteins including Galpha q, Galpha i1, and Galpha o, but not Galpha s. The mechanism of Ric-8A-mediated guanine nucleotide exchange was elucidated. Ric-8A interacts with GDP-bound Galpha proteins, stimulates release of GDP, and forms a stable nucleotide-free transition state complex with the Galpha protein; this complex dissociates upon binding of GTP to Galpha .

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Heterotrimeric guanine nucleotide-binding regulatory proteins mediate signal transduction between many membrane-bound receptors and intracellular effectors (1). Traditionally, activation of heterotrimeric G proteins1 is accomplished exclusively by the action of GPCRs, seven transmembrane-spanning proteins that typically reside in the plasma membrane. These receptors act as guanine nucleotide exchange factors (GEFs), binding the inactive GDP-bound conformation of G proteins and stimulating release of GDP from Galpha . To ensure directionality of exchange, GEFs stabilize a nucleotide-free transition state of Galpha that is disrupted by binding of GTP (2, 3). This facilitates dissociation of Galpha ·GTP from the Gbeta gamma dimer and release of these proteins from the receptor. Dissociated G protein subunits then participate in interactions with a variety of effectors.

G protein signaling is attenuated when Galpha hydrolyzes the gamma  phosphate of its bound GTP and Galpha ·GDP reassociates with beta gamma . GTPase-activating proteins (GAPs) facilitate the inactivation of many G proteins. Most of these GAPs contain a regulator of G protein signaling (RGS) domain that binds preferentially to the Galpha ·GTP transition state and accelerates GTPase activity (4, 5). More than 20 unique RGS domain-containing proteins have been discovered, and the nature of their G protein specificity and their mode of action in cells are subjects of intense interest (6, 7).

Nonreceptor activators of G proteins may operate in lieu of or in conjunction with GPCRs to enhance signaling, but their physiological role is not well understood (8-11). Activators of G protein signaling AGS1 and AGS3 were identified in a genetic screen in yeast designed to isolate expressed mammalian cDNAs that encode proteins that bypass the need for a receptor (12). AGS3 possesses Galpha guanine nucleotide dissociation inhibitor activity but may activate G proteins by liberating Gbeta gamma (10, 13). AGS1 encodes a Ras-like small GTPase that, when bound to GTP, possesses in vitro guanine nucleotide exchange activity for members of the Gi subfamily of G proteins (9).

Other newly appreciated proteins may also act as novel modulators or effectors of heterotrimeric G proteins. Genetic evidence indicates that synaptic transmission in Caenorhabditis elegans is controlled by an antagonistic Gq/Go signaling network that regulates intracellular concentrations of diacylglycerol (14-16). Galpha q is thought to enhance synaptic transmission by activation of phospholipase Cbeta , an enzyme that cleaves phosphatidylinositol 4,5-bisphosphate into diacylglycerol and inositol triphosphate (for review, see Ref. 17). Activation of Go negatively influences synaptic transmission, hypothetically by lowering concentrations of diacylglycerol (mechanism unknown). Mutant screening and suppression analyses have revealed proteins that likely operate in conjunction with Galpha q or Galpha o in this pathway (18, 19). One class of these mutations was termed RIC (resistant to inhibitors of cholinesterase). They were selected for their ability to survive the neurotoxic effects of cholinesterase inhibitors by causing a decrease in the amount of acetylcholine released at the synapse (18). The gene that complemented the ric-8 mutant allele, RIC-8/synembryn, appears to promote synaptic transmission much like Galpha q. However, the sequence of the encoded protein was not revealing (20). A more recent study also demonstrated that reduction of function mutations of C. elegans RIC-8 or GAO-1 affects a pathway that regulates the movement of centrosomes in dividing embryos (21).

In an effort to identify novel G protein signaling factors, we initiated yeast two-hybrid screens with Galpha o and Galpha s as baits and found that two mammalian homologs of Ric-8/synembryn, which we have termed Ric-8A and Ric-8B, are in fact Galpha -binding proteins. Purification and biochemical characterization of recombinant Ric-8A revealed that it is a potent guanine nucleotide exchange factor for Galpha q, Galpha i1, and Galpha o but not Galpha s. The mechanism of Ric-8A-mediated guanine nucleotide exchange was elucidated. Ric-8A interacts with GDP-bound Galpha subunits in the absence of Gbeta gamma , causing release of GDP and formation of a stable, nucleotide-free Galpha ·Ric-8A complex. GTP, but not GDP, and then binds to Galpha and disrupts the complex, releasing Ric-8A and the activated Galpha protein.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Molecular Cloning and Yeast Two-hybrid Experiments-- The open reading frames of wild type and the activated (GTPase-deficient, Q227L of Galpha s) mutants of Galpha s long, Galpha o, Galpha q, Galpha i1, and Galpha 13 were amplified using the polymerase chain reaction with oligonucleotides containing BamHI and/or SalI linkers. The respective Galpha PCR products were digested and cloned into these same sites in the LexA two-hybrid bait vector, pVJL11 (22). To initiate two-hybrid screens, the LexA-Galpha o and LexA-Galpha s bait constructs were transformed into the L40 yeast strain using the TE-lithium acetate/PEG-4000 transformation method (23). Yeast harboring these bait plasmids were then transformed with a rat brain embryonic cDNA library contained in a pVP16 activation domain prey vector (a gift of Thomas Südhof, University of Texas Southwestern Medical Center). Approximately 106 individual transformants were screened with each bait. For studies of pairwise two-hybrid interactions, yeast co-transformed with specific bait and prey fusion combinations growing in liquid culture were spotted onto complete synthetic medium plates lacking tryptophan and leucine, grown, and subjected to a beta -galactosidase filter assay as described (24).

The rat RIC-8A prey clone isolated in the two-hybrid screen lacked the first 9 coding nucleotides of the equivalent mouse and human RIC-8/synembryn sequences in the data base. These missing nucleotides were repaired by PCR with codons for the first three amino acids of mouse RIC-8/synembryn (NP_444424). These residues are identical among mouse and human Ric-8A and C. elegans Ric-8. The repaired rat RIC-8A PCR product was cloned into the baculovirus donor construct, pFastBacGSTTev. pFastBacGSTTev was created by inserting a GST tag and a Tev protease cleavage site into the vector pFastBac-1 (Invitrogen, Inc.). The open reading frame of the rat RIC-8B clone obtained in the two-hybrid screen was amplified by PCR with a 5' oligonucleotide containing a BamHI linker and an oligonucleotide that annealed 3' to the gene in the prey vector. This product was digested with BamHI and SalI and cloned into the His-tagged baculovirus donor vector pFastBacHTa (Invitrogen).

To clone the human cDNA transcripts that encode the 531 and the putative 401-amino acid Ric-8A variants (see "Results"), total RNA was prepared from HeLa cells using an RNeasy Maxiprep kit (Qiagen), and cDNA was generated using a RETROscript kit (Ambion, Inc.). The human RIC-8A cDNA encoding the 531-amino acid protein was amplified using PCR primers derived from the DNA sequence in GenBankTM (AL390088). Primers designed to amplify the hypothetical 401-amino acid variant were derived from the incomplete cDNA sequence (AK022870) found in GenBankTM, but we were not able to amplify any product from either HeLa or NTERA2 cell cDNA. This variant was generated using the 531-amino acid protein-encoding cDNA as PCR template and a 3' PCR primer that corrected the missing single base pair insertion of this putative cDNA. Both amplified products contained EcoRI and SalI linkers added by the primers. The products were digested with these restriction enzymes and cloned into those restriction sites in the mammalian expression vector pCMV5 (25), the two-hybrid prey vector pGADGH (26), pFastBacHTa (Invitrogen, Inc.), and pFastBacGSTTev.

Protein Purification-- Baculoviruses encoding GST-Ric-8A and His6-Ric-8B were produced and amplified using methods in the Bac-to-Bac Sf9 cell transfection system (Invitrogen, Inc.). To prepare the recombinant Ric-8A, Sf9 cells growing in IPL41 medium with 1% fetal bovine serum, 0.1% pluronic acid, 1% chemically defined lipid concentrate (Invitrogen), and 10 µg/ml gentamicin at a density of 2 × 106 cells/ml were infected for 48 h with amplified GST-Ric-8A baculoviruses. These cells were pelleted and lysed in lysis buffer (20 mM NaHepes, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, and protease inhibitor mixture (phenylmethylsulfonyl fluoride, Nalpha -p-tosyl-L-lysine-chloromethyl ketone, N-tosyl-L-phenylalanine-chloromethyl ketone, leupeptin, and lima bean trypsin inhibitor; Sigma)) by nitrogen cavitation. GST-Ric-8A was purified from the 100,000 × g supernatant fraction of these lysates by adsorbtion to glutathione Sepharose 4B (Amersham Biosciences). The fusion protein was eluted from the washed glutathione Sepharose with lysis buffer containing 20 mM reduced glutathione and then exchanged into lysis buffer. To remove the GST tag, the glutathione resin with attached GST-Ric-8A protein was washed with lysis buffer and incubated with Tev protease (Invitrogen) for 16 h at 4 °C. The cleaved, eluted Ric-8A protein (amino acid sequence G-E-F-Ric-8A) was further purified on a Hi-trap Q column (Amersham Biosciences) and eluted with a linear gradient of NaCl (150 mM to 1 M). The active protein eluted at ~375 mM NaCl and was exchanged into lysis buffer. Active Tev-digested Ric-8A was resolved by 11% SDS-PAGE and visualized with Coomassie Blue; it was also trypsinized and subjected to mass spectrometry. Despite the fact that Tev-digested Ric-8A co-migrated with protein standards of ~75 kDa on SDS-PAGE, the predicted mass of the purified protein (>95%) of 60.1 kDa was confirmed by mass spectrometry (C. Thomas, University of Texas Southwestern Medical Center). Attempts to purify nonaggregated His6-Ric-8B have been unsuccessful to date.

Myristoylated Galpha o and nonmyristoylated Galpha i1 were purified from Escherichia coli as previously described (27). Myristoylated Galpha i1 containing a His6 tag inserted after amino acid 121 was purified from E. coli and was a gift from Roger K. Sunahara (University of Michigan). Galpha s short was purified from E. coli and was a gift from Mark E. Hatley (University of Texas Southwestern Medical Center). Galpha q and beta 1gamma 2 dimers were purified from Sf9 cells as previously described (28).

GST-Ric-8A Binding to G Proteins in Membrane Detergent Extracts-- GST-Ric-8A affinity resin or control resin was prepared by incubating purified GST-Ric-8A (37 µg) or buffer with glutathione-Sepharose 4B (bed volume, 25 µl) (Amersham Biosciences) for 1 h at 4 °C in extraction buffer (20 mM NaHepes, pH 8.0, 150 mM NaCl, 2 mM MgSO4, 1 mM EDTA, 1 mM dithiothreitol, and 1% C12E10). The resin was collected by brief centrifugation and washed twice with 1 ml of extraction buffer.

Rat brain membrane extracts were prepared by homogenizing whole rat brains in 10 mM Tris-HCl, pH 8.0, 11% sucrose, and protease inhibitor mixture using a Dounce homogenizer. The homogenate was centrifuged at 500 × g to remove debris, and the supernatant was sequentially centrifuged at 12,000 × g and 100,000 × g. The 100,000 × g pellet (P100 membranes) was solubilized for 1 h in extraction buffer and centrifuged again at 100,000 × g. A volume of detergent extract corresponding to 8 mg of extracted membrane protein was then incubated with the GST-Ric-8A and control glutathione resins. After incubation at 4 °C for 1 h, the resins were washed twice with 1 ml of extraction buffer, suspended in 45 µl of extraction buffer and incubated for 16 h at 4 °C with 5 µl of recombinant Tev protease (Invitrogen). The resins were collected by centrifugation, and the soluble proteins released by Tev protease were resolved by SDS-PAGE; the gels also contained purified G protein subunit standards. The gels were transferred to nitrocellulose and blotted with the following G protein subunit-specific antisera: W082, Galpha q (29); S214, Galpha o (30); B087, Galpha i1 and Galpha i2 (31); 584, Galpha s (32); B860, Galpha 13 (33); and B600, Gbeta s1-4 (31). To visualize the bands recognized by G protein antisera, the blots were processed with secondary antibody conjugated to horseradish peroxidase and treated with SuperSignal West Femto chemiluminescent reagent (Pierce) diluted 3-fold in phosphate-buffered saline.

Formation of a Galpha ·Ric-8A Complex-- Purified myristoylated Galpha i1 (600 nM) was incubated in 500 µl of HEMNDL buffer (20 mM NaHepes, pH 8.0, 1 mM EDTA, 2 mM MgSO4, 150 mM NaCl, 1 mM dithiothreitol, and 0.05% C12E10) containing either 30 µM GDP; 30 µM GTPgamma S; or 10 mM NaF, 30 µM AlCl3, and 30 µM GDP for 1 h at 30 °C. Purified GST-Ric-8A was then added to a final concentration of 200 nM, and these reactions were incubated for an additional 20 min at 20 °C. Glutathione-Sepharose (bed volume, 25 µl) in HEMNDL buffer was added, and the reactions were incubated for 1 h at 4 °C with gentle agitation. The resin was collected and washed consecutively with three 1-ml aliquots of cold HEMNDL buffer containing the respective nucleotide and/or AlF<UP><SUB>4</SUB><SUP>−1</SUP></UP> and then boiled in SDS-PAGE sample buffer. The samples were resolved by 11% SDS-PAGE, and the gels were stained with Coomassie Blue.

Gel Filtration of the Ric-8A·Galpha i1 Complex-- Nonmyristoylated Galpha i1 was chosen for gel filtration analysis in lieu of myristoylated Galpha i1 protein because the myristoylated protein and its associated detergent gave aberrant elution profiles on gel filtration columns. Nonmyristoylated Galpha i1 (26 µM) was incubated for 1 h at 30 °C with either 260 µM [35S]GTPgamma S or [alpha -32P]GDP in gel filtration buffer (20 mM NaHepes, pH 8.0, 150 mM NaCl, 2 mM dithiothreitol, 2 mM MgSO4, 1 mM EDTA, and protease inhibitor mixture). The reaction mixtures were then diluted 2-fold by the addition of Tev-digested Ric-8A to a final concentration of 10 µM in gel filtration buffer and were subsequently incubated for 5 min at 22 °C. The mixtures were applied to tandem Superdex 75/200 columns (HR 10/30) and resolved at a flow rate of 0.2 ml/min. The amount of protein and radiolabeled nucleotide in each fraction was determined by Bradford assay and scintillation counting, respectively. A fixed volume of each odd-numbered fraction (based on a sample of 750 ng of protein in the peak protein-containing fraction) was resolved by SDS-PAGE, and the proteins were visualized with Coomassie Blue.

Effect of Ric-8A on GTPgamma S Binding and Steady-state GTPase Activity of Galpha Proteins-- The kinetics of GTPgamma S binding was assessed using a filter binding method (34). The reactions with myristoylated Galpha i1, myristoylated Galpha o, and Galpha s contained 20 mM NaHepes, pH 8.0, 100 mM NaCl, 10 mM free Mg2+, 1 mM dithiothreitol, and 0.05% C12E10. The reaction mixture for Galpha q was identical except that 0.05% Genapol C-100 detergent was used instead of C12E10. For each reaction, Tev-digested Ric-8A (200 nM) was first added to reaction buffer and 10 µM [35S]GTPgamma S (10,000 cpm/pmol). To initiate the reactions, each Galpha protein was added to a final concentration of 200 nM. Duplicate aliquots were removed at indicated times, and binding of radioactive nucleotide was stopped by the addition of ice-cold buffer containing 20 mM Tris-HCl, pH 7.7, 100 mM NaCl, 2 mM MgSO4, 0.05% C12E10, and 1 mM GTP. The quenched reactions were passed through BA-85 nitrocellulose filters and washed (20 mM Tris-HCl, pH 7.7, 100 mM NaCl, 2 mM MgSO4); the filters were dried and subjected to liquid scintillation counting. All of the reactions were conducted at 20 °C with the exception of those with Galpha i1, which were performed at 30 °C.

Steady-state GTPase reactions with Galpha q were conducted at 20 °C in buffer containing 0.05% Genapol C-100, 1 µM [gamma -32P]GTP (40,000 cpm/pmol), and the indicated amounts of Tev-digested Ric-8A and/or Gbeta 1gamma 2. The reactions were initiated by adding purified Galpha q or Galpha qbeta 1gamma 2 to a final concentration of 45 nM. Aliquots of the reactions were removed at 2, 4, 6, 8, and 10 min, and the amount of released Pi was quantified using a charcoal-based method (35).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mammalian Ric-8A and Ric-8B Interact with Subsets of Galpha Proteins in Yeast Two-hybrid Assays-- L40 yeast expressing LexA bait fusion proteins of the activated (GTPase-deficient) mutants of Galpha o and Galpha s long were transformed with a rat brain pVP16-cDNA fusion library. Approximately 106 individual transformants were screened with each bait using the HIS3 reporter of L40 and a beta -galactosidase filter assay (24). Sequence analysis of 240 plasmids rescued from positive Galpha o transformants revealed cDNA inserts that encode portions of several previously identified Galpha -interacting proteins, including: GRIN1 (36), GRIN2 (36), Ras GAP III (37), Rap1GAP (38), AGS3 (12), LGN (39), RGS8 (40), and RGS17 (RGSZ2) (38). One of the clones encoded an open reading frame, less the first three amino acids, that shared 97% amino acid identity with mouse Ric-8/synembryn (NP_444424) and 87% amino acid identity with the predicted sequence of a 531-amino acid human hypothetical protein (NP_068751). We have named these homologs Ric-8A. The rat Ric-8A protein found in the two-hybrid screen is compared with three different putative human Ric-8A proteins in Fig. 1A. The human RIC-8A cDNAs that encode proteins of 531 and 537 amino acids are likely the products of alternative RNA splicing. The sequence of a cDNA encoding the 531-amino acid variant was verified from a clone isolated from HeLa cells (not shown). Inspection of the complete human RIC-8A genomic sequence contained in a BAC clone (AC069287) revealed that the 531- and 537-amino acid encoding transcripts could be produced by the use of two alternative consensus splice acceptor sites of exon 7 (not shown). The hypothetical RIC-8A transcript predicted to encode the 401-amino acid Ric-8A protein is derived from insertion of a single base pair at codon 395 of the transcript encoding the 531-residue protein. Interestingly, this frameshift would result in synthesis of a protein with five different carboxyl-terminal amino acids and a new stop codon that form a consensus CAAX box of the type that would be modified with a geranylgeranyl moiety (Fig. 1A) (41). However, all attempts to verify the existence of this apparent point mutation using the cDNA source reported in GenBankTM (AK022870) have been unsuccessful.


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Fig. 1.   A, three putative human RIC-8A cDNAs are homologous to the rat RIC-8A clone isolated in the two-hybrid screen. The 531-amino acid human Ric-8A protein (human Ric-8A (531)) (NP_068751) is the equivalent of the 529-amino acid rat protein. A predicted 537-amino acid human Ric-8A protein (human Ric-8A (537)) (CAB66869) is identical to the 531-residue protein except for a 6-amino acid insertion, including a polyproline sequence, at codon 355. The third putative variant encodes a protein that consists of the amino-terminal 396 residues of the 531-amino acid protein with the addition of five amino acid residues that form a consensus CAAX box (CAAX-Ric-8A). B, the human RIC-8B cDNA (NM_018157) is predicted to encode a protein of 536 amino acid residues. The amino-terminal 483 amino acids of human Ric-8B and the protein encoded by the rat RIC-8B cDNA obtained in the two-hybrid screen are homologous. The remaining divergent 37 carboxyl-terminal amino acids encoded by the rat cDNA are likely the product of alternative splicing.

Twenty-three positive clones were obtained using Galpha s as bait, and of these, none encoded previously known Galpha -interacting proteins. One positive clone shared significant sequence identity with the human cDNA FLJ10620 (NM_018157). Human FLJ10620 encodes a protein of 536 amino acid residues that shares ~40% amino acid identity with mouse Ric-8/synembryn (Ric-8A). The rat clone obtained in the two-hybrid screen encodes a 520-residue protein. The amino-terminal 483 residues of this protein are 86% identical to the amino-terminal 483 amino acids of human FLJ10620. The remaining carboxyl-terminal amino acids of both proteins are completely divergent. We have named FLJ10620 and its rat homolog Ric-8B (Fig. 1B). Analysis of the RIC-8B genomic sequence from a compilation of BAC clones revealed that the Human cDNA FLJ10620 contains an extra 3' splice site that was not utilized in the Rat RIC-8B cDNA we obtained. This likely manner of alternative splicing explains the divergence of the carboxyl termini of rat and human Ric-8B.

To test the interactions of Ric-8A and Ric-8B with various Galpha proteins in the two-hybrid system, the full-length rat Ric-8A and Ric-8B preys were individually co-transformed with wild type and GTPase-deficient (QL) Galpha baits: Galpha i1, Galpha o, Galpha q, Galpha s long, and Galpha 13. Suspensions of these transformed yeast were spotted onto synthetic medium plates lacking tryptophan and leucine and tested for positive interactions between bait and prey using a beta -galactosidase filter activity assay (24). The Ric-8A prey yielded a strong positive signal with Galpha i1, Galpha o, and Galpha q and a weak signal with Galpha 13. No interaction was observed with Galpha s long (Fig. 2). The Ric-8A prey also showed a clear preference for interaction with the wild type Galpha i1 bait compared with its GTPase-defective counterpart but did not show this preference for the other Galpha baits tested. The Ric-8B prey showed a strong signal with Galpha q and a weak signal with the Galpha s long bait (Fig. 2). Much like the Ric-8A/Galpha i1 interaction, the Ric-8B prey interacted preferentially with the wild type Galpha s long bait. Intriguingly, the common two-hybrid interaction partner for these two mammalian Ric-8 homologs is Galpha q, the Galpha that was first shown to interact genetically with RIC-8 in C. elegans (20).


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Fig. 2.   Ric-8A and Ric-8B interact with different subsets of Galpha proteins in the yeast two-hybrid system. L40 yeast were co-transformed with rat Ric-8A or Ric-8B prey plasmids and the indicated wild type (WT) or activated (GTPase-deficient) mutant (QL) Galpha bait plasmids. The transformants were grown on a minimal medium plate lacking tryptophan and leucine and assayed for beta -galactosidase activity to assess positive interactions between bait and prey. Vec, vector.

GST-Ric-8A Interacts with Endogenous Membrane-derived Galpha Proteins-- To investigate whether Ric-8A interacts directly with membrane-derived Galpha proteins, a GST-full-length rat Ric-8A fusion protein was expressed and purified from baculovirus-infected Sf9 cells and used to bind G proteins present in a detergent extract of rat brain membranes. Three controls were used in this experiment: GST-Ric-8A incubated with glutathione Sepharose alone (Fig. 3, lane 1), GST-Ric-8A incubated with rat brain membrane extract and glutathione Sepharose (Fig. 3, lane 2), and brain membrane extract incubated with glutathione Sepharose alone (Fig. 3, lane 3). Proteins adsorbed to Sepharose were released by incubation with Tev protease, resolved by SDS-PAGE, and either transferred to nitrocellulose for Western blotting with G protein subunit-specific antisera or stained with Coomassie Blue. Specific binding of a protein to Ric-8A was indicated when unique protein bands were found only in the experiments where GST-Ric-8A and extract were incubated together (Fig. 3, lane 2). These bands were isolated from the gels, trypsinized, and analyzed by mass spectrometry. Unique protein bands of ~40 kDa were identified as Galpha o and a mixture of Galpha i1, Galpha i2, or Galpha i3 (Hongjun Shu, University of Texas Southwestern Medical Center). The immunoblots shown in Fig. 3 confirmed the mass spectrometry and also revealed the presence of immunoreactive Galpha q and Galpha 13. Neither Galpha s nor G protein beta  subunits were detected (Fig. 3, lane 2).


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Fig. 3.   Ric-8A binds a subset of brain membrane Galpha proteins. Glutathione-Sepharose was incubated with either GST-Ric-8A (lanes 1 and 2) or buffer (lane 3) and then washed. A detergent extract from rat brain membranes was incubated with the GST-Ric-8A beads (lane 2) or the buffer-treated beads (lane 3), and the beads were washed. Proteins bound to the glutathione Sepharose in each condition were released with Tev protease. The eluants from each condition were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with G protein subunit-specific antisera as described under "Experimental Procedures." Std, standard.

Ric-8A Interacts Preferentially with Nucleotide-free Galpha -- To understand the biochemical consequences of the interaction between Ric-8A and Galpha proteins, we first determined whether association was dependent on the identity of the guanine nucleotide bound to Galpha . Purified myristoylated Galpha i1 was first incubated with GDP, GDP and AlF<UP><SUB>4</SUB><SUP>−1</SUP></UP>, or GTPgamma S. These proteins were then further incubated with GST-Ric-8A and bound to glutathione Sepharose. The beads were extensively washed, and the bound proteins were eluted and resolved by SDS-PAGE (Fig. 4). GST-Ric-8A interacted preferentially with GDP-bound Galpha i1 (lanes 5 and 6), less well with Galpha i1·GDP·AlF<UP><SUB>4</SUB><SUP>−1</SUP></UP> (lanes 3 and 4), and not detectably with Galpha i1·GTPgamma S (lanes 1 and 2).


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Fig. 4.   Ric-8A binds to the GDP-bound form of myristoylated Galpha i1. Duplicated aliquots of purified myristoylated Galpha i1 (600 nM) were incubated for 1 h with GTPgamma S, GDP, or GDP and AlF<UP><SUB>4</SUB><SUP>−1</SUP></UP> as indicated. Purified GST-Ric-8A was then added to each reaction (final concentration, 200 nM). These mixtures were bound to glutathione Sepharose and washed extensively with buffer containing the indicated guanine nucleotide or GDP and AlF<UP><SUB>4</SUB><SUP>−1</SUP></UP>. The proteins were eluted from the beads with sample buffer, resolved by SDS-PAGE, and stained with Coomassie Blue.

To further explore the identity of any guanine nucleotide associated with the complex of Galpha i1 and Ric-8A, we gel-filtered Ric-8A·Galpha i1 complexes prepared by incubation of Ric-8A with Galpha i1 bound to radiolabeled GDP or GTPgamma S. The fractions were analyzed by SDS-PAGE and subjected to Bradford analysis and scintillation counting to quantify the amounts of complex, monomeric Ric-8A, monomeric Galpha i1, and guanine nucleotide present in each fraction (Fig. 5). SDS-PAGE showed that Ric-8A forms a stable, apparently stoichiometric complex with Galpha i1 that had been prelabeled with [alpha -32P]GDP (Fig. 5A) but not with [35S]GTPgamma S (Fig. 5B). All of the detectable [alpha -32P]GDP and [35S]GTPgamma S were associated with uncomplexed, monomeric Galpha i1. The Ric-8A·Galpha i1 complex was free of nucleotide (Fig. 5A). When the fractions containing the Ric-8·Galpha i1 complex shown in Fig. 5A (GDP experiment) were pooled, concentrated, and rerun over the gel filtration columns, the complex remained intact (data not shown). However, when the complex was incubated with GTPgamma S and then gel-filtered again, the majority of the complex was dissociated (data not shown). Together, these gel filtration experiments suggest strongly that Ric-8A acts as a Galpha protein guanine nucleotide exchange factor, interacting with the GDP-bound form of Galpha to form a stable nucleotide free complex that is disrupted upon binding of GTP.


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Fig. 5.   Ric-8A interacts with GDP·Galpha i1 to form a stable, nucleotide-free complex. Purified nonmyristoylated Galpha i1 (13 µM) bound to [alpha -32P]GDP (A) or [35S]GTPgamma S (B) was incubated with 10 µM purified Tev-digested Ric-8A for 5 min. The reaction mixtures were then resolved by gel filtration chromatography over tandem Superdex 75/Superdex 200 columns (HR 10/30). Fractions from the column were analyzed by SDS-PAGE to visualize proteins, by Bradford assay to assess the protein concentration of each fraction, and by scintillation counting to determine the amount of radiolabeled nucleotide in each fraction.

Ric-8A Stimulates GTPgamma S Binding to a Subset of Galpha Proteins-- To determine whether Ric-8A acts as a GEF for Galpha proteins, the kinetics of GTPgamma S binding to purified Galpha proteins was determined in the presence and absence of purified Tev-digested Ric-8A. Binding of guanine nucleotide to Galpha proteins is limited by the rate of dissociation of GDP. Ric-8A dramatically stimulated the rate of GTPgamma S binding to Galpha q from immeasurable values to 0.1 mol of GTPgamma S·mol Galpha q-1·min-1 (Fig. 6A), to Galpha i1 from 0.02 to 0.15 mol GTPgamma S·mol Galpha i1-1·min-1 (Fig. 6B), and to Galpha o from 0.16 to 0.32 mol GTPgamma S·mol Galpha o-1·min-1 (Fig. 6C) but did not stimulate binding to Galpha s short appreciably (0.085-0.1 mol GTPgamma S·mol Galpha s-1·min-1) (Fig. 6D). Ric-8A also stimulates the rate of GTPgamma S binding to Galpha 13.2


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Fig. 6.   Ric-8A is a guanine nucleotide exchange factor for a subset of Galpha proteins. Purified Galpha q (A), Galpha i1 (B), Galpha o (C), and Galpha s short (D) (200 nM each) were incubated with [35S]GTPgamma S in reactions containing 0 () or 200 (black-square) nM Tev-digested Ric-8A. Duplicate aliquots of these reaction mixtures were taken at the indicated time points, quenched, and filtered to adsorb nucleotide-bound protein. The amount of G protein-bound [35S]GTPgamma S was determined by scintillation counting.

Ric-8A Stimulates the Steady-state GTPase Activity of Galpha q-- The rate-limiting step in Galpha protein-catalyzed steady-state GTPase reactions in vitro is the release of GDP from the Galpha . Inclusion of a GEF such as Ric-8A in such reactions should enhance the observed rate of GTPase activity in a manner similar to that observed with GTPgamma S binding. Steady-state GTPase reactions containing varying amounts of Tev-digested Ric-8A were initiated by the addition of Galpha q (45 nM) (Fig. 7A). Ric-8A maximally accelerated the GTPase activity of Galpha q from immeasurable values to 0.29 mol Pi·mol of Galpha q-1·min-1. The EC50 for Ric-8A was ~160 nM. Ric-8A also stimulated Galpha i1 steady-state GTPase activity in a manner consistent with the observed kinetics of Ric-8A-stimulated Galpha i1 GTPgamma S binding shown in Fig. 6 (data not shown).


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Fig. 7.   A, Ric-8A stimulates the steady-state GTPase activity of Galpha q. Galpha q (45 nm) was incubated with the indicated concentrations of Tev-digested Ric-8A and [gamma -32P]GTP. Duplicate aliquots from each reaction were taken at 2-min intervals from 0 to 10 min. The amount of 32Pi in each aliquot was determined, and the reaction rates were calculated. B, beta 1gamma 2 inhibits Ric-8A-stimulated Galpha q GTPase activity. Galpha q (45 nM) was used to initiate GTPase reactions containing Tev-digested Ric-8A (500 nM) and the indicated concentrations of beta 1gamma 2. The samples were processed as described for A. C, Ric-8A does not stimulate the steady-state GTPase activity of heterotrimeric Galpha qbeta 1gamma 2. Galpha q (45 nM) was incubated with 200 nM (black-square) or 500 nM () beta 1gamma 2. This heterotrimeric Galpha qbeta 1gamma 2 was then used to initiate GTPase reactions containing the indicated amounts of Tev-digested Ric-8A. The samples were processed as described for A.

GPCRs interact with intact G protein heterotrimers to promote nucleotide exchange. Ric-8A is capable of promoting such exchange with individual alpha  subunits. The indicated amounts of purified G protein beta 1gamma 2 subunits and an amount of Ric-8A corresponding to approximately three times the EC50 value (500 nM) were mixed and used to initiate steady-state GTPase reactions containing 45 nM Galpha q. Gbeta 1gamma 2 clearly inhibited Ric-8A-stimulated GTPase activity at concentrations comparable with that of Galpha q in the assay (Fig. 7B). However, the inhibition did not appear to be complete.

To test the effect of Ric-8A on the steady-state GTPase activity of preformed G protein heterotrimers, Galpha qbeta 1gamma 2 was first formed by incubating purified Galpha q (45 nM) with 200 and 500 nM concentrations of purified beta 1gamma 2 for 30 min at 20 °C. These heterotrimer preparations were then used to initiate steady-state GTPase reactions containing the indicated concentrations of Ric-8A (Fig. 7C). Ric-8A was not capable of interacting with preformed G protein heterotrimers to facilitate nucleotide exchange (Fig. 7C). beta gamma apparently competes with Ric-8A for binding to Galpha proteins. Ric-8A functions as a GEF for monomeric Galpha proteins, which clearly differs from the mechanism used by conventional GPCRs.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We report the discovery of Ric-8A and Ric-8B as two homologous Galpha -binding proteins. Ric-8A is a unique guanine nucleotide exchange factor for a subset of Galpha proteins, unlike a GPCR because it does not contain transmembrane spanning sequences and because it activates only monomeric G protein alpha  subunits in vitro. Ric-8A is a G protein activator and not just an exchange factor, because the Ric-8A-mediated exchange process appears to proceed in one direction (exchange of GDP for GTP). When Galpha i1 is bound to GDP, binding of Ric-8A causes rapid release of GDP. Ric-8A does not measurably bind the GTP-bound form of Galpha i1. Upon release of GDP, Ric-8A stabilizes an otherwise extremely unstable nucleotide-free conformation of Galpha i1. GTP, but not GDP, then binds to Galpha i1, which causes the complex to dissociate and release the activated Galpha protein.

Mammalian Ric-8A and Ric-8B were identified as Galpha -binding proteins in yeast two-hybrid screens designed to search for novel interaction partners of activated, GTP-bound forms of Galpha o and Galpha s (Figs. 1 and 2). Despite the fact that Ric-8A and B were detected with these presumably GTP-bound proteins, subsequent biochemical experiments showed that Ric-8A interacts preferentially with the GDP-bound or nucleotide-free forms of Galpha proteins (Figs. 5 and 6). This discrepancy is reconciled somewhat by the fact that Ric-8 showed a clear preference for the wild type (GDP-bound) Galpha proteins in the case of the Ric-8A/Galpha i1 and Ric-8B/Galpha s long two-hybrid interactions (Fig. 2). Ric-8A and B were found to interact with unique subsets of Galpha proteins (Fig. 2). Of the Galpha s tested, Galpha q was the common interaction partner of Ric-8A and B.

Despite the fact that Ric-8A and GPCRs differ structurally and mechanistically, understanding the physical mechanism by which Ric-8A promotes guanine nucleotide exchange could provide important insights into the mechanism of action of GPCRs. When GDP-bound Galpha i1 and Ric-8A are mixed, a stable nucleotide-free complex is formed in detergent-free solution (Fig. 5). The ability to isolate substantial quantities of this nucleotide-free complex provides a unique opportunity for crystallization and determination of the structure of a G protein alpha  subunit in a nucleotide-free conformation; this has been an elusive goal.

An important aspect of Ric-8 function yet to be resolved in terms of traditional heterotrimeric G protein biology is the inability of Ric-8A to activate trimeric forms of G proteins in vitro (Fig. 7). Both GPCRs and the G protein activator AGS1 differ from Ric-8A in this regard. GPCRs and AGS1 (9) promote exchange on Galpha subunits when they exist as alpha beta gamma heterotrimers. The function of the Ric-8 proteins could thus be as signal amplifiers. After a heterotrimeric G protein is activated by a GPCR and subsequently hydrolyzes GTP but before the signal is completely attenuated by rebinding to Gbeta gamma , a Ric-8 protein could bind a monomeric G protein alpha  subunit and reactivate it. This mode of action would serve to amplify the duration of a signal that comes from an individual G protein.

An intriguing question will be to determine the specific physiological Galpha target(s) of Ric-8A and B in vivo. Genetic studies in C. elegans show that mutation of the RIC-8 gene negatively affects aspects of synaptic transmission that are positively controlled by Galpha q. C. elegans Ric-8 was predicted to act as an upstream activator of Galpha q or to operate in a pathway parallel with Galpha q (20). To date, we have not been able to observe effects of mammalian Ric-8A on Galpha q signaling in intact cells. Overexpression of Galpha q in cultured cells led to an increase in steady-state and hormone-stimulated production of inositol trisphosphate, whereas overexpression of full-length or CAAX Ric-8A or co-overexpression of Galpha q and full-length or CAAX Ric-8A did not lead to a further increase in steady-state or hormone-stimulated inositol trisphosphate accumulation.3 These results certainly do not preclude Ric-8A from activating Galpha q in vivo, but they are consistent with other cellular targets or other sites of action of mammalian Ric-8A. Recent work with C. elegans RIC-8 revealed that in addition to its role in promoting synaptic transmission via a Galpha q-coupled pathway, mutation of RIC-8 influences centrosome movements during early embryogenesis (21). This aspect of C. elegans RIC-8 function was shown genetically to be independent of the action of Galpha q and more likely to involve Galpha o or another G protein family member. Because mammalian Ric-8A potently activates both Galpha o and Galpha i in vitro, examination of the effects Ric-8A has on signaling pathways controlled by these G proteins should also be undertaken. However, our preliminary steps in this direction have also not been encouraging.

An alternative and attractive hypothesis is that the targets of Ric-8-like exchange factors are not plasma membrane-bound G protein alpha  subunits, which we expect to be heterotrimeric. Increasing evidence points to G proteins that reside on internal cellular membranes that might not contain Gbeta gamma subunits (42-44). It is unclear how these G proteins become activated, but proteins like Ric-8A and B are now obvious candidates. The possible existence of the 401-amino acid Ric-8A variant is intriguing in this regard, because it contains a consensus CAAX box at its carboxyl terminus (Fig. 1). Expression of the recombinant engineered protein in cultured cells revealed that it can reside on membranes in a CAAX box-dependent manner.3 However, we have not been able to confirm the existence of a transcript encoding this protein, and a sequencing error is a possible explanation for its existence in GenBankTM. The 531-amino acid Ric-8A protein appears to be mostly cytosolic3 and would be accessible to the cytosolic face of most organellar membranes in addition to the plasma membrane. Other questions of major importance include elucidation of the identity of the signaling pathways that are regulated by Ric-8 in vivo and the possible existence of signaling inputs that control the activities of Ric-8 proteins.

    ACKNOWLEDGEMENTS

We thank members of the Gilman laboratory for helpful discussions, Linda Hannigan for technical assistance, Dr. Mark Hatley for the gift of Galpha s, Dr. Roger Sunahara for the gift of Myr-Galpha i1, and Drs. Celestine Thomas and Hongjun Shu for mass spectroscopy analyses.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM34497 and the Raymond and Ellen Willie Distinguished Chair in Molecular Neuropharmacology (to A. G. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY177754 and AY177755.

Dagger To whom correspondence should be addressed: Dept. of Pharmacology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9041. Tel.: 214-648-2370; Fax: 214-648-8812; E-mail: Alfred.gilman@utsouthwestern.edu.

Published, JBC Papers in Press, December 30, 2002, DOI 10.1074/jbc.M211862200

2 W. D. Singer and P. C. Sternweis, personal communication.

3 G. G. Tall and A. G. Gilman, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: G protein, guanine nucleotide-binding regulatory protein; GTPgamma S, guanosine 5'-3-O-(thio)triphosphate; C12E10, polyoxyethylene 10-lauryl ether; Tev, tobacco etch virus; GST, glutathione S-transferase; GPCR, G protein-coupled receptor; GEF, guanine nucleotide exchange factor; GAP, GTPase-activating protein; RGS, regulator of G protein signaling.

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DISCUSSION
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