©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Functional Importance of the Amino Terminus of G(*)

(Received for publication, August 31, 1995; and in revised form, October 20, 1995)

John R. Hepler (1) Gloria H. Biddlecome (1) Christiane Kleuss (1) Laura A. Camp (2) Sandra L. Hofmann (2) Elliott M. Ross (1) Alfred G. Gilman (1)(§)

From the  (1)Departments of Pharmacology and (2)Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75235

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

G is palmitoylated at residues Cys^9 and Cys. Removal of palmitate from purified G with palmitoylthioesterase in vitro failed to alter interactions of G with phospholipase C-beta1, the G protein beta subunit complex, or m1 muscarinic cholinergic receptors. Mutants C9A, C10A, C9A/C10A, C9S/C10S, and truncated G (removal of residues 1-6) were synthesized in Sf9 cells and purified. Loss of both Cys residues or truncation prevented palmitoylation of G. However, truncated G and the single Cys mutants activated phospholipase C-beta1 normally, while the double Cys mutants were poor activators. Loss of both Cys residues impaired but did not abolish interaction of G with m1 receptors. These Cys residues are thus important regardless of their state of palmitoylation. When expressed in HEK-293 or Sf9 cells, all of the proteins studied associated entirely or predominantly with membranes, although a minor fraction of nonpalmitoylated G proteins accumulated in the cytosol of HEK-293 cells. When subjected to TX-114 phase partitioning, a significant fraction of all of the proteins, including those with no palmitate, was found in the detergent-rich phase. Removal of residues 1-34 of G caused a loss of surface hydrophobicity as evidenced by complete partitioning into the aqueous phase. The Cys residues at the amino terminus of G are thus important for its interactions with effector and receptor, and the amino terminus conveys a hydrophobic character to the protein distinct from that contributed by palmitate.


INTRODUCTION

Heterotrimeric guanine nucleotide binding proteins (G proteins) (^1)link cell surface receptors with intracellular effectors(1, 2, 3) . In the absence of activators, G protein alpha, beta, and subunits are associated with each other and the inner surface of the plasma membrane. Although the G protein polypeptides are not intrinsically hydrophobic, they are membrane-bound at least in part because of covalent lipid modifications. G protein subunits are prenylated and carboxymethylated at their carboxyl termini(4, 5) . These modifications promote association of the beta subunit complex with membranes and interactions of beta with alpha and effectors(6) . Members of the G(i) subfamily of alpha subunits are myristoylated at their amino termini; this modification also promotes membrane anchorage and interactions of alpha with beta and effectors(7, 8, 9) . Although members of the G(s), G(q), and G subfamilies of alpha subunits are not myristoylated, they (and G family members, excepting transducin) are palmitoylated on one or more cysteine residues near their amino termini(10, 11, 12) . Arachidonate may also be incorporated similarly(13) . In contrast to myristoylation, palmitoylation of G subunits is a dynamic and regulated process. Activation of appropriate receptors appears to stimulate depalmitoylation of cognate G subunits(14, 15, 16) .

The lipid modifications of G subunits are at or near the amino terminus, and this domain (upstream of the G(1) nucleotide binding region; roughly residues 1-35) has been implicated in a number of functions. The amino terminus is clearly important for interactions with beta(17, 18) , and it is implicated in interactions with receptors (19) and effectors (9, 18) as well. The amino and carboxyl termini of G form an associated subdomain of the GDP-bound form of the protein that becomes disordered upon activation(20, 21) . The amino termini of G and G differ from those of most other G proteins. The site of initiation of translation is not clear (Met^1 or Met^7 in G; Met^1, Met^6, or Met^7 in G). The shorter form (assuming Met^7 as initiator) is most similar to other G subunits(22) . It is not known if only one or both of these species is expressed naturally, although all currently identified G mRNAs (except that from Drosophila) encode the longer protein. G and G also have two amino-terminal Cys residues (positions 9 and 10) that both serve as sites for palmitoylation(23, 24, 25) .

The role of G protein palmitoylation is unclear, and several conflicting reports have appeared. In the case of G, one study (23) suggests that palmitoylation is essential for membrane localization, while others (14, 15) indicate that nonpalmitoylated mutants of G remain associated with the plasma membrane. Wedegaertner et al.(23) also reported that palmitoylation is necessary for association of G with membranes and subsequent activation of its physiological effector, PLC-beta1. In contrast, others have indicated that palmitoylation is not required for association of either G(q) or G with membranes(24, 25) , and Edgerton et al.(25) found that the C9A/C10A mutant of G failed to activate PLC-beta1. Published data also suggest that palmitoylation is important for coupling of G(q) with the NK2 receptor (25) but not the alpha(2)-adrenergic receptor(23) . All of these studies have utilized transfected cells expressing G protein alpha subunits carrying mutations designed to prevent palmitoylation.

We have investigated the importance of amino-terminal domains of G proteins using the m1 muscarinic cholinergic receptor/G/PLC-beta1 signaling pathway as a model system, and we have emphasized studies of purified and reconstituted proteins. We have found that the amino-terminal domain of G is clearly important for activation of PLC-beta1. Although palmitoylation of Cys residues 9 and/or 10 is not necessary for interactions of G with PLC-beta1, receptors, or beta, the Cys residues themselves are important for interactions with PLC-beta1 and, to a lesser extent, with receptor. The amino terminus of G also appears to confer a hydrophobic character on the protein, independent of its palmitoylation.


EXPERIMENTAL PROCEDURES

Materials

[^3H]Palmitoyl-Ha-ras(26) and purified recombinant palmitoylthioesterase were prepared as previously described(27) . Anti-G sera WO82 and Z811 was supplied by P. C. Sternweis (University of Texas, Southwestern), and anti-G serum 584 and common anti-G serum P960 by S. M. Mumby (University of Texas, Southwestern). Purified recombinant PLC-beta1 was kindly provided by S. G. Rhee (National Institutes of Health).

G Mutants

G tagged with six histidine residues at the carboxyl terminus (GCH6) and amino-terminal mutations of GCH6 were generated by synthesizing oligonucleotide cassettes for substitution insertion as described elsewhere(28) . The following complementary pair of oligonucleotides were synthesized to construct GCH6: 5`- GCTGAACCTGAAGGAGTACAATCTGGTCAGGCATCACCATCACCATCACTAATA and 3`-ACGTCGACTTGGACTTCCTCATGTTAGACCAAGGTCCGTTGGTAGTGGTAGTGATTATTCGA. This cassette encodes the extreme carboxyl terminus of G, the hexahistadine tag, and a stop codon, and it contains PstI and HindIII sites at its 5` and 3` ends, respectively. Following annealing, the cassette was cloned into the unique PstI site at the extreme carboxyl terminus of the G-coding region and the 3` HindIII site of the Escherichia coli expression vector (G/NpT7-5). The resulting plasmid was linearizing with HindIII, blunt-ended with Klenow fragment, and partially digested with EcoRI to yield a 1200 kilobase pair fragment encoding GCH6. This DNA was cloned into the Sf9 baculovirus expression vector pVL1392 (using the EcoRI and SmaI sites). This served as the parent for synthesis of amino-terminal mutants of GCH6. These mutants (see Fig. 3) were synthesized by insertion of oligonucleotide cassettes using the unique NotI site of the pVL1392 polycloning region and the unique EcoNI site near the initiator codon of G. Recombinant baculoviruses were generated as described previously(29, 30) . The nucleotide sequence for all G mutant cDNA constructs was confirmed by dye terminator sequencing using an Applied Biosystems 373A automated sequencer (Perkin-Elmer).


Figure 3: Palmitoylation and purification of amino-terminal mutants of G. A, amino-terminal sequences of wild type G and mutants. All constructs were hexahistadine tagged at the carboxyl terminus. B, Sf9 cells were infected with viruses encoding beta(2) and (2) subunits and either wild type G (WT-G), or amino-terminal mutants of G (C9A, C10A, and C9A/C10A (C9,10A), or G-short). Infected cells were labeled with [^3H]palmitate, and G subunits were recovered from membrane extracts by Ni-NTA affinity chromatography. Samples were resolved by SDS-PAGE and visualized by fluorography and by immunoblotting with a specific anti-G serum (WO82). C, purification of GCH6 by affinity chromatography and anion exchange chromatography. Sf9 cells (6-liter culture) were infected with viruses encoding beta(2), (2), and GCH6 subunits. Cholate extracts of cell membranes (L) were bound to Ni-NTA resin (top left) and eluted with a 150 mM imidazole bump (B). The sample was then bound to and eluted from Q-Sepharose anion exchange resin (right, top and bottom); individual fractions were analyzed for their capacity to stimulate phospholipase C-beta1 (bottom), and protein was visualized by Coomassie Blue staining (top). Phospholipase C-beta1 activity is expressed as pmol product/min/ng phospholipase C-beta1.



Labeling and Isolation of [^3H]Palmitate-labeled G and Mutants

Sf9 cells (25 ml of culture; 1 times 10^6 cells/ml) were triply infected with viruses encoding hexahistadine-tagged wild type or mutant G together with viruses encoding G and G subunits for 48 h(29) . Cells were then labeled with [^3H]palmitate (1 mCi/ml) for 3 h(10) . Cells were collected by centrifugation, washed three times in buffer A (50 mM NaHepes, pH 8.0, 20 mM beta-mercaptoethanol, 50 µM GDP, 100 mM NaCl, 10 mM NaF, 5 mM MgCl(2), 30 µM AlCl(3), and 0.2 mg/ml of phenylmethylsulfonyl fluoride), and lysed by rapid, repeated (three times) freezing and thawing. Membranes were isolated by centrifugation (55,000 rpm; Beckman TL-100), resuspended in buffer A, extracted with sodium cholate (1% w/v in buffer A) for 1 h at 4 °C, and centrifuged. The resulting supernatant was combined with 100 µl of Ni-NTA resin (Qiagen) and mixed for 1 h at 4 °C. Resin was recovered by centrifugation and washed with 3 times 1 ml of buffer B (buffer A + 1% sodium cholate, 15 mM imidazole, 500 mM NaCl) and 2 times 250 µl of buffer C (50 mM NaHepes, pH 8, 20 mM beta-mercaptoethanol, 50 µM GDP, 1% octyl-beta-D-glucopyranoside). Highly enriched, ^3H-labeled recombinant GCH6 was eluted by washing the resin five times with 100 µl of buffer D (buffer C containing 100 mM NaCl, 150 mM imidazole, and 10% glycerol). Recoveries of protein were monitored by silver staining and fluorography of SDS gels as described previously(10) .

Purification of Amino-terminal Mutants of G

GCH6 and mutants were expressed in Sf9 cells and purified to near homogeneity by Ni-NTA affinity chromatography and Q-Sepharose anion exchange chromatography. Sf9 cells (2-4 liters; 1.5-2 times 10^6 cells/ml) were triply infected with the desired GCH6 virus together with beta(2) and (2) viruses for 48-60 h. Cells were pelleted, suspended in 400 ml of buffer A, and lysed by nitrogen cavitation at 4 °C (500 p.s.i. for 1 h; Parr Instruments). Nuclei were removed from lysates by centrifugation, and supernatants were centrifuged (35,000 rpm, Beckman Ti-45) for 30 min at 4 °C. The resulting membranes were suspended in 90 ml of buffer A (3-5 mg protein/ml) and extracted with 1% sodium cholate in buffer A for 1 h at 4 °C. The resulting extract was collected by centrifugation (100,000 times g) and mixed with 5 ml of Ni-NTA resin for 1 h. After application to a 3 times 13-cm column, the resin was washed with 20 column volumes of buffer B and 3 volumes of buffer C. Protein was eluted with four 5-ml washes of buffer D. The Ni-NTA eluate was diluted 15-fold with buffer E (50 mM NaHepes, pH 7.4, 1 mM EDTA, 3 mM EGTA, 5 mM MgCl(2), 2 mM dithiothreitol, 0.1 mM GDP, 10 mM NaF, 0.03 mM AlCl(3)) and applied to a Q-Sepharose column (5 ml) that had been equilibrated with buffer E. The column was washed with 5 column volumes of buffer F (50 mM NaHepes, pH 7.4, 1 mM EDTA, 3 mM EGTA, 5 mM MgCl(2), 2 mM dithiothreitol, 0.1 mM GDP), and protein was eluted with a 40-ml linear gradient of NaCl (0-1 M) in buffer F.

Transfection of HEK-293 Cells

cDNAs encoding G, G-short, and C9A/C10A Gwere cloned into the mammalian expression vector pCMV-5(31) ; none of these constructs contained the hexahistidine tag. HEK-293 cells (150-mm dishes; 75% confluent) were transfected with 25 µg of purified DNA for 30 h using Lipofectamine (Life Technologies, Inc.) as described elsewhere(14) . Cells were then harvested in lysis buffer (50 mM NaHepes, pH 7.4, 1 mM EDTA, 3 mM EGTA, 5 mM MgCl(2), 150 mM NaCl, 100 µM GDP, and 0.2 mg/ml of phenylmethylsulfonyl fluoride) and lysed by nitrogen cavitation. Intact nuclei were removed by centrifugation, and membranes and cytosol were separated (120,000 times g, 1 h at 4 °C, Beckman TL-100). All fractions were diluted to the same volume; membrane protein (5 µg for G alone and 25 µg for G + beta) and equal volumes of normalized samples from the cytosolic and nuclear fractions were analyzed by SDS-PAGE.

Amino-terminally Truncated G

The amino terminus of GTPS-activated GCH6 was removed by tryptic digestion essentially as previously described(32) . Digested samples were recovered by Ni-NTA affinity chromatography to ensure that the carboxyl terminus was intact.

Measurement of Phospholipase C-beta1 Activity

Activation of purified phospholipase C-beta1 by purified G subunits was performed as described elsewhere(29) , as were assays of phospholipase C-beta1 activity.

Reconstitution of m1 Muscarinic Receptors and G Proteins into Phospholipid Vesicles

The m1 muscarinic receptors, G (not hexahistadine-tagged), G protein beta(1)(2) subunits, and phospholipase C-beta1 used for reconstitution assays were purified from baculovirus-infected Sf9 cells; these methods will be described elsewhere. (^2)Lipid-detergent micelles were prepared by rehydrating dried lipids (bovine liver phosphatidylethanolamine, bovine brain phosphatidylserine, and cholesteryl hemisuccinate) in buffer containing 20 mM NaHepes (pH 7.5), 100 mM NaCl, 1 mM EGTA, 2 mM MgCl(2), and 0.2% deoxycholate, followed by sonication under argon gas for 15 min at room temperature. The final concentration of lipids was 413 µM phosphatidylethanolamine, 245 µM phosphatidylserine, and 45 µM cholesteryl hemisuccinate. Micelles (25 µl) were then incubated with G(q) (5 pmol of G plus 10 pmol of beta(1)(2)) for 15 min; m1 receptors (3 pmol) were added, and the volume was brought to 50 µl with 20 mM NaHepes (pH 7.5), 100 mM NaCl, 1 mM EGTA, and 2 mM MgCl(2). The mixture was applied to a 1-ml AcA34 gel filtration column, and the protein-lipid vesicles were recovered in the void volume. BSA (0.1 mg/ml final) was added, and the vesicles were treated with dithiothreitol (5 mM) for 1 h on ice before assay.

Assay of Reconstituted Proteins

GTP hydrolysis was measured essentially as previously described(33, 34) . Briefly, receptor-G(q) vesicles were mixed with 1 µM [-P]GTP and either 1 mM carbachol or 10 µM atropine in a volume of 50 µl (final buffer: 20 mM NaHepes [pH 8.0], 100 mM NaCl, 1.1 mM EDTA, 0.2 mM EGTA, 3.9 mM total MgCl(2), and 0.26 mg/ml BSA). The mixture was incubated at 30 °C for various times, and reactions were stopped by addition of 750 µl of Norit A (5% in 50 mM NaH(2)PO(4)). Samples were centrifuged, and 400 µl of supernatant were counted in a liquid scintillation mixture. To measure GTP hydrolysis in the presence of phospholipase C-beta1, the vesicles were mixed with the enzyme prior to addition of other reagents.

m1 receptor-catalyzed GTPS binding was measured at 30 °C as described elsewhere(35) , using 100 nM [S]GTPS and 1 mM carbachol; the final buffer composition was the same as in the GTP hydrolysis assays (see above). Data describing the time course of GTPS binding were fit to a two-component equation: y = A(1 - e) + (mt + b), where A is the maximum amount of GTPS bound in response to carbachol, k is the rate constant for receptor-stimulated binding, m is the basal rate of binding (essentially constant over the assay interval), and b is the amount of GTPS nonspecifically bound at zero time (t). For C9S/C10S G, the linear component (mt + b) was omitted because there was no observable binding of nucleotide in the absence of carbachol.

Triton X-114 Phase Partitioning of Proteins

Partitioning of protein samples between aqueous and detergent-rich phases using TX-114 was performed as previously described(36) . Following appropriate treatments (see ``Results''), samples were prepared in 260 µl of a buffer consisting of 50 mM Hepes, pH 8.0, 5 mM beta-mercaptoethanol, 150 mM NaCl, 1% Triton X-114, and 5 mM MgCl(2). Of this, 200 µl were removed and applied to the top of a 300-µl sucrose cushion (6%) prepared with the same buffer containing 0.1% TX-114. The samples were then separated into detergent and water phases as previously described(36) . The resulting upper water and lower detergent phases were collected and adjusted to contain equivalent total volumes (225 µl) of the original buffer and equivalent amounts of TX-114. These were compared to the original sample by SDS-PAGE and Western blot analysis.

Miscellaneous Procedures

Labeling of Sf9 cells with [S]methionine and [^3H]palmitate and immunoprecipitation of radiolabeled recombinant G subunits were performed as described previously(10) . Fatty acids removed from G by hydrolysis were analyzed by HPLC (10) .


RESULTS

When Sf9 cells are infected with baculovirus encoding G, G, or G along with viruses encoding beta(2) and (2) subunits, newly synthesized alpha subunits accumulate in both membranes and cytosol; however, only the membrane-associated alpha subunits incorporate [(^3)H]palmitate (Fig. 1A).^3 To study the functional consequences of palmitoylation in greater detail, we generated an affinity-tagged G (hexahistadine at the carboxyl terminus) using the baculovirus expression system. When synthesized in Sf9 cells, GCH6 incorporates [^3H]palmitate and can be purified rapidly from membrane extracts by affinity chromatography with Ni-NTA resin (Fig. 1B).


Figure 1: Labeling and isolation of recombinant G protein alpha subunits from Sf9 cells by immunoprecipitation or affinity chromatography. A, Sf9 cells were infected with viruses encoding G protein beta(2) and (2) subunits and either G (ralpha), G (ralpha), or G (ralpha). Infected cells were labeled with [S]methionine (left) or [^3H]palmitate (right), fractionated into cytosol (C) and membranes (M), and subjected to immunoprecipitation using specific antisera (Z811 for alpha(q), 584 for alpha(s), and P960 for alpha). Immunoprecipitated, radiolabeled proteins were resolved by SDS-PAGE and visualized by autoradiography or fluorography. In this experiment G is visualized as two bands; see footnote 3 for explanation. B, Sf9 cells were infected with viruses encoding beta(2), (2), and hexahistadine-tagged G (GCH6). Infected cells were incubated with [^3H]palmitate and fractionated into cytosol and membranes. Cholate extracts of membranes (L) were mixed with Ni-NTA resin, the flow through (FT) was collected, and the resin was washed with high salt (W1) and low salt (W2); bound protein was eluted using 150 mM imidazole (Bump). Fractions were resolved by SDS-PAGE and visualized by silver staining (left) or fluorography (right).



Treatment with Palmitoylthioesterase

A palmitoylthioesterase capable of removing palmitate from c-Ha-ras and G has been purified from bovine brain, cloned, and expressed in Sf9 cells(26, 27) . Treatment of [^3H]palmitate-labeled G or c-Ha-ras with recombinant palmitoylthioesterase results in almost total loss of label from the proteins (Fig. 2A). HPLC analysis of the material so removed from G reveals that the label is in fact in palmitate (Fig. 2B). However, treatment of purified GCH6 with palmitoylthioesterase failed to alter the protein's capacity to activate PLC-beta1 (Fig. 2C). Similarly, such treatment failed to alter interaction of G with beta as assessed by the capacity of beta to reverse activation of G by AlF(4) (Fig. 2D). Removal of palmitate from G also failed to interfere with the protein's interactions with m1 muscarinic receptors as assessed by the capacity of the purified receptor to stimulate nucleotide exchange and GTPS binding to G (in the presence or absence of the receptor agonist carbachol). (^4)The capacity of receptor and PLC-beta1 to stimulate the GTPase activity of G (i.e. GAP activity) was also unchanged by treatment with palmitoylthioesterase (data not shown). The power of these experiments to reveal functional effects of palmitoylation is of course dependent on the stoichiometry of palmitoylation of the purified G. Although this has not been determined precisely, we believe that a significant amount of palmitate was present (see below).


Figure 2: Removal of palmitate from G by treatment with recombinant palmitoylthioesterase (rPTE). A, [^3H]Palmitate-labeled rG or c-Ha-ras purified by Ni-NTA affinity chromatography was incubated in the absence or presence of palmitoylthioesterase. Treated samples were resolved by SDS-PAGE, and labeled protein was visualized by fluorography. B, HPLC analysis of a ^3H-labeled mixture of standards of myristate, palmitate, and stearate (top), or ^3H-labeled products removed from rG synthesized in the presence of [^3H]palmitate; incubation was performed without (middle) or with (bottom) palmitoylthioesterase. C, purified rG was incubated with (bullet) or without (circle) palmitoylthioesterase for 90 min at 30 °C and then activated for 1 h with 1 mM GTPS at 30 °C. Treated rG was then mixed with purified phospholipase C-beta1 and phosphatidylinositol(4,5)-bisphosphate vesicles and assayed for phospholipase C-beta1 activity as described under ``Experimental Procedures.'' Enzymatic activity is expressed per ng of phospholipase C-beta1. D, purified G was treated with (bullet) or without (circle) palmitoylthioesterase for 90 min at 30 °C and then activated for 15 min at 20 °C with 30 µM AlCl(3), 10 mM NaF, and 5 mM MgCl(2). Treated rG (3 nM) was then mixed with the indicated concentrations of purified brain G and held for 10 min at 4 °C. Purified phospholipase C-beta1 (1 ng) and substrate vesicles were added and phospholipase C activity was measured as described. E, purified rG was treated with or without palmitoylthioesterase and then reconstituted into phospholipid vesicles with purified G and m1 muscarinic cholinergic receptors. Receptor-stimulated [S]GTPS (10 nM) binding to G was measured in the presence of either atropine (20 µM) or carbachol (100 µM) after a 10 min incubation.



Amino-terminal Mutations of G

The likely sites of attachment of palmitate to G subunits are amino-terminal cysteine residues(10, 11, 12, 15, 23) ; in the case of G, these are Cys^9 and Cys. To define the role of Cys^9 and Cys and the unique six amino acid extension at the amino terminus of G(q), we generated single and double mutants of G that substituted either Ala or Ser for Cys^9 and/or Cys. In addition, we constructed truncated forms of G that were missing either the first 6 or the first 10 amino acid residues (Fig. 3A). Some of these mutants were coexpressed with beta in Sf9 cells in the presence of [^3H]palmitate and then isolated using Ni-NTA chromatography. Consistent with previous reports(23, 25) , C9A or C10A GCH6 each retained capacity to incorporate [^3H]palmitate; however, the double mutant C9A/C10A failed to incorporate label. Unexpectedly, G that is truncated by removal of residues 1-6 (and thus retains Cys^9 and Cys) is not palmitoylated (Fig. 3B). (^5)GCH6 and five of the mutants were purified by a combination of Ni-NTA and Q-Sepharose chromatography for further characterization (Fig. 3C and Fig. 4). Yields of purified protein ranged between 0.1 and 1 mg/liter of Sf9 cell culture. G(-10) could not be purified in reasonable amounts.


Figure 4: Activation of phospholipase C-beta1 by G, G mutants, and amino-terminally cleaved G. A, purified GCH6 and amino-terminal G mutants. Each protein (500-750 ng) was resolved by SDS-PAGE and visualized by Coomassie Blue staining. B, purified proteins were activated for 1 h with 1 mM GTPS at 30 °C and activation of added phospholipase C-beta1 was assayed as described under ``Experimental Procedures.'' Data are pooled results from several experiments: GCH6 (WT-G), n = 6; G-short, n = 6; C9A, n = 2; C10A, n = 2; C9A/C10A, n = 5; C9S/C10S, n = 3. Mutant proteins were compared with GCH6 in each experiment and all values were normalized to activity stimulated by 100 nM GCH6 (100%). Inset, GCH6, C9A/C10A, and C9S/C10S were incubated with or without GTPS as before and subjected to limited tryptic digestion. Samples were then resolved by SDS-PAGE and visualized by immunoblotting with anti-G serum WO82. C, GCH6 was activated with GTPS as before and half of the sample was subjected to limited tryptic digestion. Undigested GCH6, digested G (NC-G), and C9S/C10S G were then repurified by Ni-NTA chromatography in the presence of GTPS. Recovered samples (300-500 ng) were resolved by SDS-PAGE and visualized (right) by immunoblotting with anti-G serum WO82. The indicated concentrations of each sample were assayed for phospholipase C-beta1 stimulation as described (left). Values are averages of two experiments and are normalized to the maximal value for GCH6 (100%).



The capacity of each of these proteins to stimulate purified PLC-beta1 is shown in Fig. 4. The nonpalmitoylated double Cys mutant (C9A/C10A) has a greatly reduced capacity to activate and apparent affinity for PLC-beta1. In contrast, both of the single Cys Ala mutants, which are palmitoylated, and the nonpalmitoylated truncation mutant, G-short, retain near full capacity to stimulate PLC-beta1. The Cys Ser double mutant (C9S/C10S) of G was indistinguishable from C9A/C10A. Both C9A/C10A and C9S/C10S G could be activated, based on the capacity of bound GTPS to protect the proteins from tryptic proteolysis (Fig. 4B, inset). To test the effect of removal of a larger portion of the amino terminus, we cleaved GTPS-activated GCH6 with trypsin and recovered the product (NC-G) by Ni-NTA chromatography; the hexahistidine-tagged carboxyl terminus was thus intact. Amino acid sequencing of NC-G revealed that the first 34 residues were missing. The capacity of NC-G to activate PLC-beta1 closely resembles those of C9A/C10A and C9S/C10S G (Fig. 4C).

We next studied the effects of amino-terminal mutations of G on its interactions with m1 muscarinic receptors and PLC-beta1 in reconstitution assays. Non-His-tagged G was included in these experiments to assess the effects of the tag on receptor coupling. As shown in Fig. 5A, m1 receptors stimulate nucleotide exchange and GTPS binding to GCH6, G-short, and C9S/C10S G. Although receptor-stimulated GTPS binding was nearly identical for GCH6 and G-short, the rate of nucleotide exchange was significantly reduced (5-fold) for the double Cys Ser mutant (Table 1; Fig. 5A). In contrast, the nucleotide binding properties of the single Cys Ala mutants were largely unchanged (data not shown). Similarly, carbachol stimulates steady-state GTP hydrolysis by GCH6, G-short, and non-His-tagged G, but the effect is reduced significantly with the double Cys Ser mutant (Table 1, Fig. 5B).


Figure 5: m1 Muscarinic cholinergic receptor-stimulated nucleotide exchange and phospholipase C-beta1-stimulated GTPase activity of G and amino-terminal mutants. A, receptor-stimulated GTPS binding. Purified G (not tagged), carboxyl-terminal hexahistadine-tagged G (GCH6), and tagged G(q) mutants (G-short, GC8,10S (C9S/C10S)) were reconstituted into phospholipid vesicles with purified m1 receptors. Total carbachol-stimulated [S]GTPS binding to G was measured in the presence of 100 nM [S]GTPS and carbachol (1 mM) at 30 °C for various times up to 30 min. Data shown are the average of duplicate determinations from a single experiment, which is representative of four experiments. B, receptor-stimulated GTP hydrolysis by G. G protein heterotrimers were reconstituted into phospholipid vesicles with m1 receptors and GTPase activity was assayed in the presence of either 10 µM atropine (hatched bars; A) or 1 mM carbachol (open bars; C). The assay time was either 8 min (G) or 30 min (GCH6 and mutants). C, phospholipase C-beta1-mediated potentiation of receptor-stimulated GTP hydrolysis by G. Experiments were performed as in B, except in the presence of phospholipase C-beta1 (10 nM). Data in B and C are the means ± S.D. of three separate experiments, each consisting of duplicate determinations.





Phospholipase C-beta1 is known to stimulate steady-state GTP hydrolysis by G, particularly in the presence of receptor(34) . Phospholipase C-beta1 enhances receptor-stimulated GTP hydrolysis by greater than 11-fold for both GCH6 and G-short, but only 2-3-fold for C9S/C10S G (Table 1; Fig. 5C). This is consistent with the relative inability of C9S/C10S G to activate phospholipase C-beta1. The effects of phospholipase C-beta1 on the single Cys Ala mutants were similar to those on GCH6 (data not shown). A separate point is that carboxyl-terminal hexahistadine tagging of G reduces the efficiency of receptor-G coupling; this effect is most evident with phospholipase C-beta1-stimulated GTP hydrolysis (^6)(Fig. 5C; Table 1).

Cellular Distribution of G Mutations

HEK-293 cells were transiently transfected with DNA encoding wild type G, C9A/C10A G, and G-short (Fig. 6). None of these proteins contained a hexahistadine or other tag. Cells were harvested 30 h after transfection, and nuclear, membrane, and cytosolic fractions were prepared. All of the wild type G was in the low speed pellet or the membrane fraction; none was in the cytosol. By contrast, some portion of both nonpalmitoylated proteins was found in the cytosol. Nevertheless, of the material not found in the low speed pellet, more than half (roughly 60-70%) was associated with membranes.


Figure 6: Cellular distribution of recombinant wild type G and amino-terminal mutants of G in HEK-293 and Sf9 cells. A, HEK-293 cells were transiently transfected with DNA (20 µg) encoding G, G-short, or G C9S/C10S for 30 h. None of these constructs was hexahistadine-tagged. Cells were harvested and fractionated as described under ``Experimental Procedures'' into a low speed nuclear pellet (NP), high speed membrane pellet (M), or cytosol (C). Equal volumes of normalized fractions were resolved by SDS-PAGE, and G was visualized by immunoblotting with anti-G serum WO82. B, Sf9 cells were infected with a virus encoding GCH6 or each of the indicated mutants and fractionated as described under ``Experimental Procedures'' into nuclear pellet (NP), membrane pellet (M), or cytosol (C). Equal volumes of normalized fractions were resolved by SDS-PAGE, and G was visualized by immunoblotting with anti-G serum WO82. C, same as in B, except Sf9 cells were infected with viruses encoding each indicated G subunit together with G protein beta(2) and (2) subunits.



Cellular distribution of hexahistidine-tagged wild type and mutant G proteins was also examined in Sf9 cells. In all cases, some portion of the expressed protein was found in all three cellular fractions (Fig. 6, B and C), but the majority was associated with membranes. Concurrent expression of G with beta(2) and (2) subunits did not alter the cellular distribution of the proteins but did decrease (5-fold) the accumulation of G. The majority (50-70%) of each wild type and mutant protein associated with membranes from the above samples could be extracted with sodium cholate when expressed with beta; a much smaller percentage of each G subunit (<10%) was extracted when beta was not present (data not shown).

TX-114 Detergent Partitioning

We also tested the capacity of wild type and mutant G proteins to partition between water and detergent-rich (TX-114) phases, a crude measure of hydrophobicity(36) . Bovine serum albumin is a water-soluble protein, and as expected, it partitioned exclusively into the water phase (Fig. 7A). The same was true of nonmyristoylated (E. coli-derived) G. In contrast, the majority of myristoylated G was found in the detergent phase (Fig. 7A).


Figure 7: Partitioning of G, G, and amino-terminal mutants of G into aqueous and TX-114 rich phases. A, purified proteins including bovine serum albumin (BSA, 10 µg), E. coli-derived G (nonmyristoylated (-myr), 2 µg; and myristoylated (+myr), 2 µg) were subjected to TX-114 phase partitioning as described under ``Experimental Procedures'' (L, applied sample; W, aqueous phase; and D, the detergent-rich phase). Equal volumes of normalized (see ``Experimental Procedures''), recovered fractions were resolved by SDS-PAGE and visualized by either Coomassie Blue staining (for BSA) or by immunoblotting with anti-G serum P960. B, Sf9 cells were infected with viruses encoding G protein beta(2) and (2) subunits and either GCH6, G-short, or C9A/C10A G. Infected cells were incubated with [^3H]palmitate, and G proteins were recovered using Ni-NTA affinity chromatography and elution in the presence of TX-114. Samples were then subjected to phase partitioning, and equal volumes of normalized fractions (see ``Experimental Procedures'') were resolved by SDS-PAGE and visualized either by immunoblotting with anti-G serum WO82 or fluorography. C, purified GCH6 and C9S/C10S G were activated with GTPS and then incubated without or with trypsin for limited digestion. Purified GCH6 was also treated with palmitoylthioesterase. Treated samples were recovered by Ni-NTA affinity chromatography in the presence of TX-114. Samples then were subjected to TX-114 phase partitioning, and equal volumes of normalized fractions (see ``Experimental Procedures'') were resolved by SDS-PAGE and visualized by immunoblotting with anti-G serum WO82.



Wild type and nonpalmitoylated G proteins were synthesized in Sf9 cells in the presence of [^3H]palmitate, isolated by Ni-NTA chromatography, and subjected to TX-114 partitioning (Fig. 7B). As before, only the wild type protein incorporated [^3H]palmitate. Wild type and nonpalmitoylated forms of G partitioned roughly equally between the aqueous and detergent phases when analyzed by immunoblotting. However, nearly all of the [^3H]palmitate-labeled wild type protein was found in the detergent phase. When compared with the properties of myristoylated and nonmyristoylated G, these results suggest the existence of some factor(s) in addition to palmitate that confers hydrophobicity on G. To test this hypothesis directly, we examined the behavior of purified G treated with palmitoylthioesterase and amino-terminally truncated forms of both G and CysSer G (Fig. 7C). (^7)The majority of wild type G was found in the detergent phase. A distinct and reproducible shift was observed for both C9S/C10S G and palmitoylthioesterase-treated wild type protein. Although the majority of either of these preparations was found in the aqueous phase, presumably due to loss of palmitate, a significant fraction remained associated with detergent. Of interest, however, the truncated forms of both G and C9S/C10S G were found exclusively in the aqueous phase, indicating a loss of surface hydrophobicity associated with removal of residues 1-34.


DISCUSSION

The amino-terminal domain of G and, more specifically, residues Cys^9 and Cys within this domain are important determinants of both the cellular localization of the protein and its interactions with phospholipase C-beta1 and, to a lesser extent, the m1 muscarinic receptor. The cysteine residues are important per se. Their palmitoylation is not necessary to observe characteristic interactions between G and phospholipase C-beta1, although we cannot rule out possible inhibitory effects of such modification. Both palmitoylation and some other feature of the amino terminus confer hydrophobicity on G and influence its cellular distribution.

Two prior reports describe the failure of Cys^9 and C mutants of G to activate phospholipase C-beta. In one case it was hypothesized that this was due to loss of palmitate(25) , while in the other the defect was ascribed to loss of association of G with the membrane(23) . We ascribe this phenomenon to loss of the cysteine residues themselves. Two lines of evidence indicate that palmitate is not a major direct enhancer of the interactions between G and phospholipase C-beta1 or m1 receptors. First, removal of palmitate from G with palmitoylthioesterase did not alter its observed interactions with the effector or receptor. Since we believe that the stoichiometry of palmitoylation of the purified protein is significant (but not 1 or greater, see below), we would have observed loss of a substantial stimulatory effect of palmitate (but not necessarily loss of an inhibitory one) upon removal of the fatty acid. Second, removal of the first six residues of G effectively prevents palmitoylation of the protein without interfering with its interactions with phospholipase C-beta1 or m1 receptors. However, mutation of the relevant Cys residues to either Ala or Ser impairs m1 receptor coupling and causes apparent loss of affinity of G for phospholipase C-beta1 and substantial loss of capacity to activate the enzyme as well. Cys residues rather than palmitoylated Cys residues are thus important. The role of palmitoylation of G is in some ways distinctly different from that of myristoylation of members of the G subfamily of G proteins; myristate is an important determinant of the affinity of G proteins for both effectors and the beta subunit complex(8, 9) .

There are also similarities in the effects of myristoylation and palmitoylation, in that both modifications confer hydrophobic properties on the proteins involved and facilitate their interactions with membranes(10, 14, 37, 38) . A significant fraction of both nonpalmitoylated mutants of G was found in the cytosol of transfected HEK-293 cells; in contrast, all of the wild type protein was membrane associated. Nearly all of purified G labeled with [^3H]palmitate distributed to the detergent-rich phase in TX-114 partitioning experiments. We have suggested that amino-terminal acylation of G protein alpha subunits may not simply facilitate interactions with lipid bilayers but may regulate distribution of the proteins to specialized domains of the plasma membrane such as caveoli(39) .

It has been difficult to determine the stoichiometry of palmitoylation of G. We have been unable to observe any alteration of electrophoretic mobility attributable to palmitate; myristoylated G subunits can be distinguished from their nonmyristoylated counterparts in this fashion. Mass spectrometric analysis has also been unsuccessful, apparently because of inhibitory effects of residual detergent. The best clue comes from TX-114 partitioning studies (Fig. 7). Palmitoylated G is found almost exclusively in the detergent phase. Purified G is distributed in both the aqueous and detergent-rich phases, and treatment of the protein with palmitoylthioesterase causes an observable (but not complete) shift of protein to the aqueous phase. Estimates based on the extent of this shift suggest a stoichiometry in the approximate range of 20-40%.

The precise role of Cys^9 and/or Cys is unclear. Since the loss of either of these residues is well tolerated, they are not involved in formation of a critical disulfide bond with each other. They may be involved in direct intermolecular contacts with, for example, phospholipase C-beta or receptors or in intramolecular interactions that are important determinants of G conformation. Higashijima and Ross (19) found that Cys^3 in G (the palmitoylated cysteine analogous to Cys^9 or Cys in G) interacts with Cys-substituted mastoparans, small amphipathic peptides that mimic the effects of receptors on G proteins. Similarly, Edgerton et al.(25) reported that Cys^9 and Cys mutants of G were unable to interact with the NK2 receptor. However, the same mutant G protein had a relatively modest loss of capacity to interact with m1 receptors when tested in reconstituted systems (above). In all of these scenarios one might suspect that stoichiometric palmitoylation of both cysteine residues might inhibit the function in question. This may be true; alternatively, stoichiometric palmitoylation at both sites may not be possible.

The exact site of initiation of translation of G (Met^1 or Met^7) is unknown. Direct amino-terminal sequencing of purified native protein was unsuccessful because of an unidentified block. (^8)Although both forms of the protein are capable of the characteristic interactions of G with phospholipase C-beta1 and m1 receptors, the shorter form of the protein is not palmitoylated. It is not known if residues 1-6 constitute important sites for recognition by a hypothetical protein palmitoyltransferase or if they might be required for access of G to the enzyme. Perhaps similarly, myristoylation of G proteins greatly facilitates their subsequent palmitoylation(13, 14, 40) .

Factors other than palmitate clearly contribute to the hydrophobicity of G. Nonpalmitoylated forms of the protein are still associated with membranes to a large extent and, similarly, partition partially into TX-114. Cleavage of the first 34 amino acid residues of G with trypsin eliminates the latter hydrophobic behavior. Although it is conceivable that this domain might represent a site of another lipid modification of a G subunit, we note that G synthesized in E. coli (and thus likely to be free of any such modification) has a strong tendency to aggregate(29) . However, no clues are found in the amino-terminal sequence of G; 16 of the first 34 amino acid residues are charged.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM34497, American Cancer Society Grant BE30-O, the Lucille P. Markey Charitable Trust, and the Raymond and Ellen Willie Chair of Molecular Neuropharmacology (to A. G. G.), American Heart Association, Texas Affiliate Award 94G-112 (to J. R. H.), National Institutes of Health Grant GM30355 and Robert A. Welch Foundation Grant I-0982 (to E. M. R.), and National Institutes of Health Grant CA61823 (to S. L. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 214-648-2370; Fax: 214-648-8812.

(^1)
The abbreviations used are: G proteins, heterotrimeric guanine nucleotide-binding regulatory proteins; G, the alpha subunit of a G protein; G, the beta subunit complex of a G protein; GTPS, guanosine 5`-3-O-(thio)triphosphate; HPLC, high performance liquid chromatography; BSA, bovine serum albumin; PAGE, polyacrylamide gel electrophoresis; NTA, nitrilotriacetic acid.

(^2)
G. H. Biddlecome, G. Berstein, and E. M. Ross, manuscript in preparation.

(^3)
The label from [^3H]palmitate associated with cytosolic G is myristate, not palmitate(10) . In the experiment shown in Fig. 1A, G is visualized as a pair of proteins with apparent molecular masses of 42 and 43 kDa. This is the result of unexpectedly efficient reading of the altered polyhedron initiator codon contained upstream of the inserted G sequence in the original pVL1393 expression vector(29) . This altered initiator codon was placed out of frame with the G sequence in all subsequent experiments.

(^4)
The m1 muscarinic receptor can stimulate nucleotide exchange on G in the absence of agonist, albeit at a slower rate than that observed in the presence of an agonist such as carbachol(35) .

(^5)
Much longer exposures reveal that a very small amount of label (<2% of wild type) is incorporated into truncated G.

(^6)
Prior studies (G. H. Biddlecome, G. Berstein, and E. M. Ross, unpublished results) demonstrated that addition of a hexahistidine tag at the carboxyl terminus of G decreased the capacity of phospholipase C-beta1 to stimulate steady-state GTP hydrolysis (i.e. GAP effect) by impairing the interaction of G with the m1 receptor that is necessary for rapid GDP/GTP exchange. Present studies revealed that nontagged and hexahistadine-tagged G shared similar rates of agonist-stimulated GTPS binding, whereas the tagged protein had lower rates of atropine- (basal) and carbachol-stimulated steady-state GTP hydrolysis, accounting for the higher relative level of stimulation by carbachol (Fig. 5; Table 1).

(^7)
When subjected to TX-114 phase separation analysis, purified G-short partitioned predominantly into the detergent phase. Treatment of G-short with palmitoylthioesterase failed to alter this pattern. These results provide further evidence that G-short is not palmitoylated.

(^8)
P. Sternweis and C. Slaughter, personal communication.


ACKNOWLEDGEMENTS

We thank Linda Hannigan and Karen Chapman for excellent technical assistance, Dr. Gabriel Berstein for performing the experiments presented in Fig. 2E, and Dr. Susanne Mumby for helpful comments and reading of the manuscript.


REFERENCES

  1. Simon, M. I., Strathmann, M. P., and Gautam, N. (1991) Science 252, 802-808 [Medline] [Order article via Infotrieve]
  2. Bourne, H. R., Sanders, D. A., and McCormick, F. (1990) Nature 348, 125-132 [CrossRef][Medline] [Order article via Infotrieve]
  3. Hepler, J. R., and Gilman, A. G. (1992) Trends Biochem. Sci. 17, 383-387 [CrossRef][Medline] [Order article via Infotrieve]
  4. Yamane, H. K., Farnsworth, C. C., Xie, H., Howald, W., Fung, B. K.-K., Clarke, S., Gelb, M. H., and Glomset, J. A. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 5868-5872 [Abstract]
  5. Mumby, S. M., Casey, P. J., Gilman, A. G., Gutowski, S., and Sternweis, P. C. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 5873-5877 [Abstract]
  6. Iñiguez-Lluhi, J. A., Simon, M. I., Robishaw, J. D., and Gilman, A. G. (1992) J. Biol. Chem. 267, 23409-23417 [Abstract/Free Full Text]
  7. Buss, J. E., Mumby, S. M., Casey, P. J., Gilman, A. G., and Sefton, B. M. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 7493-7497 [Abstract]
  8. Linder, M. E., Pang, I.-H., Duronio, R. J., Gordon, J. I., Sternweis, P. C., and Gilman, A. G. (1991) J. Biol. Chem. 266, 4654-4659 [Abstract/Free Full Text]
  9. Taussig, R., Iñiguez-Lluhi, J., and Gilman, A. G. (1993) Science 261, 218-221 [Medline] [Order article via Infotrieve]
  10. Linder, M. E., Middleton, P., Hepler, J. R., Taussig, R., Gilman, A. G., and Mumby, S. M. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 3675-3679 [Abstract]
  11. Parenti, M., Vigano, M. A., Newman, C. M. H., Milligan, G., and Magee, A. I. (1993) Biochem. J. 291, 349-353 [Medline] [Order article via Infotrieve]
  12. Veit, M., Nurnberg, B., Spicher, K., Harteneck, C., Ponimaskin, E., Schultz, G., and Schmidt, M. F. G. (1994) FEBS Lett. 339, 160-164 [CrossRef][Medline] [Order article via Infotrieve]
  13. Hallak, H., Muszbek, L., Laposata, M., Belmonte, E., Brass, L. F., and Manning, D. R. (1994) J. Biol. Chem. 269, 4713-4716 [Abstract/Free Full Text]
  14. Mumby, S. M., Kleuss, C., and Gilman, A. G. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2800-2804 [Abstract]
  15. Degtyarev, M. Y., Spiegel, A. M., and Jones, T. L. Z. (1993) J. Biol. Chem. 268, 23769-23772 [Abstract/Free Full Text]
  16. Wedegaertner, P. B., and Bourne, H. R. (1994) Cell 77, 1063-1070 [Medline] [Order article via Infotrieve]
  17. Navon, S. E., and Fung, B. K.-K. (1987) J. Biol. Chem. 262, 15746-15751 [Abstract/Free Full Text]
  18. Journot, L., Pantaloni, C., Bockaert, J., and Audigier, Y. (1991) J. Biol. Chem. 266, 9009-9015 [Abstract/Free Full Text]
  19. Higashijima, T., and Ross, E. M. (1991) J. Biol. Chem. 266, 12655-12661 [Abstract/Free Full Text]
  20. Coleman, D. E., Berghuis, A. M., Lee, E., Linder, M. E., Gilman, A. G., and Sprang, S. R. (1994) Science 265, 1405-1412 [Medline] [Order article via Infotrieve]
  21. Mixon, M. B., Lee, E., Coleman, D. E., Berghuis, A. M., Gilman, A. G., and Sprang, S. R. (1995) Science 270, 954-960 [Abstract]
  22. Strathmann, M., and Simon, M. I. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 9113-9117 [Abstract]
  23. Wedegaertner, P. B., Chu, D. H., Wilson, P. T., Levis, M. J., and Bourne, H. R. (1993) J. Biol. Chem. 268, 25001-25008 [Abstract/Free Full Text]
  24. McCallum, J. F., Wise, A., Parenti, M., and Milligan, G. (1995) Biochem. Soc. Trans. 23, 98
  25. Edgerton, M. D., Chabert, C., Chollet, A., and Arkinstall, S. (1994) FEBS Lett. 354, 194-199
  26. Camp, L. A., and Hofmann, S. L. (1993) J. Biol. Chem. 268, 22566-22574 [Abstract/Free Full Text]
  27. Camp, L. A., Verkruyse, L. A., Afendis, S. J., Slaughter, C. A., and Hofmann, S. L. (1994) J. Biol. Chem. 269, 23212-23219 [Abstract/Free Full Text]
  28. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  29. Hepler, J. R., Kozasa, T., Smrcka, A. V., Simon, M. I., Rhee, S. G., Sternweis, P. C., and Gilman, A. G. (1993) J. Biol. Chem. 268, 14367-14375 [Abstract/Free Full Text]
  30. Summers, M. D. and Smith, G. E. (1987) Tex. Agric. Exp. Stn. Bull. 1555
  31. Andersson, S., Davis, D. L., Dahlback, H., Jornvall, H., and Russell, D. W. (1989) J. Biol. Chem. 264, 8222-8229 [Abstract/Free Full Text]
  32. Taussig, R., Tang, W.-J., Hepler, J. R., and Gilman, A. G. (1994) J. Biol. Chem. 269, 6093-6100 [Abstract/Free Full Text]
  33. Brandt, D. R., and Ross, E. M. (1985) J. Biol. Chem. 260, 266-272 [Abstract/Free Full Text]
  34. Berstein, G., Blank, J. L., Jhon, D. Y., Exton, J. H., Rhee, S. G., and Ross, E. M. (1992) Cell 70, 411-418 [Medline] [Order article via Infotrieve]
  35. Berstein, G., Blank, J. L., Smrcka, A. V., Higashijima, T., Sternweis, P. C., Exton, J. H., and Ross, E. M. (1992) J. Biol. Chem. 267, 8081-8088 [Abstract/Free Full Text]
  36. Bordier, C. (1981) J. Biol. Chem. 256, 1604-1607 [Abstract/Free Full Text]
  37. Ying, Y.-S., Anderson, R. G. W., and Rothberg, K. G. (1992) Cold Spring Harbor Symp. Quant. Biol. 57, 593-604 [Medline] [Order article via Infotrieve]
  38. Wedegaertner, P. B., Wilson, P. T., and Bourne, H. R. (1995) J. Biol. Chem. 270, 503-506 [Free Full Text]
  39. Chang, W.-J., Ying, Y.-S., Rothberg, K. G., Hooper, N. M., Turner, A. J., Gambliel, H. A., De Gunzburg, J., Mumby, S. M., Gilman, A. G., and Anderson, R. G. W. (1994) J. Cell Biol. 126, 127-138 [Abstract]
  40. Degtyarev, M. Y., Spiegel, A. M., and Jones, T. L. Z. (1993) Biochemistry 32, 8057-8061 [Medline] [Order article via Infotrieve]

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