©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Purification of Recombinant G Proteins from Sf9 Cells by Hexahistidine Tagging of Associated Subunits
CHARACTERIZATION OF alpha AND INHIBITION OF ADENYLYL CYCLASE BY alpha(z)(*)

(Received for publication, October 27, 1994)

Tohru Kozasa Alfred G. Gilman

From the Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A method is described for purification of G protein alpha and beta subunits from Sf9 cells infected with recombinant baculoviruses. The subunit to be purified is coexpressed with an associated subunit bearing a hexahistidine tag. After adsorption of the oligomer to a Ni-containing column, the subunit to be purified is eluted specifically by promoting subunit dissociation with AlF(4). The alpha subunits of G, G(q), G(z), and G and the beta(1)(2) subunit complex were easily and efficiently purified by this method. Results were superior to established procedures in all cases.

Purified alpha was characterized for the first time. The protein has a slow rate of guanine nucleotide exchange (k = 0.01 min) and a very slow k for hydrolysis of GTP (0.1-0.2 min). GTPS (guanosine 5`-3-O-(thio)triphosphate)bulletalpha does not influence the activity of several adenylyl cyclases or phospholipases. Activated alpha(z) inhibits the activity of type I and type V adenylyl cyclases. It is a somewhat more potent inhibitor of type V adenylyl cyclase than is activated alpha.


INTRODUCTION

Heterotrimeric guanine nucleotide-binding proteins (G proteins) (^1)transduce regulatory signals from a large number of cell-surface receptors to effectors such as adenylyl cyclases, phosphodiesterases, phospholipases, and ion channels(1, 2, 3, 4, 5) . Each G protein oligomer contains a guanine nucleotide-binding alpha subunit, which can be palmitoylated and, in some cases, myristoylated, and a high affinity dimer of beta and subunits; is prenylated. Sixteen genes are known to encode G protein alpha subunits, while four beta and six subunits have been described to date. The alpha subunits are commonly classified as members of four subfamilies: alpha(s) and alpha (stimulators of adenylyl cyclases); alpha, alpha, alpha, alpha(o), alpha, alpha, alpha(z), and alpha(g) (a functionally diverse group; pertussis toxin substrates, with the exception of alpha(z)); alpha(q), alpha, alpha(14), and alpha/alpha (activators of phospholipase C-betas); and alpha and alpha. The alpha subfamily remains poorly characterized.

cDNAs that encode alpha and alpha were isolated from a mouse brain library using a homology-based, polymerase chain reaction strategy(6) . The two proteins appear to be expressed ubiquitously(6, 7, 8) . Although little is known about the identity of the receptors that activate these G proteins or the effectors that they regulate, it has been reported recently that overexpression of wild type or constitutively activated alpha causes transformation of NIH 3T3 cells(9, 10, 11) . In addition, the Drosophila gene concertina, whose product is highly homologous to both alpha and alpha, is involved in ventral furrow formation and posterior midgut invagination during gastrulation(12) .

Purification of G proteins from natural sources is problematic because of limiting quantities (in most cases) and difficulty of resolution from closely related family members. Expression of certain alpha subunits (alpha(s), alpha(i), and alpha(o)) in Escherichia coli yields large amounts of protein that can be myristoylated where appropriate (alpha(i) and alpha(o)) (13) , but the proteins are not palmitoylated and may be missing other unknown modifications (particularly alpha(s)). We (14, 15, 16, 17) and others (18) have expressed G protein alpha and beta subunits in Sf9 cells after infection with recombinant baculoviruses, but yields of appropriately modified protein are often low and purification is laborious. We describe herein a general and substantially improved method for purification of several G protein alpha and beta subunits from Sf9 cells. The protein to be purified is coexpressed with an associated hexahistidine-tagged subunit. The oligomer is adsorbed to a Ni-containing column, and the desired protein is eluted by subunit dissociation (activation with AlF(4)). This combination of specific adsorption and elution yields a highly enriched product that can be brought to essential homogeneity by one step of ion exchange chromatography. We have applied this method to permit characterization of alpha and further investigation of alpha(z).


MATERIALS AND METHODS

Plasmids and Recombinant Viruses

To construct a plasmid encoding a hexahistidine-tagged (2) subunit, the plasmid pBS(2) encoding the bovine (2) subunit (19) was digested with BglII and MscI. Synthetic oligonucleotides encoding the amino-terminal hexahistidine tag were annealed and ligated into this site. The oligonucleotides utilized had the following sequence.

The resulting plasmid was digested with BglII and XbaI, and the fragment containing the His(6)-(2) cDNA was ligated into the BglII and XbaI sites of baculovirus transfer vector pVL1392. The amino acid sequence of His(6)-(2) is MAHHHHHHA-(2)(3-71).

To construct a transfer vector for alpha, a 1.4-kilobase pair cDNA fragment that encodes murine alpha was isolated from pG12-5 (6) by digestion with EcoRI and SacI; the SacI site was blunt-ended (Klenow). This fragment was ligated into the EcoRI and SmaI sites of pVL1392. To remove two Bsu36I sites from the 5`-noncoding region of the alpha cDNA, this plasmid was digested with EcoRI and Bsu36I and self-ligated after both sites were blunt-ended (Klenow). The sequences of all constructs were confirmed by DNA sequencing.

pVLHis(6)-(2) or pVLalpha was cotransfected (Lipofectin, Life Technologies, Inc.) into Sf9 cells with BacPac6 viral DNA (Clontech) linearized with Bsu36I. Recombinant viruses were plaque-purified and amplified as described (20) . Positive viral clones were identified by immunoblotting extracts of infected cells with alpha antiserum J168 or (2) antiserum X263.

Recombinant baculoviruses encoding alpha, alpha(q), and beta(1) subunits have been described(14, 16, 21) . A virus encoding hexahistidine-tagged alpha was constructed and kindly provided by Christiane Kleuss (this laboratory). A recombinant baculovirus encoding alpha(z) was generously provided by Patrick Casey and Tim Fields (Duke University).

Purification of alpha Subunits

Membrane Preparation

Sf9 cells were cultured in suspension in IPL-41 medium (Life Technologies, Inc.) containing 1% Pluronic F68, 10% heat-inactivated fetal calf serum, and 50 µg/ml gentamicin at 27 °C with constant shaking (125 rpm). For large scale cultures, the concentration of serum was reduced to 1% and 1% lipid mix (Life Technologies, Inc.) was added.

Sf9 cells (4-liter culture; 1.5 times 10^6 cells/ml) were infected with amplified recombinant baculoviruses encoding the desired G protein alpha subunit, the beta(1) subunit, and the His(6)-(2) subunit (1 plaque-forming unit/cell for each virus). Cells were harvested 48 h later by centrifugation at 1000 times g for 10 min and suspended in 600 ml of ice-cold lysis buffer (50 mM NaHepes (pH 8.0), 0.1 mM EDTA, 3 mM MgCl(2), 10 mM beta-mercaptoethanol, 100 mM NaCl, 10 µM GDP, 0.02 mg/ml phenylmethylsulfonyl fluoride, 0.03 mg/ml leupeptin, 0.02 mg/ml 1-chloro-3-tosylamido-7-amino-2-heptanone, 0.02 mg/ml L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK), and 0.03 mg/ml lima bean trypsin inhibitor). Remaining procedures were carried out at 4 °C unless otherwise specified. Cells were lysed by nitrogen cavitation (Parr bomb) at 500 p.s.i. for 30 min. Cell lysates were centrifuged at 750 times g for 10 min to remove intact cells and nuclei. The supernatants were centrifuged at 100,000 times g for 30 min, and the resultant pellets were suspended in 300 ml of wash buffer (50 mM NaHepes (pH 8.0), 3 mM MgCl(2), 10 mM beta-mercaptoethanol, 50 mM NaCl, 10 µM GDP, and proteinase inhibitors as above) using a Potter-Elvehjem homogenizer and centrifuged again as above. The pellets (1.6-2.4 g of protein suspended in 160 ml of wash buffer) were frozen in liquid nitrogen and stored at -80 °C.

Purification of alpha

Membranes (40-60 ml, 600 mg of protein) were thawed and diluted to 5 mg/ml with wash buffer containing fresh proteinase inhibitors. Sodium cholate was added to a final concentration of 1% (w/v), and the mixture was stirred on ice for 1 h prior to centrifugation at 100,000 times g for 40 min. The supernatants (membrane extract) were collected, diluted 5-fold with buffer A (20 mM NaHepes (pH 8.0), 100 mM NaCl, 1 mM MgCl(2), 10 mM beta-mercaptoethanol, 10 µM GDP, and 0.5% CE), and loaded onto a 4-ml Ni-NTA (Qiagen) column (1.5 times 2.3 cm), which had been equilibrated with buffer A. The column was washed with 100 ml of buffer A containing 5 mM imidazole. The column was then warmed to room temperature for 15 min prior to washing with 12 ml of buffer A containing 5 mM imidazole at 30 °C and 32 ml of buffer B (20 mM NaHepes (pH 8.0), 50 mM NaCl, 10 mM beta-mercaptoethanol, 10 µM GDP, 0.5% CE, 30 µM AlCl(3), 50 mM MgCl(2), 10 mM NaF, and 5 mM imidazole) at 30 °C. Finally, the column was washed with 12 ml of buffer C (buffer A containing 150 mM imidazole). Fractions (4 ml) were collected during washing with buffers B and C. The fractions were analyzed by silver staining and immunoblotting after electrophoresis through 9% sodium dodecyl sulfate-polyacrylamide gels. alpha was eluted during washing with buffer B, while beta(1)His(6)-(2) was eluted with buffer C. The peak fractions of alpha (about 20 ml) were pooled and diluted 3-fold with buffer D (20 mM NaHepes (pH 8.0), 1 mM EDTA, 3 mM MgCl(2), 3 mM dithiothreitol, 0.7% CHAPS). This solution was then applied to a Mono S HR 5/5 cation exchange column for FPLC (Pharmacia Biotech Inc.), which had been equilibrated with buffer D. alpha was eluted (at approximately 300 mM NaCl) with a 20-ml gradient of NaCl (0-400 mM). GDP (1 µl of a 5 mM solution) was put in the tubes used to collect fractions, which were each 0.5 ml. Fractions were assayed by immunoblotting and by measurement of [S]GTPS binding activity. Peak fractions were concentrated, and the buffer was changed to buffer D containing 100 mM NaCl and 0.5 µM GDP using a Centricon 30 (Amicon). Samples were frozen in liquid nitrogen and stored at -80 °C.

Purification of alpha(z), alpha(q), and alpha

Differences from the procedure utilized for alpha are described. In the case of alpha(z), the Ni-NTA column was washed with 100 ml of buffer A containing 300 mM NaCl and 5 mM imidazole after application of the diluted membrane extract. alpha(z) was eluted from the column at room temperature with buffer E (buffer B except with 1% sodium cholate instead of 0.5% CE). alpha(z) was further purified by Mono S HR 5/5 column chromatography. The pH of buffer D was 7.4 instead of 8.0, and the NaCl gradient (25 ml) was 0-550 mM. Fractions were assayed for [S]GTPS binding activity (0.5 mM MgSO(4) total; 1 µM free Mg) and by immunoblotting (using alpha(z) antiserum P961). alpha(z) was eluted in fractions containing 400-450 mM NaCl. The pH of the final buffer was 7.4.

For purification of alpha(q), the concentration of GDP in the buffers was raised to 50 µM. The Ni-NTA column was washed with buffer A containing 300 mM NaCl and 5 mM imidazole. The column was then incubated at room temperature with buffer F (20 mM NaHepes (pH 8.0), 100 mM NaCl, 10 mM beta-mercaptoethanol, 0.2 mM MgCl(2), 5 µM GTPS, 0.2% sodium cholate, and 5 mM imidazole) and washed with 32 ml of the same buffer. This step is utilized to elute an endogenous (Sf9 cell) alpha(i)-like protein. alpha(q) was eluted with buffer E. The peak fractions from the Ni-NTA column were diluted 3-fold with buffer D and loaded onto a Mono Q HR 5/5 anion exchange column that had been equilibrated with buffer D. alpha(q) was eluted with a linear gradient of NaCl (0-400 mM; 20 ml). Fractions were assayed by immunoblotting with alpha(q)/alpha antiserum Z811 and alpha antiserum B825. Recombinant alpha(q) is recognized by both antisera and was eluted in fractions containing about 220 mM NaCl. An endogenous Sf9 cell alpha(q)-like protein is recognized by antiserum Z811 but not by antiserum B825 and eluted later in the gradient (about 280 mM NaCl)(14) . The peak fractions that contained only recombinant alpha(q) were pooled and concentrated, and the buffer was changed to buffer D containing 100 mM NaCl and 0.5 µM GDP.

For purification of alpha, the Ni-NTA column was processed as described for alpha(z). The peak fractions from the Ni-NTA column were diluted 3-fold with buffer D and loaded onto a Mono Q HR 5/5 column that had been equilibrated with buffer D. alpha was eluted with a NaCl gradient (0-400 mM; 20 ml). Fractions were assayed for [S]GTPS binding activity and by immunoblotting using alpha(s)/alpha(i) antiserum P960. alpha was eluted in fractions containing about 200 mM NaCl. The peak fractions were processed as described for alpha(q). This alpha preparation contained a very small amount of an Sf9 cell alpha(i)-like protein (about 1%).

Purification of beta(1)(2)

To purify the recombinant beta(1)(2) subunit complex, Sf9 cells were infected with baculoviruses encoding beta(1), (2), and His(6)-alpha. His(6)-alpha has an insertion of 6 histidine residues at position 121 of alpha (where the yeast alpha subunit GPA 1 has a long insert compared with mammalian alpha subunits). Ni-NTA column chromatography and Mono Q column chromatography were performed as described for purification of alpha. beta(1)(2) was eluted from the Mono Q column in fractions containing about 180 mM NaCl. The peak fractions were concentrated, and the buffer was changed to buffer D containing 100 mM NaCl.

GTPS or GTP Binding and GTPase Assays

[S]GTPS and [-P]GTP binding assays were performed as described by Northup et al.(22) . Reaction mixtures contained HEDL buffer (50 mM NaHepes (pH 8.0), 1 mM EDTA, 3 mM dithiothreitol, and 0.05% CE) with the indicated concentrations of MgSO(4) and [S]GTPS (3000-8000 cpm/pmol) or [-P]GTP (10,000 cpm/pmol). Reactions were terminated by addition of 2 ml of ice-cold 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, and 20 mM MgCl(2).

Steady state GTPase activity was measured as described in the figure legends. Reactions were terminated by addition of 20-µl aliquots to 780 µl of ice-cold 5% (w/v) Norit in 50 mM NaH(2)PO(4), and P(i) was determined as described previously(23) .

Adenylyl Cyclase Assays

Adenylyl cyclase activity was measured as described by Smigel (24) . Purified G proteins were reconstituted with 10 µg of membranes from Sf9 cells expressing type I, II, or V adenylyl cyclase in a final volume of 20 µl for 3 min at 30 °C prior to assay. All assays were performed for 7 min at 30 °C in a volume of 50 µl. The concentration of MgCl(2) was 4 mM, and that of CHAPS was 0.12%. To prepare GTPS-bound alpha subunits, proteins were incubated with 200 µM GTPS for 60 min (alpha(s), alpha(z), and alpha) or 120 min (alpha) at 30 °C in the presence of 4 mM free Mg (alpha(s), alpha, or alpha) or 5 µM free Mg (alpha(z)). Free nucleotide was removed by gel filtration through a Sephadex G-50 spin column. The concentrations of alpha subunits were calculated from [S]GTPS binding data.

Trypsin Protection Assay

alpha was diluted with HEDL buffer (to 1 µM) and was incubated with 100 µM GDP or 100 µM GDP, 30 µM AlCl(3), 50 mM MgSO(4), and 10 mM NaF at 0 °C for 10 min or with 100 µM GTPS and 10 mM MgSO(4) at 30 °C for 60 min. TPCK-treated trypsin (10% of the alpha mass) was then added, and the reaction mixtures were incubated for 10 or 30 min at 30 °C. Reactions were terminated by addition of trypsin inhibitor (10-fold mass excess of trypsin) and transfer to ice. The samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (9% gels) and immunoblotting with alpha antiserum J168.

Antisera

alpha antisera J168 and J169 were made by immunization of rabbits against the synthetic 12-amino acid peptide corresponding to the carboxyl terminus of alpha. alpha(s)/alpha(i) antiserum P960(25) , alpha(q)/alpha antiserum Z811(14) , alpha antiserum B825(26) , alpha(z) antiserum P961 (25) , and (2) antiserum X263 (27) have been described previously.

Miscellaneous Procedures

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed as described by Laemmli(28) . Protein concentrations were determined by staining with Amido Black (29) or by the method of Bradford (30) using bovine serum albumin as the standard. Protein staining with silver nitrate following polyacrylamide gel electrophoresis was performed as described by Wray et al.(31) . Immunoblotting was performed using the ECL chemiluminescense detection system (Amersham Corp.). Phospholipase C activity was assayed as described by Hepler et al.(14) . Free Mg concentrations were calculated using a K(d) of EDTA for Mg of 1 µM at pH 7.6(32) . The following reagents were kindly provided by colleagues at the University of Texas Southwestern Medical Center: alpha purified from Sf9 cells by William D. Singer; membranes from Sf9 cells expressing type I, II, or V adenylyl cyclase by Ronald Taussig; alpha(s) purified from E. coli by Wei-Jen Tang; antisera P960, P961, and X263 by Susanne Mumby; antisera Z811 and B825 by Paul C. Sternweis; and [-P]GTP by Elliott M. Ross. The cDNA encoding alpha was generously provided by Dr. Melvin I. Simon (California Institute of Technology).


RESULTS

We have previously described the purification of relatively small amounts of alpha(q), alpha, and alpha from Sf9 cells following their coexpression with G protein beta and subunits(14, 15) . Expression of the heterotrimer increases the amount of active alpha subunit in membrane fractions and prevents the aggregation of alpha(q) and alpha. However, difficulty with purification of alpha prompted the present effort to improve and generalize this technology. The resulting method for purification of G subunits takes advantage of the high specificity, affinity, and capacity of Ni-NTA resin (33, 34) for hexahistidine-tagged beta and specific elution of a coexpressed alpha subunit following activation with AlF(4).

Purification of alpha

Sf9 cells were infected with baculoviruses encoding alpha, beta(1), and His(6)-(2). A cholate extract containing the hexahistidine-tagged heterotrimer was diluted with buffer containing CE before application to the Ni-NTA column. Addition of this detergent improved the stability of the heterotrimer on the resin. After extensive washing, alpha could be activated and eluted with AlF(4) if buffers were warmed to 30 °C. Silver-stained fractions from the Ni-NTA column are shown in Fig. 1. Most of the protein in the cholate extract flows through the column (lanes 1 and 2) or is removed by washing with buffer containing low concentrations of imidazole (lanes 3-5). alpha is seen as the major protein in fractions eluted in the presence of AlF(4) (lanes 6-10). The beta(1)His(6)-(2) complex can then be eluted with 150 mM imidazole (lanes 11 and 12). beta(1) is negatively stained because of its high concentration.


Figure 1: Purification of alpha on Ni-NTA column. A cholate extract of membranes (600 mg of membrane protein) from Sf9 cells expressing alpha and beta(1)His(6)-(2) was diluted, loaded onto a 4-ml Ni-NTA column, and chromatographed as described under ``Materials and Methods.'' Fractions (4 µl) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (9% gels) and stained with silver nitrate. Lane 1, load; lane2, flow-through; lanes 3-5, wash with equilibration buffer containing 5 mM imidazole; lanes 6-10, elution with AMF plus GDP; lanes11 and 12, elution with 150 mM imidazole. The arrows at the right indicate the positions of alpha and beta(1). beta(1) is not well stained with silver nitrate; His(6)-(2) runs at the dye front.



alpha was further purified by Mono S cation exchange chromatography using CHAPS as the detergent (Fig. 2). The final sample of alpha was visualized as a homogeneous silver-stained band in polyacrylamide gels with an apparent molecular weight of 43,000 (Fig. 3, lane 1). The yield of purified protein was 600 µg from 600 mg of Sf9 cell membrane protein, which is obtained from a 1-1.5-liter culture (Table 1). The relatively low enrichment of GTPS binding activity is undoubtedly due to the presence of other GTP-binding proteins in the membrane extract. The actual stoichiometry of binding of GTPS to alpha is approximately 0.5 mol/mol (approximately 10 nmol/mg protein) (see below).


Figure 2: Mono S chromatography of alpha. The peak fractions of alpha from the Ni-NTA column were loaded onto a Mono S column and chromatographed as described under ``Materials and Methods.'' Fractions (3 µl) were assayed for GTPS binding activity with 5 µM GTPS and 10 mM MgSO(4) for 90 min at 30 °C.




Figure 3: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining of purified alpha, alpha(q), alpha(z), alpha, and beta(1)(2). alpha subunits (50 ng) purified as described in the text were subjected to electrophoresis through 9% polyacrylamide gels and were stained with silver nitrate. Lane1, alpha; lane2, alpha(q); lane3, alpha(z); lane4, alpha; lane5, beta(1)(2).





The fractions from the Ni-NTA column that were eluted with AlF(4) also contained Sf9 cell proteins that were detected with alpha(s)/alpha(i) antiserum P960 (Sf9 alpha(i)) or alpha(q)/alpha antiserum Z811 (Sf9 alpha(q)). However, both immunoreactive proteins flow through the Mono S column. When the purification procedure was followed using membranes from Sf9 cells expressing only beta(1)His(6)-(2), immunoreactive proteins were not detected with alpha antiserum J168 or broadly reactive antisera in the fractions corresponding to those where alpha is normally found. We conclude that the final preparation of alpha is essentially free of other G protein subunits.

Purification of alpha(z), alpha(q), and alpha

The beta(1)His(6)-(2) method was also used successfully to purify recombinant G subunits from two other subfamilies: alpha(z), alpha(q), and alpha. For these subunits, the Ni-NTA column was washed with buffer containing high concentrations of salt (300 mM NaCl), and the G proteins were eluted with AlF(4) in buffer containing 1% sodium cholate at room temperature. Cholate facilitates the dissociation of alpha(z), alpha(q), and alpha from beta(1)His(6)-(2). By contrast, alpha could not be eluted from the Ni-NTA column in the presence of 1% sodium cholate and AlF(4). Mono S chromatography was also used to complete the purification of alpha(z), and the yield of alpha(z) was 250 µg from 600 mg of Sf9 cell membranes (Table 1; Fig. 3, lane 3).

To purify alpha(q), the Ni-NTA column was also washed with buffer containing 5 µM GTPS to remove Sf9 alpha(i). This wash takes advantage of the relatively poor affinity of alpha(q) for GTPS in order to activate and remove endogenous G subunits with high affinity for the nucleotide. Sf9 alpha(q) was resolved from recombinant alpha(q) by Mono Q chromatography (Fig. 3, lane2). To purify alpha (Fig. 3, lane4), endogenous alpha(q) was removed by Mono Q chromatography, but it was not possible to resolve Sf9 alpha(i) from recombinant alpha. However, performance of the purification protocol for alpha in the absence of expression of this subunit suggests that the level of contamination by Sf9 alpha(i) is only about 1%. The yields of alpha(q) and alpha were 110 and 1400 µg, respectively, from 600 mg of Sf9 cell membrane (Table 1).

Purification of beta(1)(2)

Reciprocal hexahistidine tagging was also utilized to purify the beta(1)(2) subunit complex. In this case the hexahistidine tag was inserted into alpha at a position corresponding to a large insertion in the GPA1 G subunit of Saccharomyces cerevisiae. Membrane extracts containing His(6)-alpha, beta(1), and (2) were applied to the Ni-NTA column, washed with buffer containing a high concentration of NaCl, and eluted with AlF(4). beta was further purified by Mono Q column chromatography. G subunits were not detected as contaminants of this preparation. The yield of beta(1)(2) was 2 mg from 600 mg of Sf9 cell membranes (Table 1; Fig. 3, lane 5), and the product was able to support ADP-ribosylation of alpha and stimulation of alpha(s)-activated type II adenylyl cyclase activity in a manner characteristic of beta subunits purified by other methods (data not shown).

Characterization of alpha Antisera

The specificities of antisera J168 and J169 were assessed by immunoblotting (Fig. 4). A synthetic peptide corresponding to the carboxyl-terminal 12 amino acid residues of alpha was used as the antigen. This sequence has 8 amino acid residues that are identical with the corresponding sequence of alpha and 7 identities with alpha(q). Antiserum J168 reacts weakly with alpha, while antiserum J169 is specific for alpha. Neither antiserum recognizes the same amount of alpha(q), alpha, alpha(z), alpha, or alpha(s). alpha(s)/alpha(i) antiserum P960 and alpha(q)/alpha antiserum Z811 do not recognize either alpha or alpha. alpha(z) is recognized by alpha(z) antiserum P961 and very weakly by antiserum P960.


Figure 4: Immunoblot analysis of purified alpha subunits. Each purified alpha subunit (50 ng each of alpha, alpha, alpha(q), alpha, alpha(z), alpha, and alpha(s)) was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with silver nitrate (A) or immunoblotted (B) with alpha antisera J168 and J169, alpha(q)/alpha antiserum Z811, alpha(s)/alpha(i) antiserum P960, and alpha(z) antiserum P961. alpha, alpha(q), alpha(z), and alpha were purified as described under ``Materials and Methods.'' alpha was purified as described previously (15) . alpha(s) was purified from E. coli. alpha was purified from Sf9 cells and was a generous gift from Dr. William Singer(7) .



Guanine Nucleotide Binding and Hydrolysis by alpha

The time course of binding of [S]GTPS to purified alpha at 30 °C is shown in Fig. 5A. The rate of binding is slow, and maximal values are not achieved even after 150 min of incubation. The data can, however, be fit to a single exponential for estimation of maximal binding values. Binding of GTPS to alpha is slightly faster in the presence of 9 mM free Mg than at lower concentrations of the divalent cation (100 µM or 10 nM free Mg). At 10 mM Mg and 5 µM GTPS, the rate of GTPS binding is approximately 0.01 min. This rate is independent of nucleotide concentration from 1 µM to 100 µM (Fig. 5B). However, the extent of binding increases with nucleotide concentration over this range and saturates at about 10 µM GTPS (Fig. 5C). The maximum stoichiometry calculated from the data of Fig. 5B is about 0.5 mol of GTPS/mol of protein. The independence of the rate of binding of GTPS from nucleotide concentration suggests that the rate-limiting step in association of the nucleotide is dissociation of bound GDP, as observed for other G protein alpha subunits. Relatively high concentrations of GTPS are required to observe maximal binding of the nucleotide to alpha, as is also seen with alpha(q) and alpha(7, 14) . Once bound, however, GTPS dissociates from alpha very slowly. More than 90% of the labeled nucleotide remains bound 120 min after addition of a 100-fold excess of unlabeled GTPS at either 9 mM or 5 µM free Mg (data not shown).


Figure 5: Time course of binding of [S]GTPS to alpha. A, alpha (250 nM) was incubated at 30 °C in HEDL buffer with 5 µM [S]GTPS and 10 mM MgSO(4) (bullet), 1 mM MgSO(4) (circle), or 5 mM EDTA (). Aliquots (20 µl) were withdrawn at the indicated times, filtered, and counted. Data shown are the average of duplicate determinations from a single experiment that is representative of three separate experiments. B, alpha (250 nM) was incubated at 30 °C in HEDL buffer with 10 mM MgSO(4) and 1 µM (circle), 5 µM (bullet), 10 µM (), 50 µM (box), or 100 µM GTPS (). Data shown are the average of duplicate determinations from a single experiment that is representative of three separate experiments. C, the maximum stoichiometry of binding calculated from the data of Fig. 5B is plotted against the concentration of GTPS.



The capacity of various nucleotides to compete for GTPS binding to alpha is shown in Table 2. Only guanine nucleotides and, to a lesser extent, ITP compete effectively with GTPS. This pattern is typical of G protein alpha subunits(7, 25, 35) .



Most G protein alpha subunits are substantially protected from tryptic proteolysis when activated with either nonhydrolyzable analogs of GTP or AlF(4). This is also true of alpha (Fig. 6). When the protein is incubated with GTPS or AlF(4) (GDP + AMF (30 µM AlCl(3), 50 mM MgCl(2), and 10 mM NaF)), exposure to trypsin results in the generation of a stable 40-kDa fragment. In the presence of GDP, alpha is digested rapidly; one fragment with a mass of about 10 kDa is visualized with antiserum J168. A small amount of the 10-kDa band is observed after incubation with GTPS, consistent with the slow rate of nucleotide exchange. However, activation with AlF(4) appears to be complete, indicating that most of the protein in the preparation is native and capable of undergoing activator-dependent conformational changes similar to those observed with other G proteins.


Figure 6: Protection of alpha from tryptic proteolysis. alpha (1 µM) was incubated with 100 µM GDP (lanes 1-3) or AMF + 100 µM GDP (lanes 4-6) at 0 °C for 10 min or with 100 µM GTPS and 10 mM MgSO(4) (lanes 7-9) at 30 °C for 60 min. TPCK-treated trypsin (1/10 the mass of alpha) was added to each tube and incubated at 30 °C. Aliquots were withdrawn and subjected to gel electrophoresis followed by immunoblotting with antiserum J168. Lanes 1, 4, and 7, before addition of trypsin; lanes 2, 3, 5, 6, 8, and 9, after addition of trypsin and incubation at 30 °C for 10 min (lanes2, 5, and 8) or 30 min (lanes3, 6, and 9).



alpha has intrinsic GTPase activity. The steady state rate of GTP hydrolysis is 0.006 min (at 9 mM free Mg), and the rate is constant for at least 90 min after an initial lag (Fig. 7A). This rate is approximately equal to that observed for GTPS binding (GDP dissociation), and it is reduced modestly at lower concentrations of Mg. Although the slow exchange of nucleotide makes it difficult to load the protein with substrate and measure the catalytic rate constant (k) directly, we estimated the value of k indirectly in two ways. There is a delay in the release of P(i) in the steady state GTPase assay that is inversely related to the catalytic rate constant and the rate of substrate binding (Fig. 7A). This lag is equal to 1/(k + k)(23, 25) . Using a value for the lag of 5 min and k of 0.01 min, k is estimated at 0.19 min. The rate at which steady state binding of [-P]GTP is achieved is also related to the rate of nucleotide binding and k: k = k + k, since the label is released upon hydrolysis. Steady state binding of [-P]GTP is achieved with a rate of approximately 0.1 min (Fig. 7B). k is thus equal to 0.09 min. Thus, from both measurements we suggest a value for k in the range of 0.1-0.2 min.


Figure 7: GTPase activity of alpha. A, steady state GTPase. alpha was incubated in HEDL buffer at 30 °C for 3 min, and an equal volume of solution was then added to bring the final concentrations to 250 nM alpha, 10 µM [-P]GTP, and 10 mM MgSO(4) (bullet) or 0.5 mM MgSO(4) (circle). Reaction mixtures were incubated at 30 °C. At the indicated times, aliquots (20 µl) were withdrawn and release of P(i) was determined as described under ``Materials and Methods.'' Data shown are the average of duplicate determinations from a single experiment that is representative of five experiments. B, time course of [-P]GTP binding. alpha was incubated in HEDL buffer at 30 °C for 3 min. An equal volume of solution was then added to bring the final concentrations to 1 µM alpha, 5 µM [-P]GTP, and 10 mM MgSO(4). At the indicated times, aliquots were withdrawn and [-P]GTP binding was determined as described under ``Materials and Methods.'' Data shown are the average of duplicate determinations from a single experiment that is a representative of three such experiments.



Interactions of alpha with beta, Effectors, and Bacterial Toxins

The G protein beta subunit complex stabilizes the heterotrimer by slowing the rate of dissociation of GDP from G. This effect can be observed by measurement of the effect of beta on the steady state GTPase activity of G, which is limited by the rate of product dissociation. The inhibitory effect of beta(1)(2) on the steady state GTPase activity of alpha is shown in Fig. 8. It was necessary to use relatively high concentrations of alpha in these experiments because of the protein's low GTPase activity. Nevertheless, the apparent affinity of beta(1)(2) for alpha was relatively low and more than stoichiometric amounts of beta were necessary to observe inhibition. The IC for beta(1)(2) was at least 100 nM (alpha to beta ratio of 1:2); GTPase activity was inhibited by approximately 60% when the alpha to beta ratio was 1:10. Other molecular species of beta may have a higher affinity for alpha.


Figure 8: Inhibition of steady state GTPase activity of alpha by beta(1)(2). The indicated concentrations of beta(1)(2) were incubated with or without 50 nM alpha in 50 µl of HEDL buffer containing 10 µM [-P]GTP and 10 mM MgSO(4) at 30 °C for 120 min. Release of P(i) was determined as described under ``Materials and Methods.'' The difference in release of P(i) in the presence or absence of alpha is plotted. The data shown are the average of duplicate determinations from a single experiment that is representative of three such experiments. P(i) detected with 50 nM alpha or 1 µM beta(1)(2) at 30 °C for 120 min was 2.1 or 0.67 pmol/50 µl, respectively.



The availability of purified, activated alpha allowed us to test the effect of the protein on several known effector targets for G subunits. alpha did not stimulate or inhibit the activity of type I or V (Table 3) or type II (not shown) adenylyl cyclase, nor did the protein affect the activity of phospholipase C-beta1, beta2, or beta3 or phospholipase C-1 (data not shown). Similarly, alpha had no effect on ADP-ribosylation factor-sensitive phospholipase D activity (36) or phosphatidylinositol 3-kinase activity (37) (data not shown).



Possible modifications of alpha by bacterial toxins were also examined. alpha was not ADP-ribosylated by cholera toxin in the presence of ADP-ribosylation factor, nor was it ADP-ribosylated by pertussis toxin (data not shown). The latter is consistent with the fact that the cysteine residue near the carboxyl terminus that serves as the site of ADP-ribosylation of alpha(i) or alpha(o) by pertussis toxin is replaced by isoleucine in alpha.

Inhibition of Adenylyl Cyclase by alpha(z)

The capacities of alpha and alpha(z) to inhibit adenylyl cyclases were also examined (Table 3, Fig. 9). The effects of recombinant Sf9 cell-derived alpha on calmodulin-activated type I and alpha(s)-activated type V adenylyl cyclases were comparable to those seen with myristoylated alpha purified from E. coli(38) . Of interest, alpha(z) also inhibited type I and type V adenylyl cyclases to similar extents and appeared to be a somewhat more potent inhibitor of type V adenylyl cyclase activity than was alpha. Higher concentrations of GDP-alpha(z) were required to inhibit type V adenylyl cyclase than were needed for the GTPS-bound form of the protein. Similar results were reported previously for alpha and type VI adenylyl cyclase(38) . Neither alpha nor alpha(z) inhibited type II adenylyl cyclase significantly (data not shown). It thus appears that alpha(z) regulates the same types of adenylyl cyclase as do the isoforms of alpha(i)(38) . These inhibitory effects of alpha(z) were presaged by demonstration that transfection of alpha(z) could lower cyclic AMP concentrations in intact cells(39) .


Figure 9: Inhibition of type V adenylyl cyclase activity by alpha(z) and alpha. The indicated concentrations of alpha subunits were reconstituted with 10 µg of membranes from Sf9 cells expressing type V adenylyl cyclase in the presence of 50 nM GTPSbulletalpha(s). Adenylyl cyclase activity was assayed as described under ``Materials and Methods.'' Data shown are the average of duplicate determinations from a single experiment that is representative of two such experiments.




DISCUSSION

Purification of G Protein Subunits

The method described above for purification of G protein subunits by reciprocal hexahistidine tagging takes advantage of the very high affinity of Ni-NTA resin for hexahistidine (K(d) = 10) (33, 34) and extremely specific elution of the untagged subunit with AlF(4). The capacity of the column is also great (at least 5 nmol of alpha/ml). One additional ion-exchange chromatographic step is required to obtain highly purified protein. Four alpha subunits from three different subfamilies and one beta subunit complex have been purified easily and efficiently with this method. In the case of alpha(q), the final yield is 20 times greater than achieved previously using Sf9 cells(14) ; for beta(1)(2) the yield is 10 times greater than that described(16) . No adequate scheme for purification of appropriately modified alpha has been described previously. Purification of alpha(z) from brain is laborious and the yield is very low(25) . Prior purification of alpha from Sf9 cells focused on cytosolic fractions(18) . Purification of G proteins from the membrane fraction of Sf9 cells yields products that are significantly more active than their cytosolic counterparts, at least in part because of the importance of lipid covalent modifications as determinants of their affinities for associated proteins.

Based on our experience to date, we suspect that this method will also be useful for purification of alpha, alpha, alpha(o), alpha, alpha, alpha(14), and various species of beta. We make no guesses about alpha(t), alpha(g), and alpha. We were unable to purify alpha(s) to homogeneity or alpha with this method. alpha(s) appears to have a lower affinity for beta(1)His(6)-(2) on the Ni-NTA column and did not bind adequately. alpha is not activated with AlF(4)(15) and thus cannot be eluted as described. We can elute alpha with GTPS to purify the irreversibly activated subunit. Attempts to place a hexahistidine tag at the amino terminus of the beta(2) subunit gave adequate results, although the yields of purified alpha subunits were decreased by about 50%. This is perhaps due to lower levels of expression of His(6)-beta(2) than of beta(1) in Sf9 cells.

Biochemical Properties of alpha

The rate of dissociation of GDP from alpha (0.01 min) is very slow: roughly 10-20 fold slower than rates observed for alpha(s)(40) and alpha(o)(41) , but comparable to values seen with alpha(z)(25) and alpha(7) . The rate of binding of GTPS to alpha was slightly decreased with a lower concentration of Mg. Similar results have been obtained with other alpha subunits except for alpha(z); in this case high concentrations (millimolar) of Mg greatly reduce the rate of GTPS binding(25) . The observed stoichiometry for GTPS binding to alpha was only about 50%, although the results of trypsin protection assays using AlF(4) suggest that most of the protein was native. It seems likely that some alpha may denature during prolonged incubations with GTPS. We may be able to observe higher stoichiometries of binding if reconstitution with receptors permits more rapid nucleotide exchange. The estimated rate for k for alpha is 0.1-0.2 min, which is similar to values observed with alpha(z)(25) and alpha(7) but is 5-40 times slower than detected with alpha(q)(42) , alpha(s)(40) , alpha(i)(41) , and alpha(o)(41) . alpha may regulate signaling pathways with slow time constants or it may be a target for a GTPase-activating protein, as seen with alpha(q)(43) or alpha(t)(44, 45) .

alpha and alpha have significant differences in the amino acid residues that make up the guanine nucleotide binding pocket when compared with other G protein alpha subunits. The crystal structure of alpha(t) indicates that Arg, Cys, and Thr contribute to binding of the guanine and ribose rings of GTPS(46) . A similar pattern is seen with G(47) . These residues are conserved in all mammalian G subunits except alpha and alpha, where Leu, Thr, and Ile, respectively, fill these positions. (^2)These sequence differences in alpha and alpha may be responsible for the reduced affinity of GTPS binding or slow nucleotide exchange.

DNA encoding wild type alpha has been isolated as a sequence capable of highly efficient transformation of NIH 3T3 cells (9) , and a mutant of alpha that is presumed to be GTPase-deficient and therefore constitutively active can also cause cellular transformation(10, 11) . However, the effector molecule(s) that are presumably regulated by alpha have not been identified. Transient expression of alpha increases serum-activated phospholipase A(2) activity in NIH 3T3 cells (10) and stimulates a protein kinase C-dependent Na/H exchanger activity in COS-1 cells(48) . However, there is no evidence for direct interaction of alpha with these proteins. Nor is there evidence for interaction of alpha with previously identified targets for G protein alpha subunits. Expression of hexahistidine-tagged G protein subunits in appropriate cells may permit isolation of novel targets for G protein action by approaches analogous to those presented here for purification of known cohorts of these proteins.


FOOTNOTES

*
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.

(^1)
The abbreviations used are: G protein, guanine nucleotide-binding regulatory protein; GTPS, guanosine 5`-(3-O-thio)triphosphate; CE, polyoxyethylene 10-lauryl ether; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonic acid; FPLC, fast protein liquid chromatography; AMF, 30 µM AlCl(3), 50 mM MgCl(2), and 10 mM NaF; Ni-NTA, nickel-nitrilotriacetic acid.

(^2)
The sole exception is Thr, which is Val in alpha(s) and alpha.


ACKNOWLEDGEMENTS

We thank Jeffrey Laidlaw for excellent technical assistance; Melvin I. Simon (California Institute of Technology) for the alpha cDNA; Patrick Casey and Tim Fields (Duke University) for a recombinant baculovirus encoding alpha(z); William Singer for purified alpha; Alex Brown and Alan Smrcka for testing alpha in phospholipase D and phosphatidylinositol 3-kinase assays, respectively; Natsuo Ueda and Christiane Kleuss for initial contributions to the purification of beta; and other members of our laboratory for valuable comments and discussions.


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