The G Protein beta 5 Subunit Interacts Selectively with the Gq alpha  Subunit*

Julia E. Fletcher, Margaret A. Lindorfer, Joseph M. DeFilippo, Hiroshi Yasuda, Maya Guilmard, and James C. GarrisonDagger

From the Department of Pharmacology, Health Sciences Center, University of Virginia, Charlottesville, Virginia 22908

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The diversity in the heterotrimeric G protein alpha , beta , and gamma  subunits may allow selective protein-protein interactions and provide specificity for signaling pathways. We examined the ability of five alpha  subunits (alpha i1, alpha i2, alpha o, alpha s, and alpha q) to associate with three beta  subunits (beta 1, beta 2, and beta 5) dimerized to a gamma 2 subunit containing an amino-terminal hexahistidine-FLAG affinity tag (gamma 2HF). Sf9 insect cells were used to overexpress the recombinant proteins. The hexahistidine-FLAG sequence does not hinder the function of the beta 1gamma 2HF dimer as it can be specifically eluted from an alpha i1-agarose column with GDP and AlF4-, and purified beta 1gamma 2HF dimer stimulates type II adenylyl cyclase. The beta 1gamma 2HF and beta 2gamma 2HF dimers immobilized on an anti-FLAG affinity column bound all five alpha  subunits tested, whereas the beta 5gamma 2HF dimer bound only alpha q. The ability of other alpha  subunits to compete with the alpha q subunit for binding to the beta 5gamma 2HF dimer was tested. Addition of increasing amounts of purified, recombinant alpha i1 to the alpha q in a Sf9 cell extract did not decrease the amount of alpha q bound to the beta 5gamma 2HF column. When G proteins in an extract of brain membranes were activated with GDP and AlF4- and deactivated in the presence of equal amounts of the beta 1gamma 2HF or beta 5gamma 2HF dimers, only alpha q bound to the beta 5gamma 2HF dimer. The alpha q-beta 5gamma 2HF interaction on the column was functional as GDP, and AlF4- specifically eluted alpha q from the column. These results indicate that although the beta 1 and beta 2 subunits interact with alpha  subunits from the alpha i, alpha s, and alpha q families, the structurally divergent beta 5 subunit only interacts with alpha q.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

All cells possess multiple signaling pathways that transmit signals from the hormones, autacoids, neurotransmitters, and growth factors in their environment. Complex biochemical mechanisms exist to discriminate, integrate, and modulate a cell's response to this constantly changing set of stimuli. One of the best characterized signal transduction systems is the pathway used by receptors coupled to heterotrimeric G proteins1 (1, 2). Our current understanding of this signaling pathway shows it to be surprisingly complex with large families of proteins comprising the receptors, G proteins, and effectors (1, 3, 4) and important roles for both the alpha  and beta gamma subunits of the heterotrimer in activating effectors (2, 4, 5). Moreover, some ligands activate multiple G proteins (1, 2, 6), and certain receptors activate the MAP kinase pathway (6, 7) and/or other tyrosine kinase signaling pathways (8). Thus, an important unsolved question in cell signaling is how a cell selects a response from the multiple possibilities available.

Current evidence holds that specificity is determined at many levels. In addition to the tissue-specific expression of receptors, G proteins, or effectors (3), there are important protein-protein interactions involving the alpha  and beta gamma subunits of the G protein heterotrimer that determine specificity. For example, the alpha t subunit couples selectively to rhodopsin and the alpha s subunit to the beta -adrenergic receptor (1, 2, 6). Furthermore, it is clear that the beta gamma dimer is required for efficient coupling of the alpha  subunit to receptors (9, 10), and there is growing evidence supporting specific interactions of receptors with the beta gamma dimer (11-13). Both the alpha  and beta gamma subunits of transducin appear to contact rhodopsin (14, 15), and the presence of the beta gamma dimer significantly increases the affinity of the Gt alpha  subunit for rhodopsin (14). In this regard, the carboxyl terminus of the gamma  subunit and its prenyl modification have emerged as important determinants of the interaction of G proteins with receptors (12, 13). Experiments using antisense RNA to selectively remove G protein subunits in GH3 cells also support a role for the diversity of the G protein alpha beta gamma subunits in determining signaling specificity. In these cells, the Galpha o1beta 3gamma 4 heterotrimer couples preferentially to the muscarinic receptor, Galpha o1beta 2gamma 2 to the galanin receptor, and the Galpha o2beta 1gamma 3 combination to the somatostatin receptor (16, 17). Similar experiments using rat basophilic leukemia cells suggest that the m1 muscarinic receptor couples selectively to alpha q, alpha 11, beta 1, beta 4, and gamma 4 (18). Thus, the existence of multiple isoforms of the alpha , beta , and gamma  subunits and the participation of both alpha  and beta gamma subunits in receptor coupling implies that the diversity of the subunits in the G protein heterotrimer could play an important role in signal transduction.

Although a large number of studies have focused on specific interactions of the alpha  and/or beta gamma subunits with effectors (4, 19), there are few investigations of the role of the alpha -beta gamma interaction in cell signaling. Perhaps it has been assumed that because of the similarity of the known beta  subunits, all alpha  subunits would associate with all beta  subunits. Recently, two more divergent members of the beta  subunit family, beta 5 and beta 5L, have been described (20, 21). Whereas the amino acid sequences of beta 1, beta 2, beta 3, and beta 4 are 80-90% identical, beta 5 is only 52% identical and 64% similar to beta 1 (20). In addition, beta 5 has an eight amino acid extension near the amino terminus and three short amino acid insertions within the WD repeat regions of the molecule. The beta 5 subunit is expressed predominantly in the brain, with only trace amounts detected by Northern analysis in the kidney. The beta 5L subunit appears to be expressed only in retina. Both the beta 5 and beta 5L subunits can stimulate PLC-beta 2 activity when transiently transfected into COS-7 cells with the gamma 2 subunit (20, 21). However, the beta 5gamma 2 dimer fails to activate the MAP kinase pathway when transfected into these cells (22). This observation suggests that dimers containing the beta 5 subunit may have different functions from those containing other beta  subunits. In the experiments reported here, we have tested the ability of several alpha  subunits to interact with beta gamma dimers containing the beta 1, beta 2, or beta 5 subunit to determine if the variations in amino acid sequence observed for the beta  subunits are manifested as differences in affinity for alpha  subunits. Sf9 cells were co-infected with an affinity tagged gamma 2 subunit and various beta  subunits. The resulting beta gamma 2HF dimers were immobilized via the affinity tag and allowed to interact with a variety of recombinant alpha  subunits expressed in Sf9 cells. The results show that the beta 1gamma 2HF and beta 2gamma 2HF dimers interact with five different alpha  subunits from three families, whereas the beta 5gamma 2HF subunit only interacts with the alpha q subunit.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Construction of Recombinant Baculoviruses for the gamma 2HF and beta 5 Subunits-- The polymerase chain reaction was used to modify the cDNA encoding the gamma 2 subunit (23) by adding XbaI and BamHI restriction sites to the 5' and 3' ends of the gamma 2 coding region, respectively. The primers used were Sense primer: 5'-AACTCTAGAATGGCCAGCAACAACACCGC-3' XbaI; and Antisense primer: 5'-CCTGGATCCTTAAAGGATAGCACAGAAAAACTTC-3' BamHI. The products of the polymerase chain reaction were digested with XbaI and BamHI and ligated into the pDoubleTrouble (pDT) vector (24) to add the nucleotide sequences for the hexahistidine and FLAG affinity tags to the 5' end of the gamma 2 coding region. To construct useful restriction sites for subcloning into the baculovirus transfer vector pVL1393, the gamma 2HF coding sequence was excised from pDT with KpnI and BamHI and subcloned into the pCNTR shuttle vector using the Prime Efficiency Blunt-End DNA Ligation Kit (5 Prime right-arrow 3 Prime). The gamma 2HF coding region was excised from pCNTR with BamHI and ligated into the BamHI site of pVL1393 to place the ATG of the hexahistidine sequence 75 bases downstream of the polyhedron promoter. The mouse beta 5 cDNA in a Bluescript SKII vector was kindly provided by Dr. Melvin I. Simon of the California Institute of Technology. The 1803-base pair BamHI-XbaI fragment of the beta 5 cDNA was subcloned into the BamHI-XbaI sites of pVL1393. To ensure fidelity, both completed transfer vectors were sequenced in the forward and reverse directions using dye terminator sequencing on an automated sequencer (Applied Biosystems, model 377). Recombinant baculoviruses were isolated following co-transfection of the transfer vector and linearized BaculoGold viral DNA into Sf9 cells using the PharMingen BaculoGold® kit. Briefly, 2 × 106 Sf9 cells were co-transfected with 1 µg of linear BaculoGold DNA and 3-5 µg of recombinant baculovirus transfer vector DNA using calcium phosphate/DNA precipitation. Following a 4-h incubation at 27 °C, the co-transfection medium was removed, and the monolayer was rinsed with fresh TNM-FH medium (25) supplemented with 10% fetal bovine serum, 50 µg/ml gentamicin sulfate, and 2.5 µg/ml amphotericin B. The plates were incubated at 27 °C with 5 ml of fresh medium for 4-6 days. Recombinants were detected by observing swollen, extremely large cells associated with a low cell density and a large amount of cell debris. Recombinant baculoviruses were purified by one round of plaque purification using standard techniques (25). The construction of the recombinant baculoviruses coding for the alpha i1, alpha i2, alpha o, alpha s, and alpha q subunits and the beta 1 and beta 2 subunits has been described (26-28). The baculovirus encoding the avian alpha 11 protein (29) was the kind gift of Dr. T. K. Harden.

Expression and Purification of Recombinant G Protein alpha  and beta gamma Subunits-- Recombinant G protein subunits were overexpressed in suspension cultures of Sf9 insect cells as described (26, 27, 30). In most experiments, the recombinant alpha  and beta gamma subunits were extracted from cell pellets using the detergent Genapol C-100 at a concentration of 0.1% (w/v). All steps were performed at 4 °C. Frozen pellets were thawed in 15 × their wet weight in lysis buffer containing 20 mM Hepes, pH 7.5, 150 mM NaCl, 3 mM MgCl2, 1 mM EDTA, 17 µg/ml phenylmethylsulfonyl fluoride, 20 µg/ml benzamidine, and 2 µg/ml each of aprotinin, leupeptin, and pepstatin, and burst by nitrogen cavitation (600 p.s.i. for 20 min) at 0 °C. The crude lysate was mixed with an equal volume of lysis buffer supplemented with 0.2% (w/v) Genapol C-100 and stirred for 1 h. The Genapol extract was centrifuged at 100,000 × g for 60 min and the supernatant decanted, and aliquots were frozen in liquid N2. The alpha s subunit used in the adenylyl cyclase assays was prepared as above except 0.1% (w/v) CHAPS was substituted for 0.1% (w/v) Genapol C-100. Partially purified alpha i1 subunits were used in some experiments. Sf9 cell pellets overexpressing the alpha i1 subunit were extracted without detergent; a 100,000 × g supernatant was prepared and the protein purified on a DEAE column exactly as described (26). This preparation of alpha i1 subunit is approximately 95% pure, as determined by quantitation of a silver-stained gel.

Preparation of the beta gamma -Anti-FLAG Affinity Column-- All column steps were carried out at 4 °C. Typically, 1 ml of the Genapol C-100 extract of Sf9 cells overexpressing the desired beta gamma 2HF dimer was applied to a 0.5-ml anti-FLAG M2 affinity gel column equilibrated with column buffer (lysis buffer containing 0.1% Genapol C-100 and 1 mM beta -mercaptoethanol) at a flow rate of 0.2 ml/min. The resulting beta gamma 2HF-anti-FLAG affinity gel column (beta gamma 2HF affinity column) was washed three times with 3 ml of column buffer. This procedure resulted in a highly pure preparation of beta gamma dimers immobilized on the column (see Fig. 1). The amount of beta gamma dimer immobilized on the column was about 6 µg/0.5 ml of resin as judged by silver staining of the beta gamma dimer eluted from the column with 0.1 M glycine, pH 3.5. This represents about 5% of the nominally available FLAG binding sites. Usually the interaction of an alpha subunit with a particular beta  subunit was measured by applying 2 ml of a Genapol C-100 extract of Sf9 cells expressing the desired alpha  subunit to a beta gamma 2HF affinity column at a flow rate of 0.2 ml/min. The resulting alpha beta gamma 2HF affinity column was washed 4 times with 4 ml of column buffer. In some experiments, the alpha beta gamma 2HF heterotrimer was eluted with 0.1 M glycine, pH 3.5. In the experiment presented in Fig. 5A, the procedure was modified such that 2 ml of alpha i2 extract and 2 ml of alpha q extract were mixed and applied to the beta gamma 2HF affinity column. In the experiment presented in Fig. 6, a range of 17.5-525 µg of partially purified alpha i1 was mixed with 2 ml of alpha q extract and applied to the beta gamma 2HF affinity column.

Specific Elution of alpha  Subunits from beta gamma 2HF-Anti-FLAG Affinity Gel-- To demonstrate a functional interaction between alpha  subunits and the immobilized beta gamma subunits, a beta gamma 2HF affinity column (0.5 ml) was prepared at 4 °C as described above, washed three times with 3 ml of column buffer, and then equilibrated in alpha  subunit binding buffer (20 mM Hepes, pH 8.0, 100 mM NaCl, 1 mM MgCl2, 0.3% (w/v) C12E10, 10 mM beta -mercaptoethanol, and 5 µM GDP). Then, 17.5 µg of purified alpha i1 subunit diluted in 1 ml of alpha  subunit binding buffer was applied to the beta gamma 2HF affinity column at a flow rate of 0.2 ml/min. The beta gamma 2HF affinity column was washed 4 times with 4 ml of alpha  subunit binding buffer and twice with 2 ml of alpha  subunit binding buffer containing 300 mM NaCl. The column was incubated at room temperature for 15 min. The alpha i1 subunit was eluted with 4 × 0.5 ml of 20 mM Hepes, pH 8.0, 50 mM NaCl, 1 mM EDTA, 1.0% cholate, 10 mM beta -mercaptoethanol, 10 mM MgCl2, and 100 µM GTPgamma S, also at room temperature. The column was washed twice with 2 ml of alpha  subunit binding buffer containing 300 mM NaCl, before final elution with 0.1 M glycine, pH 3.5.

Slight modifications of the above procedure were used to examine the interaction of the alpha q subunit with the beta 5gamma 2HF dimer. The 0.5-ml beta 5gamma 2HF affinity column was prepared and washed as described in the previous section, except that the GDP concentration was increased to 50 µM in the alpha  subunit binding buffer and the column buffer. Two ml of a Genapol C-100 extract of Sf9 cells expressing the alpha q subunit was applied at a flow rate of 0.2 ml/min. The alpha qbeta 5gamma 2HF affinity column was washed twice with 1 ml of column buffer, four times with 1 ml of alpha  subunit binding buffer with 0.2% (w/v) C12E10, and finally twice with 2 ml of alpha  subunit binding buffer containing 300 mM NaCl and 0.2% (w/v) C12E10. The column was brought to room temperature for 15 min, and alpha q was specifically eluted with 20 mM Hepes, pH 8.0, 50 mM NaCl, 1 mM EDTA, 0.05% cholate, 10 mM beta -mercaptoethanol, 10 mM MgCl2, 10 mM NaF, 30 µM AlCl3, and 50 µM GDP. The cholate concentration was reduced to 0.05% in this buffer because higher cholate concentrations dissociated beta 5 from gamma 2HF.

Extraction of G Proteins from Bovine Brain Membranes and Activation with GDP-AMF-- Frozen bovine brains were obtained from PelFreeze and membranes prepared according to the method of Sternweis and Robishaw (31), with the addition of 0.2 µg/ml aprotinin to all buffers. The membrane preparations were stored at -80 °C. Membranes were thawed, washed once with ice-cold 20 mM Tris, pH 8.0, 1 mM EDTA, 100 mM NaCl, and pelleted at 40,000 × g for 30 min at 4 °C. The washed membrane pellet (1 g of protein) was extracted for 1 h at 4 °C with 200 ml of 0.5% (w/v) C12E10 in 50 mM Tris, pH 8.0. The extract was clarified by centrifugation at 143,000 × g for 60 min and stored at -80 °C. To examine the interaction of the G proteins in the membrane extracts with the various beta  subunits, the extracts were thawed and mixed with the different beta gamma 2HF Genapol extracts from Sf9 cells and activated by addition of concentrated stocks to give final concentrations of 3 mM MgCl2, 5 mM NaF, 15 µM AlCl3, and 2.5 µM GDP. The mixture was incubated at 30 °C for 45 min (32). The alpha  subunits were deactivated by addition of 500 mM EDTA to a final concentration of 20 mM EDTA and incubated for an additional 30 min at 30 °C. Typically, 4 ml of the deactivated mixture was applied to 0.5 ml of anti-FLAG affinity gel equilibrated with 20 mM Hepes, 0.5% (w/v) C12E10, 5 µM GDP, pH 8.0. The loaded affinity column was washed with 5 ml of equilibration buffer containing 400 mM NaCl and eluted with 1.0 ml of 0.1 M glycine, pH 3.5, as described above.

Silver Staining, Immunoblotting, and Quantitation-- Samples were prepared for electrophoresis, loaded on 0.75 mm, 12% acrylamide gels, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the gels stained with silver according to the method of Bloom et al. (33). Purified bovine brain Gi/o heterotrimer (31) was used as a standard. The protein concentrations in the gel were compared with ovalbumin concentration standards and quantitated following silver staining using a BioImage scanning densitometer and the Whole Band® software (BioImage, Ann Arbor, MI). For Western blots, gels were transferred to nitrocellulose and immunoblotted using the following primary antibodies: an anti-G protein beta  subunit antibody (NEN Life Science Products, catalog number 808), 1:1000 dilution; an anti-G protein alpha  subunit antibody (Calbiochem, catalog number 371737), 1:1000 dilution; and an anti-alpha q/alpha 11 antibody (Santa Cruz, catalog number sc-392), 1:100 dilution. The primary antibodies were detected using goat anti-rabbit IgG(Fc) alkaline phosphatase conjugate (Promega) or donkey anti-rabbit IgG F(ab')2 horseradish peroxidase conjugate (Amersham Corp.). The density of the bands on autoradiographs obtained following ECL detection was also estimated using the Whole Band® software.

Adenylyl Cyclase Assays-- Recombinant baculovirus encoding a FLAG epitope-tagged rat type II adenylyl cyclase was kindly provided by Dr. Ravi Iyengar, Mount Sinai School of Medicine. Sf9 cells were infected with the cyclase baculovirus and harvested 72 h later, when viability was approximately 80%. The cell pellet was washed three times with 6.8 mM CaCl2, 55 mM KCl, 7.3 mM NaH2PO4, 47 mM NaCl, pH 6.2, and membranes prepared according to the procedure of Taussig et al. (34). The washed membrane pellet was resuspended in 20 mM Hepes, pH 8, 200 mM sucrose, 1 mM EDTA, 2 mM DTT, 17 µg/ml phenylmethylsulfonyl fluoride, 16 µg/ml N-alpha -p-tosyl-L-lysine chloromethyl ketone, 16 µg/ml N-tosylphenylalanyl chloromethyl ketone, 2 µg/ml leupeptin, and 3 µg/ml lima bean trypsin inhibitor at a final total protein concentration of 1.5 mg/ml, as determined by the method of Bradford (35). Aliquots were frozen in liquid N2 and stored at -80 °C. A 0.1% (w/v) CHAPS extract of Sf9 cells overexpressing alpha s (see above) was activated with 100 µM GTPgamma S in 5 mM MgSO4, 1 mM DTT, and 1 mM EDTA, pH 8, for 30 min at 30 °C (34). Excess GTPgamma S was removed by centrifugation through P6 resin (Bio-Rad) equilibrated in 50 mM Hepes, pH 8, 150 mM NaCl, 5 mM MgSO4, 1 mM DTT, 1 mM EDTA, 0.1% CHAPS, as described previously (13). The first elution fraction, containing activated alpha s, was held on ice. Reaction tubes containing a total of 25 µl of type II cyclase membranes (12 µg of protein/assay tube), activated alpha s, beta gamma , and/or buffer were prepared at room temperature. The reaction was begun by addition of 75 µl of reaction mix pre-equilibrated at 30 °C. The standard reaction mixture contained 25 mM Hepes, pH 8, 10 mM phosphocreatine, 10 units/ml creatine phosphokinase, 0.4 mM 3-isobutyl-1-methylxanthine, 10 mM MgSO4, 0.5 mM ATP, and 0.1 mg/ml bovine serum albumin. Reactions were carried out for 7 min at 30 °C. Cyclic AMP production was stopped by the addition of 1.0 ml of 0.11 N HCl and cyclic AMP quantified by radioimmunoassay (36).

Expression of Results-- Experiments presented under "Results" are representative of three or more similar experiments.

Materials-- All reagents used in the culture of Sf9 cells and for the expression and purification of G protein alpha  and beta gamma subunits have been described in detail (26, 30). The baculovirus transfer vector, pVL1393, was purchased from Invitrogen; the BaculoGold® kit was from PharMingen; 10% Genapol C-100 and the anti-alpha common subunit antibody were from Calbiochem; Prime Efficiency blunt-end DNA Ligation Kit was from 5 Prime right-arrow 3 Prime; anti-FLAG® M2 affinity gel was from Eastman Kodak; polyoxyethylene 10 lauryl ether (C12E10) was from Sigma; the anti-alpha q/alpha 11 antibody was from Santa Cruz; the NEN-808 anti-beta subunit antibody was from NEN Life Science Products, and nitrocellulose was from Schleicher and Schuell. All other reagents were of the highest purity available.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

The objective of this study was to determine if selectivity in alpha -beta gamma interactions could be observed in vitro with recombinant G protein subunits isolated from baculovirus-infected Sf9 cells. We first constructed a recombinant baculovirus encoding sequential affinity tags on the amino terminus of the gamma 2 subunit, a hexahistidine tag followed by a FLAG epitope tag (24). When used in conjunction with an anti-FLAG antibody covalently linked to agarose beads, the FLAG epitope tag provides a convenient method for separating alpha  subunits bound to beta gamma 2HF from alpha  subunits free in solution. Previous work has shown that addition of a hexahistidine or FLAG affinity tag to the amino terminus of the gamma  subunit does not prohibit association with the beta  subunit (37-39) or the subsequent association of the beta gamma dimer with alpha  subunits (37). The heterotrimeric G protein crystal structure also suggests that an extension of the amino terminus of the gamma  subunit would be unlikely to interfere with alpha -beta gamma interactions (40, 41).

The silver-stained SDS-polyacrylamide gel in Fig. 1 illustrates the steps involved in the preparation of beta gamma and alpha -beta gamma affinity columns. Sf9 cells were co-infected with recombinant baculovirus encoding for the beta 1 and gamma 2HF subunits, and the recombinant beta 1gamma 2HF protein was extracted from membranes of cells harvested 48 h post-infection. Crude detergent extracts were applied to anti-FLAG M2 affinity gel columns and washed with 5-10 column volumes. The resulting product of this one-step purification is shown in lane 3 of Fig. 1. The FLAG epitope tag on the gamma 2HF subunit is available for binding to the anti-FLAG antibody and produces a dramatic increase in purity in a single step. The presence of the hexahistidine tag and FLAG epitope results in reduced electrophoretic mobility of the gamma 2HF subunit relative to gamma 2 (lane 1 versus 3). Approximately 12 µg of beta 1gamma 2HF were captured per ml of anti-FLAG M2 affinity gel suspension, as determined by quantitation of the eluted beta subunit on a silver-stained gel. In subsequent experiments designed to monitor alpha -beta gamma interaction, the beta 1gamma 2HF was captured as before and the resulting beta gamma 2HF affinity column used to specifically bind partially purified alpha i1 subunits. To determine first the amount of alpha i1 necessary for stoichiometric binding to immobilized beta 1gamma 2HF, replicate beta 1gamma 2HF affinity columns were prepared and then varying amounts of alpha i1 subunit applied. Stoichiometric binding was achieved at a 3-7-fold excess (w/w) of alpha i1 over immobilized beta 1gamma 2HF (data not shown). Lane 4 shows the alpha i1 preparation used for these experiments. Lane 5 shows the resulting alpha i1beta 1gamma 2HF eluted with 0.1 M glycine after an excess of alpha i1 was applied to the beta 1gamma 2HF affinity column.


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Fig. 1.   Establishment of function alpha -beta gamma 2HF interactions on the FLAG affinity column. Three experiments were performed using the anti-FLAG M2 affinity gel: the beta 1gamma 2HF dimer was purified from an extract of Sf9 cells; the alpha i1 subunit was bound stoichiometrically to a beta 1gamma 2HF affinity column; and the alpha i1 subunit was specifically eluted from a beta 1gamma 2HF affinity column. Elution fractions from each experiment were resolved on a 12% SDS-polyacrylamide gel and stained with silver. The migration position of the bovine brain alpha , beta , and gamma  subunits are indicated on the left. Lane 1, bovine brain standard; lane 2, pass-through (PT) after the application of a beta 1gamma 2HF Genapol C-100 extract onto the anti-FLAG M2 affinity gel; lane 3, the beta 1gamma 2HF eluted with glycine from anti-FLAG M2 affinity gel containing only immobilized beta 1gamma 2HF; lane 4, partially purified alpha i1 subunit prior to application onto the beta 1gamma 2HF affinity column; lane 5, the alpha i1beta 1gamma 2HF eluted from a alpha i1beta 1gamma 2HF anti-FLAG affinity column with 0.1 M glycine; lane 6, the alpha i1 subunit specifically eluted from beta 1gamma 2HF affinity column with 100 µm GTPgamma S; lane 7, subsequent elution of the beta 1gamma 2HF dimer after GTPgamma S elution of the alpha i1 subunit.

To demonstrate that the immobilized beta 1gamma 2HF was properly folded and functional, a 0.5-ml alpha i1beta 1gamma 2HF affinity column, prepared identically to that shown in lane 5, was treated with 100 µM GTPgamma S. The alpha i1 eluted specifically, in a volume of 0.5 ml, as shown in lane 6. Lane 7 shows the elution of the remaining beta 1gamma 2HF dimer with glycine. Thus, the alpha i1 subunit can be dissociated from the beta 1gamma 2HF subunit with GTPgamma S treatment, analogous to activation of the native heterotrimer in solution (42). Heterotrimeric G proteins can also be activated with GDP-AMF resulting in dissociation of the beta gamma dimer (43). When an alpha i1beta 1gamma 2HF affinity column similar to that described above was activated with GDP-AMF, the alpha i1 subunit was specifically eluted (data not shown).

To test the functionality of the alpha i1beta 1gamma 2HF interaction in another way, we subjected a detergent extract of beta 1gamma 2HF to our normal beta gamma purification strategy, DEAE ion exchange chromatography followed by alpha i1-agarose affinity chromatography (30). The alpha -agarose affinity chromatography exploits the ability of GDP-AMF to dissociate the beta gamma subunit from the alpha  subunit. Fig. 2A shows a Western blot, developed with an anti-beta antibody, of the DEAE pool (PL) applied to the alpha i1 column and the alpha i1 column pass-through (PT). Comparison of lanes 1 and 2 shows a typical result for beta 1gamma 2. A very high proportion of the beta 1gamma 2 present in the DEAE pool binds to the alpha i1-agarose. Lanes 3 and 4 show a very similar result obtained when a DEAE pool containing beta 1gamma 2HF was applied to the alpha i1-agarose. This observation is consistent with the result obtained with immobilized beta 1gamma 2HF and alpha i1 free in solution, as described above. To obtain further evidence of functional alpha i1beta 1gamma 2HF interaction, we treated the beta 1gamma 2HF-loaded alpha i1-agarose column with GDP-AMF. Fig. 2B shows a silver-stained SDS-polyacrylamide gel of the alpha i1-agarose column pass-through (lane 2), wash fractions (lanes 3-5), and subsequent elution of beta 1gamma 2HF by treatment with GDP-AMF (lanes 6-8). Thus, beta 1gamma 2HF binds tightly to immobilized alpha i1 and elutes upon activation of alpha i1 with GDP-AMF.


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Fig. 2.   The beta 1gamma 2HF dimer associates with an alpha i1-agarose affinity column. A, extracts of Sf9 cells overexpressing beta 1gamma 2 and beta 1gamma 2HF dimers were partially purified on a DEAE ion exchange column, and the beta 1gamma 2 and beta 1gamma 2HF dimers were applied to alpha i1-agarose affinity columns. Aliquots of the pooled DEAE fractions (PL) and alpha -column pass-throughs (PT) were resolved on a 12% SDS-acrylamide gel and transferred to nitrocellulose. Lanes 1-4 were probed with an anti-beta -common primary antibody detected using a horseradish peroxidase-conjugated secondary antibody. The migration position of the beta 1 subunit is indicated on the left; the beta gamma combinations are indicated above the appropriate lanes. B, purification of the beta 1gamma 2HF dimer on an alpha i1-agarose column. The beta 1gamma 2HF was specifically eluted from the alpha i1-agarose support with GDP-AMF. Proteins in the pass-through (PT), washes (W1-W3), and eluates (E1-E3) were resolved on a 12% SDS-acrylamide gel and stained with silver. Lane 1, bovine brain standard; lane 2, pass-through (PT) after application of the beta 1gamma 2HF DEAE pool onto the alpha i1-agarose column; lanes 3-5, wash fractions before application of GDP-AMF; lanes 6-8, the beta 1gamma 2HF dimer eluted from alpha i1-agarose column by treatment with GDP-AMF. The migration positions of the bovine brain alpha , beta , and gamma  subunits are indicated on the left. Migration positions of the beta 1 and gamma 2HF subunits are indicated on the right.

We next tested the ability of beta 1gamma 2HF to stimulate one effector, type II adenylyl cyclase. It is known that beta 1gamma 2 is a potent activator of type II cyclase in the presence of an alpha s subunit activated with GTPgamma S (44). The data in Table I compare the stimulation of type II cyclase by beta 1gamma 2 and beta 1gamma 2HF in the concentration range 0-100 nM. At 100 nM, the beta 1gamma 2HF dimer is capable of a 15-fold stimulation of cyclase over the effect of GTPgamma S-alpha s alone. However, at each concentration tested, the beta 1gamma 2 dimer activates adenylyl cyclase to a significantly greater extent than does the beta 1gamma 2HF dimer. This reduced stimulation could be due to a decreased effective concentration of beta 1gamma 2HF relative to beta 1gamma 2 at the adenylyl cyclase-containing membrane surface, or to a specific interference between the affinity tags on the gamma  subunit's amino terminus and type II cyclase. This matter is under further investigation. We conclude that the presence of the hexahistidine and FLAG epitopes on the amino terminus of the gamma 2 subunit does not abrogate interaction of beta 1gamma 2HF with at least one effector, type II adenylyl cyclase.

                              
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Table I
Stimulation of type II adenylyl cyclase by native and affinity tagged beta gamma subunits
Sf9 cells were infected with a recombinant baculovirus encoding for type II adenylyl cyclase, membranes prepared, the membranes stimulated with GTPgamma S-alpha s, and the indicated concentration of beta gamma subunit for 7 min, and the cyclic AMP produced measured using a radioimmunoassay. The beta gamma subunits were purified on a DEAE and alpha i1 affinity column. The basal rate of cAMP production without GTPgamma S-alpha s was 1.0 pmol/ml/min. See "Experimental Procedures" for details. The data are averages of 2-3 duplicate determinations.

Previous work with the adenosine A1 receptor showed little difference between the ability of alpha i1, alpha i2, and alpha i3 to support high affinity binding of agonist in the presence of beta 1gamma 2 (13, 45). Since this observation implies similar affinity of the three alpha i subunits for beta 1gamma 2, we tested the ability of another alpha i isoform, the alpha i2 subunit, to bind to a beta 1gamma 2HF affinity column. Two ml of a crude detergent extract of Sf9 cells infected with recombinant baculovirus encoding the alpha i2 subunit was applied to a 0.5-ml beta 1gamma 2HF affinity column as described above, washed extensively, and the bound alpha i2 and beta 1gamma 2HF eluted with glycine. A silver-stained polyacrylamide gel of the product is shown in Fig. 3A, lane 2. Thus, the immobilized beta 1gamma 2HF was also able to bind alpha i2, and a 2-ml volume of crude extract containing alpha i2 subunit was sufficient excess to obtain stoichiometric binding of alpha i2 to immobilized beta 1gamma 2HF.


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Fig. 3.   The alpha i2 subunit does not associate with beta 5gamma 2HF. A, a detergent extract of the alpha i2 subunit overexpressed in Sf9 cells was applied to beta 1-, beta 2-, and beta 5gamma 2HF affinity columns. After extensive washing, the alpha beta gamma 2HF heterotrimers were eluted from the anti-FLAG M2 affinity gel with 0.1 M glycine, pH 3.5, and immediately neutralized with 1 M Tris, pH 8. A beta 5gamma 2HF affinity column to which no alpha i2 extract was applied was also eluted with 0.1 M glycine (lane 5). Proteins in each eluate were resolved on a 12% SDS-acrylamide gel and stained with silver. Lane 1, bovine brain standard; lane 2, eluate from the beta 1gamma 2HF affinity column; lane 3, eluate from the beta 2gamma 2HF affinity column; lane 4, eluate from the beta 5gamma 2HF affinity column; lane 5, beta 5gamma 2HF standard. B, immunoblot of the samples in A probed with an anti-alpha -common primary antibody. The primary antibody was detected using an alkaline phosphatase-conjugated secondary antibody. The migration position of the alpha i2 subunit is indicated on the left.

Having demonstrated functional activity for beta 1gamma 2HF and its ability to bind both purified alpha i1 and a crude detergent extract of the alpha i2 subunit, we tested the ability of other beta gamma 2HF dimers to bind alpha i2. The beta 1-, beta 2-, and beta 5gamma 2HF columns were first constructed by application of appropriate crude cell extracts to anti-FLAG M2 affinity gel. Pilot experiments were performed to ensure that equivalent amounts of beta 1-, beta 2-, and beta 5gamma 2HF were bound to anti-FLAG columns by applying a sufficient excess of each beta gamma 2HF detergent extract to saturate the available FLAG binding sites. Equal volumes of a crude detergent extract of Sf9 cells infected with recombinant baculovirus encoding the alpha i2 subunit were then applied to each beta gamma 2HF affinity column. The columns were washed extensively to remove any nonspecifically bound alpha i2. Finally, the alpha i2beta gamma 2HF was eluted with glycine. The elution products were analyzed by gel electrophoresis followed by silver staining and Western blotting with an anti-alpha common antibody (Fig. 3, A and B). Under these conditions, the beta 1gamma 2HF and beta 2gamma 2HF columns captured equivalent, and roughly stoichiometric, amounts of alpha i2 (Fig. 3A, lanes 2 and 3). Since the beta 5 subunit co-migrates with the alpha i2 subunit under the electrophoresis conditions employed, it is not possible to determine from the silver-stained gel whether beta 5gamma 2HF captured alpha i2 (Fig. 3A, lanes 4 and 5). However, the Western blot in Fig. 3B demonstrates clearly that beta 5gamma 2HF bound little, if any, alpha i2 under conditions where beta 1gamma 2HF and beta 2gamma 2HF bound amounts of alpha i2 easily detectable with the same primary antibody (compare lanes 2 and 3 with 4).

To determine if beta 5gamma 2HF was unable to bind alpha i2 due to steric constraints of immobilization, we tested the ability of beta 5gamma 2HF free in solution to bind to an alpha i1-agarose column, an experiment analogous to the beta 1gamma 2HF-alpha i1-agarose system illustrated in Fig. 2. The concentration of beta 5gamma 2HF in the DEAE pool applied to the alpha i1-agarose column was compared with the concentration of beta 5gamma 2HF in the column pass-through. Both the DEAE pool and the alpha i1 column pass-through gave similar intensity when developed with an anti-beta subunit antibody (data not shown), indicating little or no binding. Furthermore, no beta 5gamma 2HF product was detected on silver-stained polyacrylamide gels after treatment of the alpha i1-agarose with GDP-AMF. Therefore, the low affinity of beta 5gamma 2HF for alpha i1 is not due to immobilization of the beta gamma 2HF.

We then selected representatives of three alpha  subfamilies, alpha i/o, alpha s, and alpha q (46), to investigate the ability of beta 5 to interact with other alpha  subunits. Crude detergent extracts of Sf9 cells infected with recombinant baculovirus encoding the appropriate alpha  subunit isoform were applied to three beta gamma 2HF affinity columns constructed as before. After extensive washing, the specifically bound alpha  subunits were eluted, along with their beta gamma 2HF counterparts, by treatment with 0.1 M glycine. The resulting products were analyzed by Western blotting with either an anti-alpha common antibody in the case of alpha i2, alpha o, and alpha s or with an anti-alpha q specific antibody for alpha q. The results are shown in Fig. 4. As observed earlier using alpha i2 (Fig. 3), beta 1gamma 2HF and beta 2gamma 2HF bind easily detectable amounts of alpha i2 under conditions where beta 5gamma 2HF does not (Fig. 4, lanes 1-3). This pattern is repeated with respect to binding alpha o and alpha s (lanes 4-9). However, when detergent extracts containing recombinant alpha q were applied to each beta gamma 2HF column, beta 1gamma 2HF, beta 2gamma 2HF, and beta 5gamma 2HF all bound alpha q equally (lanes 10-12). Thus, beta 5gamma 2HF appears to bind the alpha q subunit selectively. To determine if the beta 5gamma 2HF dimer was able to interact with other members of the Gq family, we have performed pilot experiments with a recombinant, avian alpha 11 subunit (29). This protein is 96% identical in amino acid composition to the mouse alpha 11 subunit (47) and 100% identical in the 20 amino acids shown to contact the beta  subunit in the x-ray structure of the heterotrimer (41). Preliminary results indicate that crude detergent extracts containing alpha 11 bind equally well to all three beta gamma 2HF dimers (data not shown). Thus, at least one other member of the Gq family binds to the beta 5gamma 2HF dimer. The interaction of the other members of the family (the G14-16 alpha  subunits) with the beta 5 subunit is currently under investigation.


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Fig. 4.   Interaction of four alpha  subunits with three different beta gamma 2HF affinity columns. Detergent extracts of Sf9 cells overexpressing the alpha i2, alpha o, alpha s, and alpha q subunits were applied to beta 1-, beta 2-, and beta 5gamma 2HF affinity columns. After extensive washing, specifically bound alpha beta gamma 2HF heterotrimers were eluted from the anti-FLAG M2 affinity gel with 0.1 M glycine. The proteins in each eluate were resolved on a 12% SDS-acrylamide gel and transferred to nitrocellulose. The alpha  subunits applied are indicated above each panel; the identity of the beta  subunit in each beta gamma 2HF affinity column used is indicated above each lane. Lanes 1-9 were probed with an anti-alpha -common primary antibody, and lanes 10-12 were probed with an anti-alpha q/11 primary antibody. Both primary antibodies were detected using an alkaline phosphatase-conjugated secondary antibody. The appropriate molecular weights for each alpha  subunit are indicated.

The observation of selective alpha q-beta 5gamma 2HF interaction raises the issue of relative affinities. One approach to this question is to determine the ability of other alpha  subunits to compete with alpha q for binding to beta 5gamma 2HF. Since the alpha q preparation was from a crude cell extract, we first selected a similar preparation of the alpha i2 subunit for competition experiments. The beta 1gamma 2HF and beta 5gamma 2HF columns were constructed as before, and then a mixture of equal volumes of alpha i2 and alpha q detergent extracts were applied to each. After extensive washing, the alpha beta gamma 2HF complexes were eluted with 0.1 M glycine. The alpha i2/alpha q mixture applied to the columns (L) was compared with the glycine elution fractions (E) by Western blot using anti-alpha common or anti-alpha q antibodies. Lanes 1-4 of Fig. 5A show the result obtained with beta 1gamma 2HF. As expected from the individual alpha  subunit experiments, the beta 1gamma 2HF affinity column captured both alpha i2 and alpha q subunits (lane 2 versus 4). The beta 5gamma 2HF affinity column also bound alpha q (lane 8) at a level roughly comparable to that bound by beta 1gamma 2HF (lane 4 versus 8). However, beta 5gamma 2HF bound no detectable alpha i2 subunit (lane 2 versus 6).


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Fig. 5.   The beta 5gamma 2HF dimer selectively associates with the alpha q subunit in the presence of recombinant and native G proteins. A, a mixture of alpha q and alpha i2 subunits overexpressed in Sf9 cells was prepared as described under "Experimental Procedures" and applied to beta 1gamma 2HF and beta 5gamma 2HF affinity columns. After extensive washing, specifically bound alpha beta gamma 2HF heterotrimers were eluted from the anti-FLAG M2 resin with 0.1 M glycine. The proteins in the load (L) and eluate (E) were resolved on a 12% SDS-acrylamide gel and transferred to nitrocellulose. B, the G proteins in an extract of bovine brain membranes were activated with GDP-AMF, mixed with equal aliquots of Sf9 cell extracts expressing the beta 1gamma 2HF or the beta 5gamma 2HF dimers, incubated for 45 min, and quenched with EDTA as described under "Experimental Procedures." Each mixture was applied to a separate anti-FLAG M2 affinity gel column. After extensive washing, specifically bound alpha beta gamma 2HF heterotrimers were eluted with 0.1 M glycine, pH 3.5. The proteins in the load (L) and eluate (E) were resolved on a 12% SDS-acrylamide gel and transferred to nitrocellulose. In both A and B, lanes 1, 2, 5, and 6 were probed with an anti-alpha -common primary antibody. Lanes 3, 4, 7, and 8 were probed with an anti-alpha q/11 primary antibody. Both primary antibodies were detected using an alkaline phosphatase-conjugated secondary antibody. The migration positions of the alpha i and alpha q subunits are indicated on the left.

Since all the above experiments were performed with recombinant proteins, we tested the alpha  subunit selectivity of the beta gamma affinity columns with native alpha  subunits. As bovine brain membranes are known to contain a complex mixture of alpha  subunits, including alpha i, alpha o, and alpha q (3, 48), we used an extract of bovine brain membranes as a starting material for these experiments. Crude detergent extracts of Sf9 cells infected with recombinant baculovirus encoding for the beta 1gamma 2HF or beta 5gamma 2HF dimers were mixed with brain membrane extract and the alpha  subunits activated by treatment with GDP-AMF as described under "Experimental Procedures." The mixtures were deactivated by addition of excess EDTA, resulting in the association of a fraction of the brain alpha  subunits with the recombinant beta gamma 2HF dimers. The deactivated mixture was applied to an anti-FLAG affinity column and the alpha beta gamma 2HF complexes eluted. The proteins in the mixtures applied to the affinity columns and the glycine elution fractions were resolved on acrylamide gels, transferred to nitrocellulose, and probed with anti-alpha subunit antibodies. The beta 1gamma 2HF dimer bound alpha  subunits which gave positive signals with anti-alpha common antibodies and anti-alpha q/11 antibodies (Fig. 5B, lanes 2 and 4). However, the beta 5gamma 2HF dimer only associated with alpha  subunits detected by the anti-alpha q/11 antibody (lane 6 versus 8), in agreement with the selectivity observed with recombinant alpha  subunits (Fig. 5A). Interestingly, the beta 5gamma 2HF dimer binds a clearly resolved doublet from the brain extract (Fig. 5B, lane 8). The anti-alpha q/11 antibody used in these experiments does not cross-react with alpha i/o subunits, and therefore this doublet is most likely alpha q and/or alpha 11. Thus, when the beta 5gamma 2HF dimer was presented with a complex mixture of native heterotrimeric G proteins, it selectively bound the alpha q/11 subunits. Moreover, it did not appear to interact with the alpha i or alpha o subunits which are present at high concentrations in brain membranes.

Because the alpha q, alpha i2, and bovine brain preparations are all crude detergent extracts, it is not possible to estimate the molar ratio of competing alpha  subunit to alpha q subunit applied to the immobilized beta gamma 2HF. To address this issue in part, we employed the purified alpha i1 preparation described previously. The alpha i1 subunit in this preparation represents approximately 95% of the intensity on a silver-stained gel. Increasing amounts of this alpha i1 stock were diluted into a fixed, larger volume of alpha q crude extract. Based on the amount of beta 5 subunit immobilized on the column as estimated from silver-stained gels, a 3-100-fold excess of alpha i1 was added to the alpha q extract. These mixtures were applied to immobilized beta 5gamma 2HF, washed, and eluted with 0.1 M glycine. The loading mixture, the last wash, and the elution fractions were examined by Western blot using an anti-alpha q/11 antibody (Fig. 6A) and an anti-alpha common antibody (Fig. 6B). Even at the largest excess of alpha i1 over the immobilized beta 5gamma 2HF, there was no detectable competition by alpha i1 for alpha q binding to beta 5gamma 2HF. The ECL signal representing bound alpha q in Fig. 6A was quantitated on a scanning densitometer. Fig. 6C shows a plot of this integrated intensity versus excess alpha i1 present. Note that there is no apparent diminution of alpha q binding at ratios of alpha i1 to beta 5gamma 2HF far in excess of the ratio required for stoichiometric binding of alpha i1 by beta 1gamma 2HF (about 3:1).


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Fig. 6.   The beta 5gamma 2HF dimer selectively associates with the alpha q subunit in the presence of an excess of partially purified alpha i1 subunit. A, 2 ml of an extract of Sf9 cells overexpressing the alpha q subunit was combined with increasing amounts of partially purified alpha i1 (17-525 µg, a 3-100-fold excess of alpha i1 over beta 5 (w/w)) and applied to separate beta 5gamma 2HF affinity columns. After extensive washing, specifically bound alpha :beta gamma 2HF heterotrimers were eluted from the anti-FLAG M2 affinity gel with 0.1 M glycine. Proteins in the load (L), wash (W), and eluate (E1, E2, and E3) were resolved on a 12% SDS-acrylamide gel and transferred to nitrocellulose. Samples were probed with an anti-alpha q/11 primary antibody and detected using a horseradish peroxidase-conjugated secondary antibody. The migration position of the alpha q subunit is indicated on the left. B, immunoblot of the same column samples as in A but probed with an anti-alpha -common antibody. The migration position of the alpha i1 subunit is indicated on the left. Lanes 1-5 contained no alpha i1; lanes 6-10, 3 × alpha i1; lanes 11-15, 10 × alpha i1; lanes 16-20, 30 × alpha i1; lanes 21-25, 100 × alpha i1. C, a plot of the integrated intensity of the alpha q subunit signal from fractions E1-E3 shown in A versus 3-100-fold excess of the alpha i1 subunit over the immobilized beta 5 (w/w).

To demonstrate that the alpha qbeta 5gamma 2HF interaction was functional, we constructed an alpha qbeta 5gamma 2HF affinity column as described in Fig. 4 and treated the immobilized heterotrimer with GDP-AMF to activate and thereby dissociate alpha q. Fig. 7A presents a silver-stained gel of the GDP-AMF elution product (E1-E3-AMF, lanes 4-6). Because alpha q and beta 5 co-migrate under these electrophoresis conditions, we verified the identity of the GDP-AMF and glycine elution products by Western blot with an anti-alpha q/11 antibody (Fig. 7B). Comparison of lanes 4 and 7 in Fig. 7B shows that the majority of the alpha q bound to beta 5gamma 2HF eluted specifically with GDP-AMF. Thus, the alpha q subunit is associating with the immobilized beta 5gamma 2HF in a manner that permits the alpha q subunit to be activated and to dissociate from the beta 5gamma 2HF.


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Fig. 7.   Functionality of the Gqalpha subunit-beta 5 interaction. The alpha qbeta 5gamma 2HF affinity column was prepared as described under "Experimental Procedures." The alpha q subunit was specifically eluted from the immobilized beta 5gamma 2HF using GDP-AMF, and the beta 5gamma 2HF was eluted from the anti-FLAG M2 affinity gel using 0.1 M glycine. Proteins in each eluate were resolved on a 12% SDS-polyacrylamide gel and stained with silver (A) or transferred to nitrocellulose and probed with anti-alpha q/11 antibody (B). A, lane 1, bovine brain standard; lane 2, pass-through (PT) after the application of the alpha q subunit Genapol C-100 extract onto a beta 5gamma 2HF affinity column; lane 3, final wash fraction (W) before application of GDP-AMF; lanes 4-6, the alpha q subunit eluted from the alpha qbeta 5gamma 2HF affinity column by treatment with GDP-AMF; lane 7, subsequent elution with 0.1 M glycine of residual alpha q subunit and beta 5gamma 2HF dimer. The migration position of the bovine brain alpha , beta , and gamma  subunits are indicated on both the left and the right. B, corresponding Western blot. The migration position of the alpha q subunit is indicated on the left.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The data presented in this report provide clear evidence that the diversity of the subunits in the G protein heterotrimer can have important functional consequences for the interaction of certain alpha  and beta  subunits. Although all the alpha  subunits examined interact with the beta 1 or beta 2 subunit, the structurally different beta 5 subunit interacts selectively with the alpha q subunit and the nearly identical alpha 11 subunit. We inspected two heterotrimeric crystal structures for sites of intersubunit contact which might be responsible for the observed selectivity (40, 41). These structures show that nine locations involving 16 amino acids on the beta 1 subunit are primarily responsible for interacting with the Switch I, Switch II, and the amino-terminal regions of the alpha  subunit. Of these 16 amino acids, only 3 are different in the beta 5 subunit (Leu55 right-arrow Gly, Tyr59 right-arrow Leu, and Ser98 right-arrow Thr, based on the beta 1 sequence). Although the essential residues necessary for a WD repeat (20, 49) are conserved in the beta 5 subunit, the overall amino acid sequence of the protein is only 52% identical and 62% similar to that of the beta 1 subunit. Thus, there are amino acid differences in the sequences surrounding the direct alpha  subunit contact sites and other regions of dissimilarity distributed throughout the entire beta 5 sequence. Similarly, examination of the sequences of the alpha  subunits shows multiple differences in the amino acids contacting the beta  subunits in the alpha i, alpha o, alpha s, and alpha q subunits, but there is only one site where the alpha q/11 subunit is unique (41). The alpha i, alpha o, and alpha s subunits have a Phe at position 195 in the beginning of the Switch II region, and the alpha q/11 subunit share a Val at this position (41). Since there are multiple differences in sequence in both the alpha  and beta  isoforms under consideration relative to the isoforms that have been crystallized, it is not possible to suggest a molecular basis for the selective interaction of the beta 5gamma 2HF dimer with the alpha q subunit. However, the net effect of the various differences in alpha -beta contacts must be substantial, as we have found that a large excess of the alpha i1 subunit does not measurably compete with the alpha q subunit for binding to the beta 5 subunit (see Fig. 6).

In evaluating the selectivity of the beta 5gamma 2HF dimer for alpha  subunits in the Gq family, it is important to consider the fidelity with which Sf9 cells modify recombinant proteins. The alpha  subunits of most G proteins are modified with myristoyl and/or palmitoyl groups at their amino terminus, and the gamma  subunits are modified with a prenyl group at their carboxyl terminus (50). These modifications markedly affect the affinity of the alpha  subunits for the beta gamma dimers (51). The available evidence suggests that the proteins used in this work are properly modified. Recombinant Gi and Go alpha  subunits have been shown to be myristoylated (26), and the Gq and G11 alpha  subunits are able to activate phospholipase C-beta equally with native proteins (52). The Gs alpha  subunit produced in Sf9 cells fully activates adenylyl cyclase and is 50-fold more potent than the protein expressed in Escherichia coli but is not as potent as alpha s purified from liver (53). The carboxyl terminus of the gamma 2 subunit expressed in Sf9 cells appears to be properly and fully processed (54). Thus the available experimental evidence supports the hypothesis that recombinant proteins isolated from Sf9 cells are properly modified, and therefore the interactions reported here with recombinant proteins mimic those in intact cells. Most importantly, the major result of the study is considerably strengthened by the data shown in Fig. 5B demonstrating that the beta 5gamma 2HF dimer also selectively associates with the alpha q/11 subunits in a mixture of native G proteins extracted from brain membranes.

Little is known about the biological role of the six different beta  subunits in determining the specificity of cellular signaling. The beta 1-beta 4 subunits are widely expressed, each contain 340 amino acids and are 80-90% identical in sequence (3). In contrast, Northern analysis of various murine tissues shows the beta 5 subunit to be expressed predominantly in the brain (20), but more recently beta 5 subunit expression has been detected in rat portal vein (55). Expression of the similar beta 5L subunit which has a 42-amino acid amino-terminal extension appears restricted to certain areas of the retina (21). These two beta  subunits do appear to be localized to the membrane (21) and are thus presumed to be involved in G protein-mediated signaling in sensory and nervous tissue. The data in this report suggest that the beta 5 subunit (and possibly the beta 5L subunit) participates in signaling via alpha q-linked receptors. Interestingly, treatment of rod outer segment membranes with GTPgamma S failed to release the beta 5L subunit (21). Because members of the alpha q family are slow to exchange guanine nucleotides and are more readily activated by AMF (52), this observation is consistent with our finding of a specific interaction between the beta 5 and alpha q subunits.

The biological implications of the restricted tissue distribution and the divergent sequences of the two beta 5 subunits are not fully understood. The beta gamma dimer has multiple roles in G protein-mediated signaling. In addition to being required for the alpha  subunit to couple to receptors (9, 10, 14), the dimer can regulate the activity of multiple effectors including certain isoforms of PLC-beta , K+, and Ca2+ channels, phosphatidylinositol 3-kinase, adenylyl cyclase, the MAP kinase pathway and can help translocate receptor kinases to the plasma membrane (4). The functional roles of the two beta 5 subunits have not been fully explored, but they have been demonstrated to form functional dimers with the gamma 2, gamma 3, gamma 4, gamma 5, and gamma 7 subunits (20, 21). Analysis of the interaction of the beta  and gamma  subunits using the yeast two-hybrid technique also shows an interaction between the beta 5 subunit and multiple gamma  subunits (56). Moreover, the beta 5gamma 2 and beta 5Lgamma 2 dimers markedly increase inositol phosphate breakdown in COS-7 cells transfected with the cDNAs for either beta 5 subunit, the gamma 2 subunit, and PLC-beta 2 (20-22). Although the beta 5gamma 2 dimer can activate PLC-beta 2 in transfected COS cells, it does not activate the MAP kinase or JNK kinase pathways in these cells (22). In contrast, transfection of the beta 1gamma 2 dimer is able to activate both PLC-beta and the kinases (22, 57, 58). Our preliminary experiments show that the beta 5gamma 2HF dimer is not able to activate type II adenylyl cyclase. Thus, the beta 5 subunit (and possibly the beta 5L subunit) may not interact with certain important effectors.

The data described above combined with the data in this report suggest a number of possibilities for the biological role of the beta 5 subunits in signaling. First, heterotrimers containing the beta 5 subunit are most likely to couple to the alpha q subunit, and thus only alpha q-linked receptors may generate a beta 5gamma dimer to regulate effectors. The ability of other members of the Gq family to couple to the beta 5 subunit needs to be explored. Second, beta gamma dimers containing the beta 5 subunit may only be capable of interacting with a subset of the effectors regulated by other beta gamma dimers. In the retina, the alpha q-linked pathways have been assumed to play a minor role in visual signal transduction (59), but recent studies of mouse retina using immunological techniques have demonstrated the presence of the alpha 11 subunit and PLC-beta 4 (60). Thus, a function for this signaling pathway may emerge. A wide variety of alpha q-linked receptors exist in neural tissue (61). One interesting pathway regulated by m1 or bradykinin receptors via the alpha q subunit involves inhibition of M-type potassium currents (62, 63). The known ability of the beta gamma dimer to regulate K+ and Ca2+ channels via multiple mechanisms (4, 61, 64) suggests interesting potential roles for dimers containing the beta 5 subunit in the regulation of ion channel activity. As multiple G protein-mediated signals are often integrated by a single neuron (61, 64), selective inputs by different beta gamma dimers may allow distinct cellular responses. The observation that the beta 5gamma 2 dimer does not appear to activate the MAP kinase pathway (22) reinforces this possibility and indicates that dimers containing the beta 5 subunit may regulate a limited range of effectors. Thus, there may be an advantage to a more restricted beta gamma signal in retina and neurons. Since recombinant beta gamma dimers of defined composition have not been tested against all the known effectors regulated in this manner, it will be important to determine which effectors are regulated by dimers containing the beta 5 subunit. This information may help explain the restricted tissue distribution of these proteins.

In summary, the data in this report provide partial understanding for the large diversity of the proteins comprising the G protein heterotrimer. The finding that the beta 5 subunit interacts selectively with the alpha q subunit suggests that it will be important to examine this issue in a number of signaling systems using recombinant proteins.

    ACKNOWLEDGEMENTS

We thank Dr. Joel M. Linden and Anna Robeva for the pDoubleTrouble (pDT) expression vector, Dr. Melvin I. Simon for the cDNA for the beta 5 subunit, Dr. Ravi Iyengar for the baculovirus encoding type II adenylyl cyclase, and Dr. T. K. Harden for the baculovirus encoding the alpha 11 subunit. We also acknowledge Kate Kownacki for technical assistance, the University of Virginia Biomolecular Research Facility for DNA sequencing, and the Diabetes Core Facility for cAMP assays.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants PO1-CA-40042 and RO1-DK-19952.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Box 448 Health Sciences Center, University of Virginia, Charlottesville, VA 22908. Tel.: 804-924-5618; Fax: 804-982-3878; E-mail: jcg8w{at}virginia.edu.

1 The abbreviations used are: G proteins, guanine nucleotide-binding regulatory proteins; Sf9 cells, Spondoptera frugiperda cells (ATCC number CRL 1711); DTT, dithiothreitol; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; C12E10, polyoxyethylene 10 lauryl ether; Genapol C-100, polyoxyethylene, 10 dodecyl ether; ECL®, enhanced chemiluminescence; TLCK, N-alpha -p-tosyl-L-lysine chloromethyl ketone; FLAG antibody, anti-FLAG® M2 antibody; GTPgamma S, guanosine 5'-3-O-(thio)triphosphate; GDP-AMF, a mixture of GDP, MgCl2, NaF, and AlCl3, at the indicated concentrations; MAP kinase, mitogen-activated protein kinase; PLC, phospholipase C.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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