Reconstitution of Receptors and GTP-binding Regulatory Proteins (G Proteins) in Sf9 Cells
A DIRECT EVALUATION OF SELECTIVITY IN RECEPTOR·G PROTEIN COUPLING*

(Received for publication, September 19, 1996, and in revised form, November 4, 1996)

Alastair J. Barr Dagger , Lawrence F. Brass § and David R. Manning

From the Department of Pharmacology and the § Departments of Medicine and Pathology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

The selectivity in coupling of various receptors to GTP-binding regulatory proteins (G proteins) was examined directly by a novel assay entailing the use of proteins overexpressed in Spodoptera frugiperda (Sf9) cells. Activation of G proteins was monitored in membranes prepared from Sf9 cells co-expressing selected pairs of receptors and G proteins (i.e. alpha , beta 1, and gamma 2 subunits). Membranes were incubated with [35S]guanosine 5'-(3-O-thio)triphosphate (GTPgamma S) ± an agonist, and the amount of radiolabel bound to the alpha  subunit was quantitated following immunoprecipitation. When expressed without receptor (but with beta 1gamma 2), the G protein subunits alpha z, alpha 12, and alpha 13 did not bind appreciable levels of [35S]GTPgamma S, consistent with a minimal level of GDP/[35S]GTPgamma S exchange. In contrast, the subunits alpha s and alpha q bound measurable levels of the nucleotide. Co-expression of the 5-hydroxytryptamine1A (5-HT1A) receptor promoted binding of [35S]GTPgamma S to alpha z but not to alpha 12, alpha 13, or alpha s. Binding to alpha z was enhanced by inclusion of serotonin in the assay. Agonist activation of both thrombin and neurokinin-1 receptors promoted a modest increase in [35S]GTPgamma S binding to alpha z and more robust increases in binding to alpha q, alpha 12, and alpha 13. Binding of [35S]GTPgamma S to alpha s was strongly enhanced only by the activated beta 1-adrenergic receptor. Our data identify interactions of receptors and G proteins directly, without resort to measurements of effector activity, confirm the coupling of the 5-HT1A receptor to Gz and extend the list of receptors that interact with this unique G protein to the receptors for thrombin and substance P, imply constitutive activity for the 5-HT1A receptor, and demonstrate for the first time that the cloned receptors for thrombin and substance P activate G12 and G13.


INTRODUCTION

The actions of numerous cell-surface receptors on enzymes and ion channels are mediated by heterotrimeric (alpha beta gamma ) GTP-binding regulatory proteins (G proteins)1 (1). Four families of G proteins have been defined, represented by Gs, Gi, Gq, and G12 (2). The activity of each G protein is tightly linked to the binding and hydrolysis of GTP (3). Agonists working through 7-transmembrane domain receptors promote the release of GDP from the alpha  subunit and thus an exchange for GTP present in the cytoplasm. Correlates of the exchange are an altered conformation of the alpha  subunit and dissociation of the subunit from beta gamma . The alpha  subunit as a monomer and beta gamma as a heterodimer, or both operating conjointly, are responsible for the regulation of effectors. G proteins can also be activated by nonhydrolyzable analogues of GTP, for example GTPgamma S, a process similarly promoted by agonists.

Considerable effort has been expended on identifying the G proteins activated by various receptors. Notwithstanding work with purified proteins, interactions have been defined mostly through inferences drawn from effectors regulated. The stimulation of adenylyl cyclase is almost always achieved through activation of Gs, for example, while the inhibition is linked to members of the Gi family (4). Stimulation of the phosphoinositide-specific phospholipase C-beta can be accomplished through Gi (pertussis toxin (PTX)-sensitive) or through members of the Gq family (PTX-insensitive) (5). No effector has yet been identified for the G12 family, although Na+/H+ exchange is a tightly linked correlate (6). The use of effector regulation to deduce receptor·G protein linkages, however, is less than perfect. There is often no resolution among individual members of a particular G protein family, and the nature of effector regulation itself has become increasingly complicated with the appreciation that subunits released from many G proteins can modulate the activation achieved by one (4). Effectors are also subject to numerous forms of feedback or cross-regulation that further limit the extent to which they faithfully mirror receptor·G protein communication (4, 6, 7).

A number of sophisticated techniques have been used to gain either a greater degree of resolution among G proteins regulating a particular effector or a measurement of receptor·G protein communication directly. Antisense constructs (8, 9), PTX-resistant analogues of Gi and Go (10), and antibodies that disrupt interactions between receptors and G proteins (11, 12) are examples of the former. Co-purification of receptor·G protein complexes (13, 14), agonist-promoted photoaffinity labeling with azidoanilido-GTP (15, 16), and agonist-promoted GTPgamma S binding (17) have been used to examine interactions directly. None of these techniques, however, is used widely, and many suffer from practical limitations. Even the use of purified receptors and G proteins to assess the potential for communication has led to some debate regarding the fidelity of interactions (18).

Spodoptera frugiperda (Sf9) cells have recently been established as an intact cell setting for reconstitution of the human 5-hydroxytryptamine1A (5-HT1A) receptor with mammalian G protein subunits (19). Receptors endogenous to Sf9 cells have yet to be characterized, but so far have not interfered with the analysis of numerous mammalian receptors introduced through recombinant baculoviruses. The levels of G proteins endogenous to Sf9 cells, moreover, are quite low relative to those of mammalian G proteins that can be similarly introduced. Sf9 cells also carry out processing events that support the normal targeting of receptors and G protein subunits to the cell membrane, thus providing a relatively normal phospholipid milieu for interaction. In the previous study (19), the 5-HT1A receptor was demonstrated to couple to various members of the Gi family, i.e. Gi, Go, and Gz, as assessed by Gpp(NH)p-sensitive increases in affinity for radiolabeled agonists.

A valuable index of coupling is the process of G protein activation itself. An assay of activation permits an evaluation of coupling without resort to radiolabeled agonists and circumvents presumptions regarding changes in agonist affinity. More importantly with respect to many other assays, the requirement for effector activity as a means of monitoring coupling is eliminated. In the present study, we have investigated the selectivity of receptor·G protein coupling by measuring directly the activation of G proteins introduced into Sf9 cells. The measurement is based on agonist-promoted binding of [35S]GTPgamma S to G protein alpha  subunits isolated subsequently by immunoprecipitation. We have used this methodology to study the coupling of four different receptors (the 5-HT1A, beta 1-adrenergic, neurokinin-1 (NK1), and thrombin receptors) to members of each class of G protein. The expected selectivity in coupling, whereas only intimated previously, was confirmed, and novel interactions between receptors and G proteins were identified.


EXPERIMENTAL PROCEDURES

Baculoviruses

Recombinant baculoviruses encoding alpha s-s, alpha i1, alpha q, beta 1, and gamma 2 (20-22) were kindly provided by Drs. T. Kozasa and A. Gilman at Southwestern Medical Center (Dallas), and those for alpha 12 and alpha 13 were a gift from Dr. N. Dhanasekaran at Temple University School of Medicine (Philadelphia). Baculoviruses for the rat beta 1-adrenoreceptor, human NK1 receptor (23), and human thrombin receptor were gifts from Drs. E. Ross at Southwestern Medical Center (Dallas), T. Fong at Merck & Co., and Drs. S. Seiler and P. Rose at Bristol-Myers Squibb (Princeton, NJ), respectively. Those encoding the 5-HT1A receptor and alpha z were constructed in this laboratory (19).

Cell Culture and Membrane Preparation

Sf9 cells were cultured as described previously (19) but with charcoal-treated serum. For infection with recombinant baculoviruses, the cells were subcultured in monolayer and infected with one or more viruses at a multiplicity of infection of at least one for each virus. The medium was replaced 16 h following infection with Sf900II optimized serum-free medium (Life Technologies, Inc.). The cells were harvested at 48 h and homogenized in ice-cold 20 mM HEPES (pH 8.0), 1 mM EDTA, 0.1 mM phenylmethysulfonyl fluoride, 10 µg/ml leupeptin, and 2 µg/ml aprotinin by repeated passage through a 26-gauge needle. The homogenate was centrifuged at 100 × g for 5 min, and the resulting supernatant fraction was centrifuged at 16,000 × g for 30 min to pellet the membranes. The membranes were washed and resuspended at ~3 mg/ml protein in homogenization buffer for storage at -70 °C. In experiments where the thrombin receptor was expressed, the thrombin protease inhibitor D-Phe-Pro-Arg chloromethyl ketone (1 µM) and Nalpha -tosyl-Lys chloromethyl ketone (100 µM) were included throughout the period of infection and subsequent expression of receptor.

Assay of [35S]GTPgamma S Binding

Membranes (20 µg of protein) from Sf9 cells expressing receptors and/or G protein subunits were resuspended in 55 µl of 50 mM Tris-HCl (pH 7.4), 2 mM EDTA, 100 mM NaCl, 1 µM GDP, and a concentration of MgCl2 calculated to give the desired concentration of free Mg2+. [35S]GTPgamma S (1300 Ci/mmol, DuPont NEN) was added to a final concentration of 30 nM, and the incubation was allowed to proceed for 5 min at 30 °C in the absence or presence of a selected agonist. The incubation was terminated by adding 600 µl of ice-cold 50 mM Tris-HCl (pH 7.5), 20 mM MgCl2, 150 mM NaCl, 0.5% Nonidet P-40 (Calbiochem), 1% aprotinin, 100 µM GDP, and 100 µM GTP. After 30 min, the extract was transferred to an Eppendorf tube containing 2 µl of non-immune serum pre-incubated with 150 µl of a 10% suspension of Pansorbin® cells (Calbiochem). Nonspecifically bound proteins were removed after 20 min by centrifugation. The extract was then incubated for 1 h at 4 °C with 10 µl of a G protein alpha  subunit-directed antiserum, pre-immune serum, or non-immune serum, all of which had been pre-incubated with 100 µl of a 5% suspension of protein A-Sepharose. With the exception of the alpha 12-directed antiserum, generated toward the peptide QENLKDIMLQ, the antisera have been described previously (19, 24). Immunoprecipitates were collected and washed three times in the extraction buffer, once in the buffer without detergent, and then boiled in 0.5 ml of 0.5% SDS followed by addition of 4 ml of Ecolite+TM (ICN, Costa Mesa, CA). The samples were analyzed directly by scintillation spectrometry.

Other Immunological Procedures

Immunotransfer blotting and quantitation of alpha z was accomplished as before (19). Immunoprecipitation of [35S]methionine-labeled proteins was also accomplished as before (19) but under the conditions of extraction and immunoprecipiation used for the nucleotide-binding assays above. Efficiency of immunoprecipitation for antiserum 6354 under these conditions was calculated by comparison to the amount of [35S]methionine-labeled alpha z immunoprecipitated by antiserum 8645 following denaturation (presumed to be 20% (25)).


RESULTS

Activation of G proteins in membranes prepared from Sf9 cells was examined first for the combination of Gz and the human 5-HT1A receptor. Gz is a member of the Gi family and was chosen based on experiments indicating that the binding of [35S]GTPgamma S to alpha z is strictly dependent on co-expression of receptor (see below). Gz also exhibits the capacity to couple to the 5-HT1A receptor, as do other members of the Gi family (19). Fig. 1 represents a set of experiments in which Sf9 cell membranes containing Gz (i.e. alpha z, beta 1, and gamma 2) and the 5-HT1A receptor were incubated with [35S]GTPgamma S ± serotonin (5-HT) over a range of Mg2+ concentrations. alpha z was subsequently immunoprecipitated with antiserum 6354 (generated toward residues 24-33), and bound [35S]GTPgamma S was counted directly. As shown in the figure, the binding of [35S]GTPgamma S to alpha z was dependent on Mg2+ and was optimum in the range of 0.5-10 mM of the divalent cation. Binding was enhanced by serotonin but also occurred in the apparent absence of agonist. The use of non-immune serum instead of antiserum 6354 confirmed specificity of binding for alpha z, as did pretreatment of antiserum 6354 with the peptide used for immunization (not shown). Similar results were achieved with antiserum 2921, directed toward the C terminus of alpha z (residues 346-355).


Fig. 1. Binding of [35S]GTPgamma S to the alpha  subunit of Gz co-expressed with the 5-HT1A receptor. Sf9 cells were infected with recombinant baculoviruses expressing the 5-HT1A receptor, alpha z, beta 1, and gamma 2, and membranes were prepared 48 h thereafter for the analysis of [35S]GTPgamma S binding to alpha z as described under "Experimental Procedures." The incubation with [35S]GTPgamma S was conducted in the presence (black-square) or absence (bullet , open circle ) of 1 µM serotonin (5-HT) at varying concentrations of Mg2+. Immunoprecipitation was performed with the alpha z-directed antiserum 6354 (solid symbols) or with non-immune (i.e. normal) serum (open symbol). Data points are the means ± S.E. of three independent experiments.
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Having established that Gz communicates with the 5-HT1A receptor as monitored by [35S]GTPgamma S binding, we examined further the requirements of the binding assay for receptor and G protein subunits (Fig. 2). As above, a significant degree of binding of [35S]GTPgamma S to alpha z was evident when the 5-HT1A receptor, alpha z, and beta 1gamma 2 were co-expressed, and inclusion of serotonin in the assay increased binding further. Identical results were obtained when the 5-HT1A receptor and alpha z were expressed together but without addition of beta 1gamma 2. Co-expression of the receptor and beta 1gamma 2 without alpha z confirmed that the binding was specific for alpha z. Omission of the receptor demonstrated that Gz alone did not bind [35S]GTPgamma S.


Fig. 2. Binding of [35S]GTPgamma S as a function of 5-HT1A receptor and Gz subunit expression. Membranes were prepared from Sf9 cells expressing recombinant proteins as indicated. [35S]GTPgamma S binding was quantified following incubation with or without 1 µM serotonin (5-HT) at 3 mM free Mg2+ using the alpha z-specific antiserum 6354 or non-immune serum. Data points represent means ± S.E. of five independent experiments.
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The amount of alpha z following infection of Sf9 cells with recombinant viruses encoding the 5-HT1A receptor, alpha z, beta 1, and gamma 2 was 15-30 pmol of subunit per mg of membrane protein, and the efficiencies of extraction and immunoprecipitation were 90 and 60%, respectively. We calculated that 0.5 pmol of [35S]GTPgamma S was immunoprecipitated with alpha z per mg of membrane protein (following treatment of membranes with agonist). Thus, the amount of bound [35S]GTPgamma S was 5-10% of immunoprecipitated alpha z. Binding was not enhanced by increasing the concentration of [35S]GTPgamma S nor by omitting GDP from the assay (GDP was used to suppress agonist-independent binding). Binding of [35S]GTPgamma S could be increased 2-3-fold by increasing the time of incubation to 60 min.

The assay was next extended to G proteins from other families and to other receptors. Fig. 3 illustrates the ability of the chosen antisera to immunoprecipitate the respective alpha  subunits. The antisera were generated with peptides corresponding the C-terminal 10 amino acid residues of alpha s (1191), alpha q (0945), alpha 12 (121), and alpha 13 (120). Results obtained with the two antibodies specific for alpha z are shown for comparison. No subunit was evident when non-immune serum was substituted or when the Sf9 cells were not infected. The appearance of alpha q as two bands has been reported previously (21). Western blots using the "consensus" antisera 8645 and 1398 reveal that expression levels of the different subunits were within severalfold of each other (alpha 12 could not be analyzed by this means). Binding of [35S]GTPgamma S to each of the alpha  subunits co-expressed with beta 1gamma 2 but not receptors is shown as a function of Mg2+ in Fig. 4. No binding occurred for alpha z, alpha 12, or alpha 13 at any concentration of Mg2+. alpha s and alpha q, in contrast, bound [35S]GTPgamma S in a Mg2+-dependent fashion, as did alpha i1 (not shown). Subsequent experiments were carried out at 3 mM free Mg2+.


Fig. 3. Immunoprecipitation of [35S]methionine-labeled G protein alpha  subunits. Sf9 cells expressing the indicated recombinant alpha subunits plus beta 1 and gamma 2, or non-infected cells, were harvested 44 h after infection and incubated for a further 4 h in methionine-free Grace's medium containing [35S]methionine (50 µCi/ml). Solubilization and immunoprecipitation of alpha  subunits was achieved as described under "Experimental Procedures." A, antisera were generated with peptides corresponding the C-terminal 10 amino acid residues of alpha s (1191), alpha q (0945), alpha 12 (121), and alpha 13 (120). Immunoprecipitated proteins were resolved by SDS-polyacrylamide gel electrophoresis and analyzed for incorporated label by autoradiography. The exposure time was 2-3 days. B, antisera were generated with peptides corresponding to amino acid residues 24-33 (antiserum 6354) or the C-terminal 10 amino acid residues (2921) of alpha z. Where indicated, the antisera were pre-incubated with 1 µg of cognate peptides. Visualization of immunoprecipitated alpha z was carried out as described for the previous panel.
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Fig. 4. Binding of GTPgamma [35S] to G protein alpha  subunits in the absence of receptors. Membranes from Sf9 cells expressing the indicated recombinant alpha  subunits plus beta 1 and gamma 2 were incubated with [35S]GTPgamma S at various concentrations of free Mg2+. The amount of [35S]GTPgamma S bound to each subunit was quantified following immunoprecipitation with the relevant antiserum. The data for each alpha  subunit represents a single experiment and are consistent with the data from two other experiments.
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The potential for coupling of Gz to beta 1-adrenergic, thrombin, and NK1 (substance P) receptors was next assessed (Fig. 5). The 5-HT1A receptor, as noted previously, caused an increase in [35S]GTPgamma S binding in the absence of added agonist and promoted a further increase when agonist (serotonin) was added. Binding was also promoted by the thrombin and NK1 receptors, though to a lesser extent. In the latter instances, agonists were required. The beta 1-adrenergic receptor failed to activate Gz.


Fig. 5. Receptor-promoted binding of [35S]GTPgamma S to alpha z. Membranes were prepared from Sf9 cells expressing recombinant proteins as indicated and then incubated with [35S]GTPgamma S ± agonist at 3 mM free Mg2+. Agonists were serotonin (1 µM) for the 5-HT1A receptor, isoproterenol (1 µM) for the beta 1-adrenergic receptor, the peptide SFLLRN (30 µM) for the thrombin receptor, and [Sar9,Met(O2)11]Substance P (100 nM) for the NK1 receptor. In the absence of expressed receptor, serotonin was used as the agonist. Vehicles were ascorbic acid (0.003%) for serotonin and isoproterenol, and water for the other agonists. Immunoprecipitation was performed with the alpha z-directed antiserum 6354 or non-immune serum. Data points represent the means ± S.E. from three experiments. Statistically significant increases from values obtained with alpha zbeta 1gamma 2 without receptor (far left set of bars) are noted (*, p < 0.05; **, p < 0.01).
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A completely different order of selectivities was evident for Gs (Fig. 6). As noted above, alpha s binds some [35S]GTPgamma S regardless of receptor. Omission of alpha s revealed that the binding was relatively specific for the introduced subunit. Activation of the beta 1-adrenergic receptor with isoproterenol caused a 3-4-fold increase in binding. No increase was evident without agonist. Co-expressed 5-HT1A, thrombin, and NK1 receptors, with or without agonists, had no effect. The level of binding for the Gs/beta 1-adrenergic receptor combination (~90,000 cpm) was considerably higher than that achieved for Gz with any receptor, despite equivalent expression of the two G proteins.


Fig. 6. Receptor-promoted binding of [35S]GTPgamma S to alpha s. Membranes were prepared from Sf9 cells expressing recombinant proteins as indicated and then incubated with [35S]GTPgamma S ± agonist at 3 mM free Mg2+ as described for Fig. 5. Immunoprecipitation was performed with the alpha s-directed antiserum 1191 or non-immune serum. In the absence of expressed receptor, isoproterenol (1 µM) was used as the agonist. Data points are the means ± S.E. from three experiments. Statistically significant increases from values obtained with alpha sbeta 1gamma 2 without receptor (far left set of bars) are noted (**, p < 0.01).
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As with Gs, a significant level of receptor-independent binding was observed for Gq (Fig. 7). Omission of the recombinant alpha q indicated that some of the binding could be accounted for by alpha q (or a cross-reactive subunit) endogenous to Sf9 cells. Binding to the endogenous subunit was enhanced by activation of the NK1 receptor. However, the signal provided by endogenous subunit was well below that achieved with the mammalian subunit. A modest degree of [35S]GTPgamma S binding was elicited by the activated 5-HT1A receptor and was dependent on the mammalian subunit (not shown). A much higher level of binding was attained with activated thrombin and NK1 receptors. The receptor-enhanced binding in all three instances was agonist-dependent. The beta 1-adrenergic receptor, regardless of agonist, did not promote binding.


Fig. 7. Receptor-promoted binding of [35S]GTPgamma S to alpha q. Membranes were prepared from Sf9 cells expressing recombinant proteins as indicated and then incubated with [35S]GTPgamma S as before. Immunoprecipitation was performed with the alpha q-directed antiserum 0945 or non-immune serum. In the absence of expressed receptor, [Sar9,Met(O2)11]substance P (100 nM) was used as the agonist. Data points are the means ± S.E. from three experiments. Statistically significant increases from values obtained with alpha qbeta 1gamma 2 without receptor (far left set of bars) are noted (**, p < 0.01).
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Communication of the receptors with G13 is shown in Fig. 8. The data for G12 were identical (not shown). In the case of these two G proteins, only two of the receptors (the thrombin and NK1 receptors) promoted binding of [35S]GTPgamma S. No binding was observed without agonists. The -fold enhancement in binding achieved by agonists, in fact, was the highest for any of the receptor/G protein combinations (greater than 6-fold). As noted previously, GDP was included in all binding assays to suppress receptor-independent binding, an action we had confirmed for Gz and Gs. However, GDP suppresses [alpha -32P]GTP azidoanilide incorporation into alpha 12 and alpha 13 in platelet membranes (16). Consistent with this report, we found that removal of GDP, while having no effect on the negligible receptor-independent binding of [35S]GTPgamma S by alpha 12 and alpha 13, increased binding promoted by agonists by about 50%.


Fig. 8. Receptor-promoted binding of [35S]GTPgamma S to alpha 13. Membranes were prepared from Sf9 cells expressing recombinant proteins and then incubated with [35S]GTPgamma S as before. Immunoprecipitation was performed with the alpha 13-directed antiserum 120 or non-immune serum. In the absence of expressed receptor, [Sar9,Met(O2)11]substance P (100 nM) was used as the agonist. Data points are the means ± S.E. from three experiments. Statistically significant increases from values obtained with alpha 13beta 1gamma 2 without receptor (far left set of bars) are noted (**, p < 0.01).
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DISCUSSION

We have examined the potential of various receptors to couple to members of each of the four families of G proteins. The method used, receptor-promoted binding of [35S]GTPgamma S to G protein alpha  subunits expressed in Sf9 cells, is powerful. Purification of receptors and G protein subunits is not required for reconstitution, nor is the assay compromised by the heterogeneity in both types of proteins inherent to mammalian expression systems. Importantly, receptor·G protein coupling can be analyzed without resort to effector activity. This latter property allows the modeling of receptor·G protein interactions directly (but nevertheless in a native milieu) and a definition of coupling for those G proteins whose effectors are not yet known. We have demonstrated here that the beta 1-adrenergic receptor couples selectively to Gs and not to other G proteins, that the 5-HT1A receptor is selectively coupled to Gz (of the G proteins tested) but shows some activity toward Gq, and that the thrombin and NK1 receptors couple to G12 and G13, as they do to Gq and Gz, but not Gs. Selectivities in interactions previously intimated are now demonstrated directly, and novel interactions are identified.

Gz was used as a representative of the Gi family and as a prototype in the design of the assay. Among the most important traits exhibited by Gz was an inability to bind [35S]GTPgamma S without co-expression of receptor. This property was evident at all concentrations of Mg2+ and was subsequently found to be shared with G12 and G13. The inability to bind [35S]GTPgamma S under the constraints of the assay was consistent with the low rates of exchange of GDP for GTPgamma S established previously for purified alpha z, alpha 12, and alpha 13 (26-28). Gs and Gq both displayed significant levels of receptor-independent binding at millimolar concentrations of Mg2+. Although GDP/GTPgamma S exchange has not been fully analyzed for Gq (21), purified Gs exchanges GDP for GTPgamma S quite rapidly at high concentrations of Mg2+ (29).

Gz is activated to the greatest extent by the serotonin-activated 5-HT1A receptor. The activation by this receptor was predicted based on the communication between the two proteins implied previously (19) and the fact that the 5-HT1A receptor is coupled to members of the Gi family in mammalian neurons (30, 31). We found no evidence for activation of Gs through the 5-HT1A receptor, as once implied (32, 33), but did document a modest activation of Gq. The latter finding is consistent with activation of phosphoinositide hydrolysis in several types of cells expressing the 5-HT1A receptor at high concentrations of agonist (34) and is also reminiscent of a somewhat paradoxical affect of alpha q on ligand affinity previously noted in Sf9 cells (19). Activation of Gz by the agonist-activated 5-HT1A receptor was clearly greater than the activation achieved by the thrombin and NK1 receptors, while the converse was true for activation of Gq.

Only a small proportion of immunoprecipitated alpha z from Sf9 membranes containing the 5-HT1A receptor and exposed to serotonin was complexed with [35S]GTPgamma S. The low level of binding may simply be related to a normal low rate of GDP/GTPgamma S exchange. Alternatively, it may reflect some instability of the [35S]GTPgamma S·subunit complex through extraction and immunoprecipitation. We were somewhat surprised that introduction of beta gamma had no influence on [35S]GTPgamma S binding to alpha z. We had determined previously that alpha z alone could increase the affinity of the 5-HT1A receptor for agonist but that a greater degree of coupling was achieved upon co-expression with beta 1gamma 2 (19). To some extent, the apparent inactivity of beta gamma might be explained by the fact that expression of the two additional subunits causes an approximately 2-fold suppression in levels of alpha z (not shown). However, we also suspect that beta gamma endogenous to Sf9 cells may act catalytically with respect to the activation process.

A significant activation of Gz occurred in the presence of the 5-HT1A receptor but without serotonin. On the one hand, serotonin may have been carried over from the serum in which the cells were initially cultured. However, we employed serum-free medium during the time at which 5-HT1A receptors were expressed and washed the cells and membranes extensively. The receptor, instead, may exhibit constitutive activity. G protein-coupled receptors can convert between active and inactive conformations, a process especially evident in overexpression systems (35). Of the four types of receptors studied here, however, only the 5-HT1A receptor exhibits a readily identified activity.

The greatest degree of selectivity in the interaction of a receptor with a G protein was encountered at the level of the beta 1-adrenergic receptor and Gs. Gs was activated only by the beta 1-adrenergic receptor, and the receptor had no action on any G protein but Gs. The pairing of the beta 1-adrenergic receptor and Gs was obviously expected (36). Another possible interaction with Gs, involving the NK1 receptor, was also sought, since an earlier report had linked this receptor not only to the activation of phosphoinositide hydrolysis but, at very high concentrations of agonist, to the stimulation of adenylyl cyclase (37). However, we were unable to observe any interaction between the NK1 receptor and Gs. We suspect that the stimulation of adenylyl cyclase observed previously was indirect.

With respect to Gq, the activation by thrombin and NK1 receptors was anticipated, though, as for all other pairings but that of the beta 1-adrenergic receptor and Gs, had not been measured directly in previous work. The stimulation of phosphoinositide hydrolysis by thrombin in most types of cells is largely insensitive to PTX, implying the use of a Gq-like protein (38). Sensitivity to PTX, though partial, is common, however, suggesting at least some contribution by Gi. That thrombin can communicate with Gi is quite clear from its ability to inhibit adenylyl cyclase through a PTX-sensitive element in a large number of cells. Conjoint utilization by thrombin of Gq and Gi is consistent with our data, wherein Gz is used as a representative of the Gi family. Substance P also stimulates phosphoinositide hydrolysis in a PTX-insensitive fashion (39), and the addition of Gq/11 to phospholipid vesicles containing NK1 receptors results in conversion of the receptors to a high affinity state (40). Utilization of Gi (and hence Gz in our experiments here) by NK1 receptors is less well documented. However, the NK1 receptor appears to regulate a large conductance Cl- channel through a member of the Gi family (41). The list of receptors that activate Gz can now be extended to those for thrombin and substance P.

We were most interested in the potential of the different receptors to activate G12 and G13. Receptors normally linked to these G proteins are poorly characterized, and effectors have not yet been identified. Our results clearly demonstrate that thrombin and NK1 receptors link to the activation of G12 and G13, while beta 1-adrenergic and 5-HT1A receptors do not. That thrombin should utilize these G proteins is consistent with its ability to support incorporation of [alpha -32P]GTP azidoanilide into alpha 12 and alpha 13 in platelet membranes (16) and with the block of thrombin-stimulated DNA synthesis with an antibody directed toward alpha 12 (42). Our results substantiate the capacity of the cloned receptor for thrombin (43), as distinct from a recently deduced second receptor (44), to achieve the activation. It is intriguing that the two receptors that activate G12 and G13 here, the cloned thrombin and NK1 receptors, also activate Gq and Gz. Whether activation of the latter two G proteins conjointly is predictive of coupling to G12 and G13 is worth pursuit. We were also interested to note that the beta 1-adrenergic receptor does not couple to G12 or G13, as several reports had indicated that beta -adrenergic receptors activate Na+/H+ exchange, a correlate of G12 or G13 activation (6), independently of Gs (45, 46). The fact that the 5-HT1A receptor does not couple to G12 and G13 is also notable. Serotonin operating through 5-HT1A receptors is not viewed to be a complete mitogen, but thrombin is (47). It is conceivable that the mitogenic properties of thrombin are linked to the activation of G12 and G13. Overexpression of alpha 12 or alpha 13, or expression of GTPase-deficient mutants, is linked to unregulated cell growth (6).

Sf9 cells constitute a well defined, intact cell setting upon which the expression of mammalian receptors and G proteins can be superimposed. Our data support the conclusion that GTPgamma S binding is an effective means of monitoring activation of G proteins by receptors. Our data also define the interactions of several receptors with representatives of each family of G protein. Novel interactions have been identified, and their authenticity is supported by the selectivity in interactions otherwise predicted from measurements of second messenger regulation. We anticipate that the Sf9 reconstitution system will lend itself to the analysis of inverse agonism and the coupling achieved by orphan receptors. We also propose the use of the Sf9 reconstitution system for developing or otherwise optimizing techniques to map receptor·G protein communication in mammalian cells.


FOOTNOTES

*   These studies were supported by National Institutes of Health Grants GM51196, MH48125, and HL45181. 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    Recipient of a post-doctoral fellowship from the American Heart Association, Southeastern Pennsylvania Affiliate.
   To whom correspondence should be addressed: Dept. of Pharmacology, University of Pennsylvania School of Medicine, 3620 Hamilton Walk, Philadelphia, PA 19104-6084; 215-898-1775; Fax: 215-573-2236.
1    The abbreviations used are: G proteins, GTP-binding regulatory proteins; GTPgamma S, guanosine 5'-(3-O-thio)triphosphate; NK1 receptor, neurokinin-1 (substance P) receptor; PTX, pertussis toxin; 5-HT1A receptor, the 1A subtype of 5-hydroxytryptamine receptor; Gpp(NH)p, guanosine 5'-(beta ,gamma -imino)triphosphate.

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