©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Expression of the Human 5-Hydroxytryptamine Receptor in Sf9 Cells
RECONSTITUTION OF A COUPLED PHENOTYPE BY CO-EXPRESSION OF MAMMALIAN G PROTEIN SUBUNITS (*)

(Received for publication, May 9, 1995)

Paul Butkerait Yejia Zheng Hazem Hallak Timothy E. Graham Heather A. Miller Kevin D. Burris (§) Perry B. Molinoff (§) David R. Manning (¶)

From the Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6084

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The possibility that Spodoptera frugiperda (Sf9) cells can provide an intact cell setting for reconstitution of the human 5-hydroxytryptamine (5-HT) receptor with mammalian G protein subunits was explored. The 5-HT receptor was found to assume an uncoupled phenotype when expressed alone in Sf9 cells at relatively high levels (5-34 pmol of receptor/mg of membrane protein), i.e. agonist-binding to the receptor was characterized by a relatively high K and an insensitivity to GTP. Co-expression of the receptor with members of the alpha(i) ``family'' together with various combinations of beta(1) and subunits increased the affinity for agonists to that observed for the coupled form of receptor in mammalian cells, concomitant with conferrance of guanosine 5`-(beta,-imino)triphosphate sensitivity. The agonists employed were [^3H]8-hydroxy-N, N-dipropyl-2-aminotetralin ([^3H] 8-OH-DPAT) and [I]R(+)-trans-8-hydroxy-2-[N-n-propyl-N-(3`-iodo-2`-propenyl)amino]tetralin ([I]8-OH-PIPAT). The binding of an antagonist, [I]4-(2`-methoxyphenyl)-1-[2`-[N-(2"-pyridinyl)-p-iodobenzamido]ethyl] piperazine ([I]p-MPPI), was unaffected by co-expression of G protein subunits. Both alpha and beta subunits were required for optimal coupling. No differences were evident among alpha, alpha, alpha, alpha(o), and alpha(z) when expressed with beta(1)(2) in this regard, nor among most permutations of beta(1) subunits when expressed with alpha (beta(1)(2) approx beta(1)(3) approx beta(1)(5) > beta(1)(1)). alpha(s) and alpha(q) expressed with beta(1)(2) did not participate in coupling. These data support the conclusion that normal interactions between a mammalian receptor and a select array of G proteins can be established in intact Sf9 cells, and extend previous observations of 5-HT receptor coupling to G(o) and the pertussis toxin-insensitive G protein G(z).


INTRODUCTION

Receptors for serotonin (5-hydroxytryptamine, or 5-HT) (^1)have been classified according to properties of ligand binding and molecular structure(1) . Of the several subtypes of 5-HT receptors, the 5-HT subtype is among the most extensively characterized. Assays using the agonist [^3H]8-hydroxy-N,N-dipropyl-2-aminotetralin ([^3H]8-OH-DPAT) describe the properties of 5-HT receptors in various regions of the rat forebrain, where the receptor is postsynaptic, and in the dorsal raphe nucleus, where it is somatodendritic(2) . Activation of postsynaptic 5-HT receptors leads to inhibition of adenylyl cyclase and to stimulation of K channels, while activation of presynaptic receptors leads only to stimulation of K channels(3, 4, 5, 6) . Both actions are inhibited by pertussis toxin (PTX), implying a role for one or more forms of the G proteins G(i) and G(o)(7, 8, 9, 10) . Cloned DNAs for the 5-HT receptor reveal, as anticipated, a sequence consistent with a seven transmembrane segment motif common to G protein-linked receptors(11, 12) . Transfection of cells normally lacking 5-HT receptors with the receptor DNA establishes the expected sensitivity of adenylyl cyclase and K channels to serotonin(10, 13) . Activation of a phosphoinositide-specific phospholipase C can also be demonstrated in reconstituted systems, but requires especially high concentrations of agonist and may not be a physiological response(10, 14, 15) .

The 5-HT receptor can exhibit different affinities for [^3H]8-OH-DPAT depending on whether the receptor is coupled to a G protein(s). As assessed with membranes from rat brain or various transfected cells, the G protein-coupled form of receptor exhibits a K for [^3H]8-OH-DPAT of about 1 nM, whereas the K of the uncoupled form is about 20 nM(16, 17) . Conversion of the high- to the low-affinity form can be achieved with GTP or non-hydrolyzable analogues. GTP promotes dissociation of the G protein into monomeric alpha and heterodimeric beta subunits, which in turn destabilizes the interaction between the G protein and receptor supporting high-affinity binding of agonists(18) .

Mulheron et al.(19) were the first to express the human 5-HT receptor in Sf9 cells. Using [^3H]8-OH-DPAT, these investigators documented a B(max) of 150 fmol of receptor/mg of Sf9 cell membrane protein and a K of 3 nM. The relatively low value for the K is consistent with the coupling of the receptor to endogenous G proteins, an inference confirmed by a GTP-induced shift in K to 23 nM and by serotonin-promoted labeling of an alpha(o)-like subunit with [P]GTP-azidoanilide. 5-HT, dopamine D4, and Substance P receptors, when similarly present at low levels in Sf9 cells, also exhibit GTP-sensitive binding of agonists(20, 21, 22) . However, seven transmembrane segment receptors expressed at higher levels do not(23, 24, 25) , consistent with the hypothesis that endogenous G proteins can be limiting. Parker et al.(26) , expressing the 5-HT receptor in Sf9 cells at a density of 3 pmol/mg of membrane protein, found that only 7% of the receptors bound [^3H]8-OH-DPAT with high affinity.

We verify here a predominantly uncoupled phenotype of the human 5-HT receptor when expressed at high levels in Sf9 cells. Using this phenotype as a starting point, we explored the possibility that Sf9 cells can provide an intact cell setting for reconstitution of the receptor with mammalian G protein subunits. Sf9 cells carry out a variety of co- and post-translational processing events which, although not as extensive as those occurring in mammalian cells, support a viable targeting of receptors and G protein subunits to the cell membrane. In this respect, Sf9 cells provide a level of organization beyond that achieved in vitro with proteins whose purification is difficult or (in the case of bacterially expressed proteins) that lack relevant covalent modifications. In distinction to mammalian cell models, Sf9 cells are free of potentially interfering seven transmembrane segment receptors, and the low level of endogenous G proteins permits more rigorous testing of mammalian subunits for their ability to interact with co-expressed receptors. We report here that human 5-HT receptors expressed in Sf9 cells can be induced to assume a coupled phenotype upon co-expression with an appropriate array of G protein subunits. Both alpha and beta subunits are required for optimal coupling, with the selectivity for alpha subunits restricted to those of the alpha(i) family, i.e. alpha, alpha, alpha, alpha(o), and alpha(z). An interaction of the 5-HT receptor with G(o) and G(z), the distributions of which overlap extensively in mammalian brain, has not been previously reported.


EXPERIMENTAL PROCEDURES

Recombinant Baculoviruses

The XbaI/BamHI fragment of the human 5-HT receptor DNA (the gift of M. Caron and R. Lefkowitz, (27) ) subcloned into the pDP5 vector was provided by Dr. Dolan Pritchett (University of Pennsylvania). A BglII site was introduced near the initiating methionine codon, and most of the 5`-untranslated sequence was eliminated, by the polymerase chain reaction using 5`-GAAGATCTGGCGCGCAGGCATGGAT-3` as a forward primer and 5`-AACCACAACTAGAATGCAGTG-3` (pDP5-specific) as the reverse primer (the BglII site is underlined, and the initiating codon is double underlined). The polymerase chain reaction product was digested with BglII and EcoRI to yield a 1.3-kilobase fragment with 11 authentic nucleotides 5` to the initiating codon and 34 authentic (plus 11 pDP5-related) nucleotides 3` to the stop codon. The BglII/EcoRI fragment was subcloned into the transfer vector pVL1392. Sequencing by the dideoxy chain termination method verified that no mutations had been introduced by polymerase chain reaction. Spodoptera frugiperda (Sf9) cells (Invitrogen) were transfected with a combination of the recombinant transfer vector and a modified AcNPV DNA (BaculoGold) according to the protocol provided by PharMingen (San Diego, CA). Baculovirus containing recombinant DNA was plaque-purified and amplified.

Baculoviruses containing recombinant DNA encoding alpha, alpha, alpha(q), beta(1), beta(2), (1), (2), (3), (5), and (7)(28, 29, 30) were kindly provided by Drs. T. Kozasa and A. Gilman at Southwestern Medical Center, Dallas. Those encoding alpha, alpha, and alpha(31) were the gift of Dr. J. Garrison (University of Virginia). Recombinant baculovirus DNA encoding alpha(z) was constructed in our laboratory by subcloning the EcoRI fragment of the human alpha(z) cDNA (32) into pVL1393 and transfecting Sf9 cells with a combination of the recombinant transfer vector and linearized AcMNPV DNA (Invitrogen, San Diego). Baculovirus containing recombinant DNA was isolated as described above.

Cell Culture and Membrane Preparation

Sf9 cells were maintained in suspension culture in TNM-FH media containing 10% fetal calf serum, 0.1% pluronic F-68, and 0.01 mg/ml gentamycin at 27 °C. For expression, cells were subcultured in monolayer and grown to 60% confluency, at which time they were infected with one or more recombinant viruses at a multiplicity of infection of at least 1 for each virus. Cells were harvested 48 h following infection, washed twice with 0.9% NaCl, and resuspended in 1 ml of ice-cold 20 mM HEPES (pH 8.0), 2 mM MgCl(2), 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 2 µg/ml aprotinin. After 5 min on ice, cells were homogenized by repeated passage through a 26-gauge needle. Homogenates were centrifuged at 100 g for 5 min, and the resultant supernatants at 16,000 g for 30 min. The final pellets (membrane) were washed twice with the above buffer and resuspended at 3 mg/ml protein.

[^3H]8-OH-DPAT Binding Assay

Binding of [^3H]8-OH-DPAT was carried out as described previously(33) . Each tube contained 20 µg of membrane protein in 500 µl of 50 mM Tris (pH 7.7), 0.1% ascorbic acid, 20 µM pargyline, and 0.1-50 nM [^3H]8-OH-DPAT (220 Ci/mmol, Amersham). Incubations were carried out for 30 min at 25 °C, at which time the contents were filtered under vacuum on Whatman GF/B filters presoaked in 0.5% polyethylenimine. The filters were washed 3 times with 5 ml of ice-cold buffer, placed in 5 ml of Ecolite liquid scintillation mixture, and counted by scintillation spectrometry. Specific binding was defined as the difference in radioactivity measured in the presence and absence of 10 µM 5-HT. The percentage of specific binding at the K was 90%. Maximum binding and K values were determined by Scatchard (34) transformation of saturation binding data using unweighted linear regression analysis.

[I]8-OH-PIPAT and [I]p-MPPI Binding Assays

Membranes (0.1-1.1 µg of protein per assay tube) were incubated for 30 min at 37 °C with 9-800 pM [I]R(+)-trans-8-hydroxy-2-[N-n-propyl-N-(3`-iodo-2`-propenyl)amino]tetralin ([I]8-OH-PIPAT) (2200 Ci/mmol) or 12-1200 pM [I]4-(2`-methoxyphenyl)-1-[2`-[N-(2"-pyridinyl)-p-iodobenzamido]ethyl]piperazine ([I]p-MPPI) (2200 Ci/mmol) in a total volume of 100 µl. When using [I]8-OH-PIPAT, assays were carried out in 50 mM Tris (pH 7.7), 2 mM MgCl(2), and 0.1% bovine serum albumin. Assays with [I]p-MPPI were carried out in 50 mM Tris (pH 7.7), 100 µM Gpp(NH)p, and 0.1% bovine serum albumin. Assays were terminated by the addition of 5 ml of ice-cold wash buffer (20 mM Tris, 7.4). Filtration was carried out using a Brandel cell harvester with glass fiber filters (Schleicher and Schuell No. 32, previously soaked in 0.3% polyethylenimine) followed by washing with 15 ml of ice-cold wash buffer. Specific binding was defined with 10 µM ([I]8-OH-PIPAT assay) or 100 µM ([I]p-MPPI assay) 5-HT. The percentage of specific binding at the K was 67% for [I]8-OH-PIPAT and 72% for [I]p-MPPI. Maximum binding and K values were determined by Scatchard transformation as described above.

Metabolic Labeling

Sf9 cells expressing 5-HT receptors and/or G protein subunits were harvested 44 h following infection and resuspended in methionine-free Graces insect cell media containing 10% fetal calf serum. [S]Methionine was added (50 µCi/ml), and the cells incubated for an additional 4 h at 27 °C. For studies of phosphorylation, cells were incubated for 4 h in TNM-FH media containing 10% fetal bovine serum and [P]orthophosphate (50 µCi/ml). For studies of palmitoylation, the cells were incubated for 4 h in serum-free TNM-FH media containing [^3H]palmitic acid (250 µCi/ml).

Immunological Procedures

Antibodies used are listed in Table 1. Antibodies recognizing G protein alpha and beta subunits have been described previously(35, 36, 37, 38, 39) . 5-HT receptor-directed antibodies were generated using keyhole limpet hemocyanin-conjugated synthetic peptides as antigens. Immunotransfer blotting was accomplished by solubilizing membranes prepared from uninfected or infected Sf9 cells (50 µg of membrane protein) in Laemmli sample buffer at 37 °C for 30 min. Extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), proteins transferred to nitrocellulose membranes, and the membranes incubated sequentially with rabbit antisera (1:100), biotinylated goat anti-rabbit IgG coupled to horseradish peroxidase-streptavidin, and H(2)O(2) plus 4-choro-1-napthol(37) . In some cases, I-protein A was used for detection.



For immunoprecipitation, Sf9 cell membranes were solubilized in 0.5% SDS, 50 mM sodium phosphate (pH 8.0), and 2 mM EDTA at 25 °C for 30 min. Samples were supplemented at 4 °C to achieve 50 mM sodium phosphate (pH 7.2), 0.5% SDS, 1% sodium deoxycholate, 1% Triton X-100, 150 mM NaCl, 2 mM EDTA, 1% aprotinin, and 200 mg/ml leupeptin. Selected rabbit antisera were added (1:10), followed 18 h later by protein A-Sepharose(37) . Precipitates were collected and washed in buffered 0.5% Triton X-100.

Quantitation of G Protein Subunits

alpha subunits were quantitated where indicated by Western blots using antiserum 8645. 8645 was generated with a peptide common to alpha, alpha, alpha, alpha, and alpha(t), and also present in alpha(z) and alpha(q) except for a valine in place of phenylalanine at the N terminus. G(t) purified from bovine retinal rod outer segments (40) was typically added to membranes of uninfected Sf9 cells just prior to SDS-PAGE to provide an external standard. Colorimetric intensities were linear and bracketed the intensities of subunits expressed following infection. G(t) contains beta(1) as its sole beta subunit, permitting quantitation of beta(1) expression in Sf9 cells with antiserum 8136. Cholate extraction of membranes was accomplished where indicated by addition of sodium cholate to resuspended membranes to a concentration of 1%, followed by incubation for 1 h on ice. The extract was clarified by centrifugation at 16,000 g for 30 min.


RESULTS

Expression of the human 5-HT receptor in Sf9 cells was initially evaluated using the agonist [^3H]8-OH-DPAT. Forty-eight hours following infection of the cells with recombinant baculovirus encoding the receptor, substantial levels of the receptor were found to exist (Fig. 1). B(max) values ranging from 5 to 22 pmol/mg membrane protein were observed, with K values between 7 and 18 nM. Binding of [^3H]8-OH-DPAT did not occur prior to infection, and the binding at 48 h was insensitive to GTP (Fig. 2). In separate experiments with rat hippocampal membranes (B(max) = 230 fmol/mg protein; K of the high-affinity site = 0.4 nM), GTP caused a 54% reduction in binding, consistent with published results(41) . The high Kof the receptor in Sf9 cells and the GTP insensitivity of agonist binding to this receptor were consistent with expression of the receptor in a predominantly uncoupled state.


Figure 1: Binding of [^3H]8-OH-DPAT to human 5-HT receptors in Sf9 cells. Saturation experiments using [^3H]8-OH-DPAT (2.5-50 nM) were performed on membranes prepared from Sf9 cells 48 h following infection with recombinant baculovirus encoding the human 5-HT receptor. The figure represents data obtained in one of several independent experiments, in which the B(max) was 5-22 pmol/mg membrane protein and the K was 7-18 nM. Each point in the figure represents the mean of triplicate determinations. Inset, Scatchard transformation of the data. B/F, bound/free.




Figure 2: Effects of GTP on binding of [^3H]8-OH-DPAT. Specific binding of [^3H]8-OH-DPAT (2.2 nM) was determined in the presence and absence of 100 µM GTP for membranes isolated from noninfected Sf9 cells (``-5-HT receptor'') and from Sf9 cells 48 h following infection with recombinant baculovirus encoding the 5-HT receptor (``+5-HT receptor''). Data represent the mean ± S.E. of three independent experiments, each performed in triplicate.



To ascertain the extent to which the 5-HT receptor in Sf9 cells was subject to co- or post-translational forms of processing, expression was analyzed by Western blots and metabolic labeling. Antibodies recognizing the 5-HT receptor were generated using sequences present in three different regions of the receptor, intracellular loop 2 (antiserum ``DP''), outer loop 2 (``RT''), and intracellular loop 3 (``EV'') (Table 1). Each of the three antibodies revealed four closely spaced protein bands in Western blots (bands a-d (39-45 kDa), Fig. 3). None of the four bands was detected with preimmune sera, nor were any detected in uninfected Sf9 cells, Sf9 cells expressing beta(3)-adrenergic or dopamine D3 receptors, or Sf9 cells infected with wild-type virus alone. Thus, recognition is specific for the 5-HT receptor. The appearance in Sf9 cells of closely spaced bands near the molecular weights deduced from cDNAs has been described previously for beta-adrenergic (24, 42) and N-formyl peptide (23) receptors, and is presumed to represent incomplete, heterogeneous processing of oligosaccharides (23) . The four bands were also identified by immunoprecipitation of [S]methionine-labeled protein (Fig. 4). To determine whether any of the protein species represented by these bands were substrates for phosphorylation or palmitoylation, cells were incubated with [P]orthophosphate or [^3H]palmitate. All four bands were evident as substrates for phosphorylation. Only two (bands a and b) were substrates for palmitoylation.


Figure 3: Identification of the 5-HT receptor by immunotransfer blotting. Membranes isolated from Sf9 cells expressing the human 5-HT receptor (``+5-HT receptor'') or not (``-5-HT receptor'') were subjected to SDS-PAGE (25 µg of protein/lane) followed by transfer of the resolved proteins to nitrocellulose for detection using a 1:100 dilution of antiserum DP, RT, or EV, or respective preimmune sera. Four bands, designated a-d, were observed consistently with DP, RT, and EV in membranes from cells expressing the 5-HT receptor.




Figure 4: Biosynthetic labeling of the 5-HT receptor. Sf9 cells expressing the 5-HT receptor (44 h post-infection) were incubated for 4 h in media containing [S]methionine, [P]orthophosphate, or [^3H]palmitate, and immunoprecipitation was subsequently achieved using solubilized cell membranes and the EV antiserum. Immunoprecipitates were analyzed by fluorography (^3H; 30-day exposure using ENHANCE) or autoradiography (S and P; 7- and 10-day exposures, respectively, using an intensifying screen for the latter). None of the radiolabeled bands in the 39-45-kDa range were detected when preimmune serum was used.



We suspect, based on previous observations(19, 26) , that a small proportion of the 5-HT receptor expressed in Sf9 cells is coupled to endogenous G proteins, but that the vast majority is not. In confirmation of the findings by Mulheron et al.(19) , a G protein alpha subunit that resembles alpha(o) was found in Sf9 cells. The 40-kDa subunit(s) was detected by Western blotting using antiserum 1398, which recognizes alpha(i) and alpha(o), and 9072, which is specific for alpha(o) (Fig. 5). No protein was detected with 8730, which recognizes alpha, alpha, and, to a lesser extent, alpha, thus ruling out appreciable quantities of these mammalian forms of alpha(i) in Sf9 cells. An alpha-like protein, having an apparently larger molecular size than the alpha(o) protein, was detected with 0946. Two proteins react with the alpha(s)-directed antibody 1190. No protein was detected with antisera 2919 (alpha(z)), 120 (alpha), and 130 (alpha) (not shown). Surprisingly, a 40-kDa protein was recognized by 1521. 1521 is directed toward alpha and also recognizes alpha(z). Given the absence of alpha and alpha(z) as deduced with 8730 and 2919, 1521 must either cross-react with the alpha(o)-like subunit present in Sf9 cells or recognize another protein (subunit) altogether.


Figure 5: Detection of Sf9 cell G protein alpha subunits. Membranes from uninfected Sf9 cells (50 µg of membrane protein per lane) were analyzed by immunotransfer blotting with the indicated alpha subunit-directed antisera. A description of the antisera is provided in Table 1.



Having established expression of the 5-HT receptor in Sf9 cells, and an essentially uncoupled phenotype, the effects of alpha(s) (alpha), alpha, alpha(z), and alpha(q) on the affinity for [^3H]8-OH-DPAT were assessed. In these experiments, a low concentration of agonist was used (0.5 nM) so that an increase in affinity concomitant with coupling would be detected as an increase in binding. All four subunits were expressed in Sf9 cells together with 5-HT receptors as detected with the relevant antibodies (Fig. 6). alpha(z) was found to promote a small increase in the binding of [^3H]8-OH-DPAT, while alpha(q) caused a small decrease (Fig. 7). alpha(s) and alpha were without effect.


Figure 6: Introduction of mammalian G protein subunits into Sf9 cells. Expression of alpha(s), alpha, alpha(z), alpha(q), and beta(1) were analyzed 48 h following coinfection with recombinant baculoviruses encoding the subunits and the 5-HT receptor. Shown are immunoblots using membranes (25 µg of protein/lane) prepared prior to and following infection (left and right lanes of each panel), using antisera 1190, 8730, 2921, 0946, and 5357.




Figure 7: [^3H]8-OH-DPAT binding to the 5-HT receptor co-expressed with G protein subunits in Sf9 cells. Specific binding of [^3H]8-OH-DPAT at 0.5 nM was determined for membranes prepared from Sf9 cells expressing the 5-HT receptor, alpha subunits, and beta(1)(2) as indicated. Data represent the mean ± S.E. of 5-24 individual experiments assayed in triplicate. As a point of reference, the dashed line indicates [^3H]8-OH-DPAT binding where the 5-HT receptor is expressed alone. Statistical significance (*, p < 0.01) was determined using Student's t test.



In view of the small effects of alpha subunits on binding of [^3H]8-OH-DPAT, we explored the possibility that the inclusion of beta and subunits would promote more substantive coupling. beta can stabilize alpha subunit conformation(29) , help to target alpha subunits to membranes(29, 43, 44) , and otherwise facilitate receptor-alpha subunit interactions(45, 46, 47, 48) . In preliminary experiments, we had not detected beta subunits in Sf9 cells with antibodies generated against bovine beta (5357; Fig. 6) or a peptide sequence present in beta(1) and beta(2) (and, with a one-residue difference, in beta(3) and beta(4)) (8136; not shown). We therefore introduced beta(1) and (2) subunits, a combination of subunits expressed in mammalian brain. beta(1) and (2) co-expressed with the 5-HT receptor resulted in a small increase in the binding of [^3H]8-OH-DPAT (Fig. 7). When alpha(i) or alpha(z) were expressed in addition to beta(1)(2), binding of [^3H]8-OH-DPAT was increased 3-4-fold above control and well above that observed with alpha or beta subunits alone. The potentiating effect of beta(1)(2) was not observed for alpha(s) or alpha(q). As a point of reference, alpha in these experiments was expressed at about 30 pmol of subunit/mg of Sf9 cell membrane as assessed by 8645 reactivity. Greater than 90% of the subunit was cholate-extractable. Levels of alpha(z) and alpha(q) were the same or severalfold higher. Levels of alpha(s) were not calculated, since this subunit is not equivalently recognized by 8645. beta(1) is expressed at much higher levels, about 320 pmol of subunit/mg of Sf9 cell membrane as assessed by 8136 reactivity, but only about 10% of the subunit was cholate-extractable, indicating that the remainder was denatured and/or nonspecifically aggregated(30) . (2) expression was not quantitated.

To more rigorously define the effects of alpha and beta subunits on agonist binding to the 5-HT receptor, Scatchard analysis was carried out using alpha expressed with or without beta(1)(2). Expression of alpha alone caused a moderate 3-fold increase in the affinity of the receptor for [^3H]8-OH-DPAT (Table 2). Co-expression of alpha with beta(1)(2) resulted in a 7-fold increase in the affinity of the receptor for [^3H]8-OH-DPAT. Changes in the B(max) were not observed. In separate experiments, we found alpha(q) to have the opposite effect on affinity. Consistent with the results shown in Fig. 7, alpha(q) alone caused a 35% increase in the K of the receptor for [^3H]8-OH-DPAT with no change in B(max) (data not shown).



To determine the potential of other members of the alpha(i) family to couple with the 5-HT receptor, and to assess the effects of different beta combinations, we tested the ability of selected combinations of subunits to increase binding of [^3H]8-OH-DPAT (0.5 nM, Table 3). By Western blots with 8645, membrane levels of the different alpha subunits were within severalfold of each other, generally in the rank order of alpha geq alpha approx alpha geq alpha. All the alpha subunits tested (but for alpha(q), used as a negative control) increased binding of [^3H]8-OH-DPAT to a comparable extent. All combinations of beta(1) tested with alpha supported increases in binding of [^3H]8-OH-DPAT. The effect of beta(1) combinations was beta(1)(2) approx beta(1)(3) approx beta(1)(5) approx beta(1)(7); beta(1)(2), beta(1)(3), beta(1)(5) > beta(1)(1). Functional comparisons of beta(1) and beta(2) were not pursued since beta(2) was expressed considerably less well than beta(1) in our hands.



Coupling of G proteins to receptors can also be measured by GTP-sensitivity of agonist-binding. [^3H]8-OH-DPAT binding observed with the 5-HT receptor alone was unaffected by Gpp(NH)p (Fig. 8). Co-expression of alpha(s) with the receptor had no effect on this response. Also consistent with previous data is the reversal by Gpp(NH)p of the slight decrease in binding induced by alpha(q) (Fig. 7), evident here as a Gpp(NH)p-induced increase relative to the control of alpha(q) alone. Similarly, the increases in binding promoted by alpha and alpha(z) were reversed by Gpp(NH)p. The small increase in binding supported by beta(1)(2) was reversed by Gpp(NH)p. When alpha and alpha(z) were expressed together with beta(1)(2), 60-70% of [^3H]8-OH-DPAT binding became sensitive to Gpp(NH)p.


Figure 8: Gpp(NH)p sensitivity of [^3H]8-OH-DPAT binding to the 5-HT receptor co-expressed with G protein subunits. Specific binding of [^3H]8-OH-DPAT at 0.5 nM was determined for membranes from Sf9 cells expressing the 5-HT receptor, alpha subunits, and beta(1)(2) as indicated. Data represent the mean ± S.E. for binding obtained in the presence of 100 µM Gpp(NH)p expressed as percent of binding obtained in the absence of the nucleotide for each permutation of receptor and subunit(s). The dashed line represents 100% (i.e. no change in binding with Gpp(NH)p). The data were obtained from three to ten individual experiments performed in triplicate. Comparisons were made using the two tailed Student's t test. * (p < 0.05) and** (p < 0.01) refer to differences in binding in the absence and presence of Gpp(NH)p; (p < 0.05) and (p < 0.01) refer to differences in binding between the receptor plus alpha subunit alone and the receptor plus the combination of alpha subunit and beta(1)(2), both in the presence of Gpp(NH)p.



Experiments with [^3H]8-OH-DPAT suggest that the increase in binding as a result of co-expression of G protein subunits was due to an increase in the affinity of 5-HT receptors for the agonist. To corroborate this interpretation, experiments were carried out using two newly developed radioligands, [I]8-OH-PIPAT and [I]p-MPPI. [I]8-OH-PIPAT was developed as a 5-HT receptor agonist, and [I]p-MPPI as a receptor antagonist (49, 50, 51) . Specific binding of [I]8-OH-PIPAT was not detected on membranes obtained from Sf9 cells expressing the 5-HT receptor alone (Fig. 9). When the 5-HT receptor was co-expressed with alpha, beta(1), and (2) subunits, however, binding of the agonist was clearly evident. The B(max) was 14-34 pmol/mg of Sf9 cell membrane protein, and the K was 0.35 ± 0.06 nM (n = 4). In contrast, binding of the antagonist [I]p-MPPI was observed regardless of G protein subunit expression. The density and affinity of binding sites for [I]p-MPPI were the same in the presence or absence of alpha, beta(1), and (2) subunits (Fig. 9, inset). These data confirm those obtained with [^3H]8-OH-DPAT that introduction of G protein subunits does not alter the level of expression of 5-HT receptors, but that the increase in agonist binding is attributable to a shift in affinity.


Figure 9: Binding of [I]8-OH-PIPAT and [I]p-MPPI to 5-HT receptors in Sf9 cells. Increasing concentrations of [I]8-OH-PIPAT were incubated with membranes prepared from Sf9 cells expressing 5-HT receptors alone or together with alpha, beta(1), and (2) subunits as indicated. Shown is a representative Scatchard plot. Each data point represents the mean of three determinations. Nonspecific binding was determined using 10 µM 5-HT. The experiment was repeated four times with similar results. The inset shows the effect of co-expressing alpha, beta(1), and (2) subunits with the 5-HT receptor on the density of [I]p-MPPI binding sites. The data is expressed as a percentage of the density determined in membranes prepared from Sf9 cells expressing only 5-HT receptors. Shown is the mean ± S.E. of four independent experiments performed with triplicate determinations. By Scatchard analysis the K values for binding of [I]p-MPPI to 5-HT receptors in the absence and presence of co-expressed G protein subunits were 0.63 ± 0.12 and 0.57 ± 0.14 (n = 4), respectively. Nonspecific binding was determined using 100 µM 5-HT.




DISCUSSION

Using the human 5-HT receptor and co-expressed G protein subunits, we have tested the hypothesis that Sf9 cells can serve as an intact cell reconstitution system and have determined that the 5-HT receptor can interact not only with subtypes of G(i), but with G(o) and G(z). Sf9 cells, unlike mammalian cells or tissues, have so far proven to be free of endogenous seven transmembrane segment receptors and to contain only small amounts of G proteins in relation to the amount of receptor or G protein subunits that can be introduced. Sf9 cells therefore constitute a relatively well defined setting upon which the expression of mammalian receptors and G protein subunits can be superimposed. The absence of potentially interfering receptors becomes particularly important in the classification of receptor ligands. The specificity of a ligand for a particular receptor can be determined unambiguously, and its properties as an agonist or antagonist can be inferred without knowledge of the effectors that are regulated. Although not explored here in detail, reconstitution in Sf9 cells can also be used to determine if the properties of a receptor vary according to the G protein present. Reconstitution of this nature may also be used to validate emerging techniques of mapping receptor-G protein pathways in mammalian cells, e.g. co-immunoprecipitation, agonist-promoted binding of [S]GTPS, or photoaffinity labeling.

The B(max) for the 5-HT receptor expressed here was 5-34 pmol of receptor/mg of membrane protein. This value was higher than those reported elsewhere, i.e. 0.15 and 3 pmol/mg(19, 26) . The reason for the differences in expression among the different laboratories is unclear, but may be related to the nature of the DNA constructs employed. For example, small changes in nucleotide sequences, particularly near the initiation codon, can have a marked effect on protein expression(52) . In the present study, the 5`-untranslated sequence was shortened to 11 authentic bases to which 5 bases required for introduction of a BglII site were added. In the work by Mulheron et al.(19) , nucleotides encoding a FLAG epitope together with an NheI site were introduced adjacent to the normal initiation codon. The amount of 5`-untranslated sequence in the construct employed by Parker et al. (26) was not described. Regardless of the reason for the differences in expression, sufficiently high levels of receptor were achieved in our studies to permit reconstitution with co-expressed mammalian G proteins.

SDS-PAGE was found to resolve the 5-HT receptor into several closely migrating bands. The heterogeneity in migration probably represents differences in the extent of glycosylation(23) . Whether the different receptor species differ in functionality was not ascertained. Several closely spaced bands were observed for the turkey beta-adrenergic receptor, for example, but only one could be labeled with the photoaffinity analogue iodocyanopindolol-diazirine(24) . Similar results were obtained in studies of human beta(2)-adrenergic receptors expressed in Sf9 cells(42) . Only substrates for photoaffinity labeling in the latter studies were subject to palmitoylation. In the present study, two of the four resolved species of 5-HT receptor were palmitoylated. The palmitoylation perhaps reflects transport of an appropriately folded receptor to a membrane compartment. The two other forms may represent biosynthetic precursors or improperly folded/processed receptors.

Coupling of 5-HT receptors to G proteins endogenous to Sf9 cells was not evident with the assays used in the present study, i.e. the receptors exhibited a K for [^3H]8-OH-DPAT similar to that of the low affinity form of brain 5-HT receptors(16) , and the binding of the agonist was not sensitive to GTP. These observations are consistent with data obtained for beta-adrenergic, m1- and m2-muscarinic cholinergic(24) , dopamine D3(25) , and N-formyl peptide receptors(23) . Given the results of Mulheron et al.(19) and Parker et al.(26) , we nevertheless suspect that a small fraction of the 5-HT receptors are coupled to one or more G proteins but that the coupling is obscured by the large number of uncoupled receptors. The work by Parker et al.(26) , in particular, would place an upper limit of about 200 fmol/mg of membrane protein for coupled receptor. Mulheron et al.(19) identified an agonist-activated Sf9 G(o)-like protein through [P]GTP-azidoanilide labeling. Our results with G protein-directed antibodies confirm the existence of an alpha(o)-like subunit and the probable absence of at least three species of alpha(i) in Sf9 cells. The array of endogenous subunits probably also includes homologs of alpha(s) and alpha(q). G(s) would account for isoproterenol-stimulated adenylyl cyclase activity in Sf9 cells expressing beta-adrenergic receptors(24) , and G(q) may underlie the reported Gpp(NH)p sensitivity of agonist binding to expressed substance P receptors(22) . Although Sf9 cells contain beta as demonstrated following purification of the subunit on alpha(o)-agarose(30) , the inability to detect the beta subunit by Western blotting with antibodies such as 5357 and 8136 (the latter antiserum is comparable to K-521(30) ) suggests that the subunit is not beta(1) or beta(2), and probably not beta(3) or beta(4). Alternatively, a homolog may exist in quite small amounts.

A coupled phenotype was achieved for the 5-HT receptor by co-expression of the receptor with mammalian G protein subunits, i.e. a substantial increase in affinity of the receptor for agonist (together with Gpp(NH)p sensitivity in binding, see below) was supported by co-expression of appropriate combinations of alpha, beta, and subunits. Thus, the desired functional reconstitution of receptor and G protein was realized. These results are consistent with the large body of data, obtained with native and reconstituted mammalian membranes, and with purified receptors and G proteins reconstituted into liposomes, that supports a ternary complex model of interaction (18, 53, 54) . Interestingly, when an alpha subunit was expressed alone with the 5-HT receptor, the receptor assumed an affinity for [^3H]8-OH-DPAT intermediate between that of its high- and low-affinity forms. That alpha subunits alone can interact to some extent with receptors has been demonstrated previously for alpha(t) and rhodopsin in retinal rod outer segment disc membranes(45, 55) . Alternatively, the alpha subunit may utilize endogenous beta, although with a lower affinity or effectiveness. In the former instance, consistent with our data, the interaction is stabilized by introduction of beta(55) . In terms of mechanism, the alpha and beta subunits may form a heterotrimeric complex or may act separately to enhance interaction with the receptor. It is also possible that beta prevents denaturation of alpha subunits, or promotes a preferred placement of the subunits with respect to the receptors.

Gpp(NH)p sensitivity of agonist-binding is another index of a coupled phenotype. Partial sensitivity was conferred by alpha or beta subunits alone. Maximal sensitivity to the nucleotide (60%) was achieved with the coordinate expression of both alpha and beta. These data are consistent with the measured increases in affinity for agonist described above, and with the Gpp(NH)p sensitivity observed by us and others for 5-HT receptors in membranes from rat brain.

Under the conditions of the protocols used for infection and assay, we found that alpha, alpha, alpha, alpha, and alpha(z), when expressed together with beta(1)(2), all have the capacity to interact with the 5-HT receptor. The data with respect to subtypes of alpha(i) are in general agreement with those of Raymond et al. (56) and Bertin et al.(57) , in which coupling to all three subtypes could be demonstrated for the receptor expressed in HeLa cells and Escherichia coli, respectively. A selectivity for coupling to G(56) , however, was not evident. In contrast to Bertin et al.(57) , moreover, we find that G(o) has the capacity to couple with the co-expressed receptor. This result is important in that it provides direct evidence for an interaction that has, at best, only been intimated for the mammalian protein. The reason for the difference between our observation and that achieved with bacterially expressed receptor/G protein subunits is not clear, but may be related to covalent modification. Our results are consistent with the fact that 5-HT receptors can activate an alpha(o)-like subunit endogenous to Sf9 cells(19) , and the recent reports that G(o) underlies several serotonin-controlled behaviors exhibited by Caenorhabditis elegans(58, 59) . Transduction pathways employed by the 5-HT receptor in mammalian cells have so far proven to be sensitive to PTX, consistent with the utilization of G(i) or G(o)(8, 9, 10, 14, 15) . Interactions of the receptor with alpha(z), which is PTX-insensitive, however, were not unanticipated. Wong et al.(60) , by co-transfecting 293 cells with DNA encoding stimulatory and inhibitory receptors and alpha(z), demonstrated an interaction between G(z) and receptors that normally communicate with G(i) when the latter was inactivated with PTX. Parker et al.(24) have similarly demonstrated an interaction between the m2-muscarinic cholinergic receptor and G(z) when both are reconstituted into liposomes. It is interesting to note that the amount of G(z) is relatively high in the hippocampus, where a high density of 5-HT receptors is also found(61) . The behavior of alpha(q), which tends to suppress high-affinity agonist binding, is unusual. alpha(q) may interact directly with the receptor to lower affinity for agonist in a fashion reversed by Gpp(NH)p and beta. It is also possible that alpha(q) sequesters beta from an endogenous alpha subunit that interacts (albeit poorly) with the 5-HT receptor.

We additionally examined the influence of different subunits on the coupling of 5-HT receptors to G proteins. Kisselev and Gautam (62) had demonstrated that beta(1)(1), beta(1)(2), and beta(1)(3) could interact equally well with alpha(t), but only beta(1)(1) supported interaction of alpha(t) with rhodopsin. Iiguez-Lluhi et al.(30) and Ueda et al.(63) had demonstrated, at the level of effectors, that beta(1)(1) is less good than beta(1)(2), beta(1)(3), beta(1)(5), and (when examined) beta(1)(7) at inhibiting calmodulin-stimulated type-I adenylyl cyclase, potentiating alpha(s)-stimulated type-II adenylyl cyclase, and stimulating phospholipase Cbeta(3). Four of the subtypes of subunit tested here (in combination with alpha and beta(1)), (2), (3), (5), and (7), proved to be equivalent to each other in their support of coupling to the 5-HT receptor. (1) was less effective. In this regard, the interaction between beta(1)(1) and alpha may not be particularly strong, as suggested in experiments testing the ability of beta combinations to support PTX-catalyzed ADP-ribosylation(30, 63) . This may be attributable to the fact that (1), unlike (2) (and presumably the other subunits(64, 65) ), is in part farnesylated instead of uniformly geranylgeranylated in Sf9 cells(66) , (^2)or to differences in structure. The pattern established by beta(1) combinations to support coupling of the 5-HT receptor to alpha is the converse of that reported for the coupling of rhodopsin to alpha(t)(62) but is analogous to that observed for effector regulation(30, 63) . In our studies, we did not test permutations involving beta(2) since expression of this subunit was far less robust than that of beta(1). Differences in expression between beta(1) and beta(2) have been reported previously(63) .

Previous work with rat hippocampal membranes showed that [I]8-OH-PIPAT, but not [I]p-MPPI, displays guanine nucleotide-sensitive binding to 5-HT receptors, consistent with identification of [I]8-OH-PIPAT as an agonist and [I]p-MPPI as an antagonist(49, 50) . p-MPPI completely antagonizes the inhibition of forskolin-stimulated adenylyl cyclase activity caused by 8-OH-DPAT and 8-OH-PIPAT in hippocampal membranes(51) . Detectable binding of [I]8-OH-PIPAT in Sf9 cells was observed only when 5-HT receptors were co-expressed with G protein subunits. In contrast, binding of [I]p-MPPI occurred regardless of G protein subunit expression, as would be anticipated for an antagonist. The results with [I]p-MPPI corroborate that co-expression of infected Sf9 cells with additional recombinant virus did not alter the level of expression of the 5-HT receptor.

These data support the conclusion that normal interactions can be established between a mammalian receptor and a select array of G proteins when expressed in intact Sf9 cells. Similar observations can presumably be extended to any seven transmembrane segment receptor that can be expressed in excess of the small endogenous pool of G protein to which it might couple. For those receptors whose functions remain obscure, this type of reconstitution represents a useful starting point for exploring potential pathways of transduction. Because Sf9 cells appear to be deficient in seven transmembrane segment receptors, expression of receptors and reconstituion with G protein subunits represents an ideal means to define the specificity of ligands. The reconstitution paradigm is especially germane to the characterization of agonists, whose high-affinity interactions with receptors exhibit a strong dependence on the presence of appropriate G proteins. The overt uniformity in populations of expressed receptors and G proteins, moreover, lends itself to the development and optimization of techniques to map receptor-G protein interactions in mammalian systems.


FOOTNOTES

*
These studies were supported by National Institutes of Health Grants MH48125, GM51196, NS09245, and MH14654. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: CNS Drug Discovery, Bristol-Myers Squibb, Wallingford, CT.

To whom correspondence should be addressed: Dept. of Pharmacology, University of Pennsylvania School of Medicine, 36th and Hamilton Walk, Philadelphia, PA 19104-6084. Tel.: 215-898-1775; Fax: 215-573-2236.

^1
The abbreviations used are: 5-HT, 5-hydoxytryptamine; 8-OH-DPAT, 8-hydroxy-N,N-dipropyl-2-aminotetralin; 8-OH-PIPAT, R-(+)trans-8-hydroxy-2-[N-n-propyl-N-(3`-iodo-2`-propenyl)amino]tetralin; Gpp(NH)p, guanosine 5`-(beta,-imino)triphosphate; p-MPPI, 4-(2`-methoxyphenyl)-1-[2`-[N-(2"-pyridinyl)-p-iodobenzamido]ethyl]piperazine; PTX, pertussis toxin; PAGE, polyacrylamide gel electrophoresis.

^2
J. Robishaw, personal communication.


ACKNOWLEDGEMENTS

We acknowledge Drs. T. Kozasa and A. G. Gilman (Southwestern Medical Center, Dallas), and Dr. J. C. Garrison (University of Virginia), for the kind gifts of G protein recombinant baculoviruses, Drs. M. Caron and R. Lefkowitz (Duke University) for the human 5-HT receptor DNA, and Drs. H. F. Kung, M.-P. Kung, and Mu Mu (University of Pennsylvania) for [I]8-OH-PIPAT and [I] p-MPPI.


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