INTRODUCTION

Top Abstract Introduction Procedures Results Discussion References

G protein-coupled receptors (GPCRs)¹ initiate vectorial signal transduction cascades via activation of heterotrimeric G proteins and the subsequent regulation of effector enzymes (1, 2). These function to amplify cellular response to the presence of low concentrations of extracellular mediators. As part of this process, agonist-occupied GPCRs have the capacity to catalytically activate G proteins. Some 20 distinct G protein -subunits are known, and in many cases they are highly similar in sequence. Many cells co-express a considerable number of distinct but highly related G proteins. As a novel means to examine the functional interactions of a GPCR with a single G protein, we recently generated a fusion protein between the _2A-adrenoreceptor and the -subunit of G_i1 (3, 4). Since G_i1 is a member of the subfamily of G proteins that can be modified by ADP-ribosylation catalyzed by pertussis toxin (5) and a number of these G proteins are routinely co-expressed by all cells (5), we used a modified version of G_i1 (C351G G_i1) that is resistant to the action of pertussis toxin (6) to generate the fusion protein. Following transient expression of the fusion protein in COS-7 cells, we were able to treat the cells with pertussis toxin to modify the endogenous G_i-like G proteins and thus eliminate any potential interactions between the GPCR of the fusion protein and the endogenously expressed forms of G_i (4). Despite this precaution, there was no obvious indication that the GPCR constrained within the _2A-adrenoreceptor-C351G G_i1 (_2AR-C351G G_i1) fusion protein had significant capacity to activate endogenous G proteins as well as the fusion partner G protein in an agonist-dependent manner (3, 4). However, GPCRs that have had other proteins such as green fluorescent protein linked to their C terminus (7, 8) still appear to be capable of interacting with and activating cellular G proteins (7, 8). We now examine interactions of the _2AR-C351G G_i1 fusion protein with endogenous G proteins following stable expression in Rat 1 fibroblasts. We demonstrate that in this setting agonist-occupation of _2AR-C351G G_i1 results in stimulation of the high affinity GTPase activity of both endogenous G_i and the fusion protein partner G protein and that the activation of endogenous G protein families is selective. Effective regulation of downstream signaling cascades occurred only via activation of endogenous G proteins rather than via activation of the GPCR-linked G protein. Despite this, the relative contribution of the GPCR-linked and endogenous G_i proteins to agonist-stimulated GTPase activity measured at V_max provided a highly novel means to demonstrate that the addition of adrenaline was able to activate some 6 mol of G protein/mol of receptor. Furthermore, studies that have assumed that agonist-dependent prevention of proliferation of cancer cell lines following expression of a ₂-adrenoreceptor-G_s fusion protein reflects activation of the receptor-attached G protein (9) may require careful reanalysis.

	EXPERIMENTAL PROCEDURES

Top Abstract Introduction Procedures Results Discussion References

Materials

All materials for tissue culture were supplied by Life Technologies, Inc. (Paisley, Strathclyde, Scotland). [³H]RS-79948-197 (90 Ci/mmol) was purchased from Amersham Pharmacia Biotech. [-³²P]GTP (30 Ci/mmol) was obtained from NEN Life Science Products. Pertussis toxin (240 µg/ml) was purchased from Speywood. All other chemicals were from Sigma or Fisons plc and were of the highest purity available. Oligonucleotides were synthesized on a Millipore Expedite Nucleic Acids Synthesis System.

Generation of the _2A-Adrenoreceptor-C351G G_i1 Fusion Construct

A pertussis toxin-resistant C351G form of rat G_i1 was generated (6) and linked to the porcine _2A-adrenoreceptor (10). Briefly, the ORF of the _2A-adrenoreceptor DNA was amplified by PCR using the following oligonucleotides: sense, 5'-TTGGTACCATGTATCCTTACGACGTTC-3'; antisense, 5'-AAGAATTCCATGGCGATCCGTTTCCTGTCCCCACGGC-3' (restriction sites for KpnI, EcoRI, and NcoI are underlined). The PCR-amplified fragment was digested with KpnI and EcoRI and ligated to pBluescript (pBS) (Stratagene) through these restriction sites. Introduction of the NcoI site at the 3'-end of the ORF resulted in the C-terminal amino acid of the receptor being altered from Val to Ala and removal of the stop codon. The rat C351G G_i1 cDNA contains two NcoI sites: one straddling the ATG start codon and the other 268 bp downstream from this. This 268-bp fragment was removed from C351G G_i1 in pBS by digestion with NcoI, and the remaining C351G G_i1 pBS cDNA was religated. The shortened cDNA was excised from pBS with EcoRI and cloned into the EcoRI site of the _2A-adrenoreceptor in pBS, adjacent to the 3'-end of the receptor ORF. The 268-bp fragment was then inserted between the NcoI sites at the 3'-end of the _2A-adrenoreceptor ORF and at the 5'-end of the C351G G_i1 ORF. This resulted in production of an in-frame construct whereby the 3'-end of the _2A-adrenoreceptor ORF was exactly adjacent to the 5'-end of the C351G G_i1 ORF. The full fusion construct was then excised from pBS with KpnI and EcoRI and ligated into the eukaryotic expression vector pcDNA3.

Cell Culture and Transfection

Generation of Stable Cell Lines Expressing the _2A-Adrenoreceptor-C351G G_i1 Fusion Protein-- Rat-1 fibroblasts were transfected with _2AR-C351G G_i1 in pcDNA3 using DOTAP^TM (Boehringer Mannheim), according to the protocol provided by the manufacturer, on cells plated in 10-cm² dishes at a confluency of 50-60%. Cells were allowed to grow for 3 days before being split 1:10 in medium containing 0.7 mg/ml Geneticin sulfate (Calbiochem). Individual clones were isolated and expanded in Geneticin-containing medium.

Preparation of Membranes-- Plasma membrane-containing P2 particulate fractions were prepared from cell pastes that had been stored at 80 °C following harvest. Cell pellets were resuspended in 0.5 ml of 10 mM Tris-HCl, 0.1 mM EDTA, pH 7.5 (buffer A), and rupture of the cells was achieved with 50 strokes of a hand-held Teflon on-glass homogenizer followed by passage (10 times) through a 25-gauge needle. Cell lysates were centrifuged at 1000 × g for 10 min in a Beckman TJ-6 centrifuge to pellet the nuclei and unbroken cells, and P2 particulate fractions were then recovered by centrifugation of the supernatant at 200,000 × g for 30 min in a Beckman TL 100 bench-top ultracentrifuge using a Beckman TLA 100.2 rotor. P2 particulate fractions were resuspended in buffer A and stored at 80 °C until required.

[³H]RS-79948-197 Binding Studies-- Binding assays were initiated by the addition of 2-4 µg of protein to an assay buffer (75 mM Tris, 1 mM EDTA, 12.5 mM MgCl₂, pH 7.4) containing [³H]RS-79948-197 (11) (1 nM). K_d for this ligand at the fusion protein was 0.35 nM (3). For saturation experiments, [³H]RS-79948-197 was used between 0.1 and 10 nM. Nonspecific binding was determined in the presence of 100 µM idazoxan. Reactions were incubated at 30 °C for 45 min, and bound ligand was separated from free by vacuum filtration through GF/C filters. The filters were washed with 3 × 5 ml of assay buffer, and bound ligand was estimated by liquid scintillation spectrometry.

High Affinity GTPase Assays-- These assays were performed essentially as described in Refs. 3 and 4 using [-³²P]GTP (0.5 µM, 60,000 cpm) and varying concentrations of agonists (up to 10 µM). Nonspecific GTPase was assessed by parallel assays containing 100 µM GTP. In a range of experiments, GTPase activity was measured at V_max as described previously (3).

Adenylyl Cyclase Activity-- Intact cell adenylyl cyclase activity was measured as described by Wong (12) and Mercouris et al. (13). Briefly, cells in 12-well plates were loaded for 16 h. with 1 µCi/ml [³H]adenine, in the presence or the absence of 25 ng/ml pertussis toxin. Cyclic AMP formation was stimulated by the addition of 50 µM of forskolin, and regulation of forskolin-induced cAMP formation was assayed in the presence of adrenaline or UK14304 (100 µM).

Reverse Transcriptase-PCR (RT-PCR)-- RT-PCR was performed on RNA preparations from a range of clones expressing different levels of the _2AR-C351G G_i1 fusion protein as determined from binding studies, as well as the parental Rat 1 fibroblast cell line.

Regulation of p44 MAP Kinase Phosphorylation and Mobility-- The phosphorylation of p44 MAP kinase was determined by an electrophoretic mobility shift assay (14). Cells were stimulated with the appropriate ligands for 5 min following maintenance in serum-free medium for 48 h and subsequently lysed at 4 °C in a buffer containing 25 mM Tris-HCl, 40 mM p-nitrophenol, 25 mM NaCl, 10% (v/v) ethylene glycol, 10 µM dithiothreitol, 0.2% (v/v) Nonidet P-40, 1 µg/ml aprotinin, 1 mM sodium orthovanadate, 3.5 µg/ml pepstatin A, and 200 µM phenylmethylsulfonyl fluoride at pH 7.5. Following centrifugation of the lysed samples in a microcentrifuge (13,000 rpm, 5 min), SDS-polyacrylamide gel electrophoresis loading buffer was added to a sample of the supernatant and applied to SDS-polyacrylamide gel (10% (w/v) acrylamide, 0.063% (w/v) bisacrylamide, containing 6 M urea) following heating of the sample in a boiling water bath for 5 min. Samples were transferred from the gel to nitrocellulose and immunoblotted with an antipeptide antiserum raised against amino acids 325-345 of p44 MAP kinase (14). These gel conditions provide excellent "gel shift" of this polypeptide upon ligand regulation, a feature that is synonymous with its phosphorylation and activation (14).

p70 S6 Kinase Activity Measurements-- Cells were grown to confluency in 100-mm cell culture dishes and serum-starved for approximately 16 h before treatment. After treatment with agonist for 10 min, the cells were washed twice with ice-cold phosphate-buffered saline before scraping into 1 ml of lysis buffer (55 mM Tris, pH 8.0, 4 °C, 132 mM NaCl, 22 mM NaF, 1.1 mM EDTA, 5.5 mM EGTA, 11 mM sodium pyrophosphate, 33 mM 4-nitrophenyl phosphate, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 2 µg/ml pepstatin A, 2 µg/ml leupeptin). 20 min later, the lysates were centrifuged for 10 min at 14,000 × g. The pellet was discarded, and an aliquot of supernatant was retained for Western blot analysis and protein determination. The remainder of the supernatant was precleared for 1 h with 20 µl (packed volume) of protein A-agarose that had been equilibrated with lysis buffer.

Data Analysis-- Analysis of data curves was performed using the Kaleidagraph curve fitting package driven by an Apple Macintosh computer.

	RESULTS

Top Abstract Introduction Procedures Results Discussion References

A fusion protein (_2AR-C351G G_i1) in which the N terminus of a pertussis toxin-resistant (C351G) mutant of the -subunit of the G protein G_i1 was fused to the C-terminal tail of the porcine _2A-adrenoreceptor was stably expressed in Rat 1 fibroblasts. ³H-Labeled ligand binding studies using the high affinity and highly selective ₂-adrenoreceptor antagonist [³H]RS-79948-197 were performed in membranes derived from a number of individual clones and demonstrated the presence of an apparently uniform population of sites. In clone RAGI 17, this was expressed at 1.2 ± 0.2 pmol/mg of membrane protein and bound [³H]RS-79948-197 with K_d = 4.5 ± 0.7 × 10¹⁰ M and in clone RAGI 77 at 3.3 ± 0.3 pmol/mg membrane protein with K_d = 7.8 ± 0.3 × 10¹⁰ M (means ± S.E., n = 3 in each case). RT-PCR of RNA derived from these cells using a primer pair in which the sense primer was designed to anneal with sequence contributed from the _2A-adrenoreceptor and the antisense primer from the sequence of G_i1 allowed amplification of a 465-bp fragment (data not shown). A fragment of equivalent size was generated by PCR of the fusion protein cDNA using the same primer pair, but no equivalent product was obtained from RT-PCR of RNA from parental Rat 1 cells (data not shown).

The addition of the natural ligand adrenaline (100 µM) or the synthetic agonist UK14304 (100 µM) to membranes of clone RAGI 77 cells resulted in a substantial stimulation of high affinity GTPase activity (Table I). When equivalent experiments were performed on membranes derived from pertussis toxin-treated (25 ng/ml, 24 h) cells, agonist-stimulated GTP hydrolysis was still marked although reduced (Table I). This level of pertussis toxin treatment has been shown to fully attenuate functional interactions between the _2A-adrenoreceptor and endogenously expressed G_i proteins (6, 17) in Rat 1 cells. Membranes from a clonal cell line (TAG WT17) derived from Rat 1 cells that express some 4 pmol/mg protein of the isolated porcine _2A-adrenoreceptor produced similar levels of stimulation of high affinity GTPase upon the addition of adrenaline or UK14304 (data not shown). As such, these results demonstrated both that adrenaline and UK14304 were capable of causing guanine nucleotide exchange and subsequent hydrolysis of GTP by the GPCR-linked G protein and that the GPCR within the fusion protein also had the capacity to activate endogenous G_i-like G proteins as well as the linked G protein. Trivial explanations for these data, related to potential reduction in cellular levels of the _2AR-C351G G_i1 fusion protein during pertussis toxin treatment, were eliminated by performing [³H]ligand binding studies (data not shown). Equivalent experiments performed on membranes of clone RAGI 17 also resulted in agonist stimulation of high affinity GTPase activity, which was partially pertussis toxin-sensitive (data not shown). However, since the level of stimulation was lower than in RAGI 77, clone 77 was used for all subsequent studies.

View this table: [in this window] [in a new window]   Table I 2-Adrenoceptor agonist activation of high affinity GTPase activity in membranes of RAGI 77 cells is only partially sensitive to pertussis toxin Basal high affinity GTPase activity and its regulation by adrenaline and UK14304 was measured in membranes of clone RAGI77 cells that had been pretreated or not with pertussis toxin (25 ng/ml, 24 h). Data are presented as means ± S.E. from n independent experiments.

Time courses of agonist stimulation of high affinity GTPase activity at 37 °C demonstrated that adrenaline allowed hydrolysis of GTP by the GPCR-associated C351G G_i1 to proceed in an essentially linear manner for at least 60 min. By contrast, the stimulated GTPase activity derived from the endogenous G_i population was noted to decay at time points beyond 30 min (Fig. 1), and no further stimulation was observed at times beyond 60 min. Using a time period at which the GTPase activity of both the GPCR-linked and endogenous G proteins was functioning at an essentially maximal rate in response to adrenaline, when using 0.5 µM GTP as substrate, 31.7 ± 1.3% (mean ± S.E., n = 11) of the total stimulated GTPase activity was derived from the fusion protein-linked G protein. Concentration-response curves demonstrated that half-maximal activation of the endogenous signal was achieved with 3.6-fold lower concentrations (EC₅₀ for adrenaline = 6.4 ± 0.6 × 10⁷ M) of either adrenaline or UK14304 than required to cause half-maximal activation of the receptor-linked G protein (EC₅₀ for adrenaline was 2.3 ± 0.4 × 10⁶ M) (Fig. 2A and data not shown). Such data may appear to indicate that the agonists were less potent in catalyzing GTPase activity of the fusion protein partner. However, following transient expression in COS-7 cells of either _2AR-C351G G_i1 or _2AR-C351 G_i1, adrenaline displayed 4.8 ± 1.0-fold (mean ± S.E., n = 3) greater potency to stimulate the high affinity GTPase activity of the fusion protein containing the wild type G protein sequence (Fig. 2B).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Time course of adrenaline-stimulated high affinity GTPase activity in membranes of untreated and pertussis toxin-treated clone RAGI 77 cells. Membranes were prepared from untreated and pertussis toxin-treated (25 ng/ml, 24 h) clone RAGI 77 cells. High affinity GTPase activity was then measured at various times after the addition of adrenaline (100 µM). Stimulation above basal level produced by adrenaline in pertussis toxin-treated cells (open circles) represents activity of the 2AR-C351G Gi1 construct. These values were subtracted from the stimulation above basal level produced by adrenaline in membranes of untreated cells to determine the contribution of endogenous G proteins (filled symbols) to the total signal. Data are means ± S.E. of triplicate observations from an experiment that was representative of three performed.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   The effect of the C351G mutation in Gi1 on the potency of adrenaline to stimulate high affinity GTPase activity. A, varying concentrations of adrenaline were used to stimulate high affinity GTPase activity in membranes of untreated and pertussis toxin-treated clone RAGI 77 cells. The effects of agonist on endogenous G proteins (filled symbols) and the 2AR-C351G Gi1-associated G protein (open symbols) were resolved as in Fig. 1. B, 2AR-C351G Gi1 (open symbols) or 2AR-C351 Gi1 (filled symbols) were expressed transiently in COS-7 cells. Following the addition of varying concentrations of adrenaline to membranes prepared from these cells, high affinity GTPase activity was measured. Data are means ± S.E. of triplicate observations from an experiment that was representative of three performed.

By performing agonist-stimulated high affinity GTPase measurements on membranes of untreated and pertussis toxin-treated clone RAGI 77 cells, the efficacy of a range of ligands could be assessed. Adrenaline, noradrenaline, and -methylnoradrenaline all produced a similar and maximal stimulation of high affinity GTPase activity. The other agonists tested produced less stimulation at maximally effective concentrations and functioned as partial agonists. By comparing the capacity of each ligand to stimulate high affinity GTPase activity relative to the stimulation produced by adrenaline, measurements of agonist efficacy could be obtained. The same rank order of efficacy, adrenaline = noradrenaline = -methylnoradrenaline > UK14304 > BHT933 = dexmeditomidine = xylazine > clonidine  guanabenz > oxymetazoline, was obtained for both the receptor-associated G_i1 and the endogenous G_i population (Fig. 3). However, when compared with adrenaline the efficacy of all of the partial agonist ligands to stimulate the endogenous G_i population was greater than for stimulation of the GPCR-associated G protein (Fig. 3). Indeed, in the cases of guanabenz and oxymetazoline, there was no significant level of stimulation of the GPCR-linked G protein. The efficacy assessments for agonist stimulation of the GPCR-associated C351G G_i1 in membranes of clone RAGI 77 cells were, however, almost identical to those obtained following transient expression of _2AR-C351G G_i1 in COS-7 cells (18).

View larger version (51K): [in this window] [in a new window]   Fig. 3.   Partial agonists display higher efficacy to activate endogenous Gi proteins than 2AR-C351G Gi1. The capacity of a range of agonists at the 2A-adrenoreceptor to stimulate the high affinity GTPase activity of endogenous Gi-like proteins (filled bars) and 2AR-C351G Gi1 (hatched bars) in membranes of clone RAGI 77 cells was assessed as in Fig. 1. All results were then compared relative to natural ligand adrenaline (1). The ligands were -methylnoradrenaline (2), noradrenaline (3), UK14304 (4), BHT933 (5), dexmeditomidine (6), xylazine (7), clonidine (8), guanabenz (9), and oxymetazoline (10). Results represent mean ± S.E. from three independent experiments.

Maximal GTPase activity of the GPCR-linked G_i1 and the endogenous G_i pool was assessed by measuring adrenaline-stimulated GTPase activity in membranes of untreated and pertussis toxin-treated clone RAGI 77 cells at varying concentrations of GTP. The large increase in high affinity GTPase activity caused by adrenaline in membranes of untreated cells was confirmed to represent an increase in V_max without an alteration in K_m for GTP (Fig. 4A). Although pertussis toxin treatment reduced V_max of the basal high affinity GTPase activity (Fig. 4B), this was also produced without a measurable alteration in the measured K_m for GTP (Fig. 4B). However, resolution of agonist-induced stimulation of the GTPase activity of the fusion protein-coupled and endogenous G_i populations demonstrated the K_m for GTP of the fusion protein-coupled G_i1 to be some 2-fold lower (260 ± 7 nM) than for the endogenous G_i (520 ± 50 nM) (mean ± S.E. n = 3) (Fig. 4B). Since the levels of expression of the _2AR-C351G G_i1 fusion protein could be measured directly from saturation [³H]antagonist binding studies, this allowed calculation of the catalytic center activity of the adrenaline-stimulated fusion protein (3) as 4.2 ± 0.5 min¹ (mean ± S.E., n = 3). Since amounts of endogenous G protein activated by the fusion protein cannot be assessed directly, it was not possible to measure catalytic center activity. However, if this is similar to that of the fusion protein-linked G protein, then it could be estimated from the V_max calculations that the adrenaline-occupied _2AR-C351G G_i1 fusion protein was capable of causing activation of some 5 mol of endogenous G_i/mol of receptor-linked G protein at saturating concentrations of GTP (Fig. 4).

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Vmax for stimulation of endogenous Gi-like G proteins by adrenaline is higher than for stimulation of 2AR-C351G Gi1, but the Km for GTP is lower. Basal high affinity GTPase activity (open symbols) and its regulation by adrenaline (filled symbols) was measured at varying concentrations of GTP in membranes of untreated clone RAGI 77 cells. The data from a representative experiment of three performed are presented as an Eadie-Hofstee transformation (A). Equivalent experiments were performed on membranes from pertussis toxin-treated cells. Using the strategy of Fig. 1, basal high affinity GTPase and its stimulation due to activation of endogenous Gi-like proteins (triangles) and 2AR-C351G Gi1 (squares) were resolved (B).

Following stable expression in Rat 1 fibroblasts, G_i-linked receptors including the _2A-adrenoreceptor (19, 20) and the -opioid receptor (14) have been shown to be able to mediate the phosphorylation and activation of the extracellularly regulated kinase (ERK) MAP kinases in response to agonist ligands. The addition of UK14304 (100 µM, 7. 5 min) to serum-deprived clone RAGI 77 cells resulted in the phosphorylation of virtually the entire cellular complement of p44 MAP kinase as measured by a reduced mobility of the protein through SDS-polyacrylamide gel electrophoresis (Fig. 5). By contrast, UK14304 was unable to cause phosphorylation of p44 MAP kinase in pertussis toxin-treated clone RAGI 77 cells (Fig. 5). This was not due to an alteration in the time frame of p44 MAP kinase phosphorylation and dephosphorylation (data not shown). As anticipated from previous studies (14), both epidermal growth factor (10 nM, 7.5 min) (Fig. 5) and platelet-derived growth factor (10 nM, 7.5 min) (data not shown) were able to cause effective phosphorylation of p44 MAP kinase in both untreated and pertussis toxin-treated cells. The _2A-adrenoreceptor is also able to stimulate the activity of p70 S6 kinase in Rat 1 cells by a pathway that is independent of activation of the ERK MAP kinases (15, 21). UK14304 (100 µM, 10 min) produced robust activation of p70 S6 kinase in serum-deprived RAGI 77 cells that could subsequently phosphorylate a peptide substrate (Fig. 6). The same was true of PDGF (10 nM, 10 min). As with p44 MAP kinase phosphorylation, this capacity of UK14304, but not that of PDGF, was completely attenuated following pertussis toxin treatment (Fig. 6).

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Occupation of 2AR-C351G Gi1 by UK14304 results in stimulation of p44 MAP kinase via endogenous Gi proteins but not via 2AR-C351G Gi1. Clone RAGI 77 cells were deprived of serum for 48 h, and then vehicle (lanes 1 and 4), UK14304 (100 µM) (lanes 2 and 5), or epidermal growth factor (10 nM) (lanes 3 and 6) was added for 7.5 min. The cells were also treated with either vehicle (lanes 1-3) or pertussis toxin (25 ng/ml) (lanes 4-6) for 24 h before the addition of receptor agonist. Cell lysates were resolved by SDS-polyacrylamide gel electrophoresis containing 6 M urea, and the presence of p44 MAP kinase was detected immunologically (13). Phosphorylation and activation of this polypeptide is correlated with its reduced mobility through this gel system.

View larger version (48K): [in this window] [in a new window]   Fig. 6.   Occupation of 2AR-C351G Gi1 by UK14304 results in stimulation of p70 S6 kinase only via endogenous Gi proteins and not via 2AR-C351G Gi1. Clone RAGI 77 cells were deprived of serum for 48 h, and then vehicle (bars 1 and 4), PDGF (10 nM) (bars 2 and 5), or UK14304 (100 µM) (bars 3 and 6) was added for 10 min. The cells were also treated with either vehicle (bars 1-3) or pertussis toxin (25 ng/ml) (bars 4-6) for 24 h before the addition of receptor agonist. p70 S6 kinase was immunoprecipitated as described (14, 15), and activity was measured by the capacity of the immunoprecipitated samples to cause phosphorylation of a substrate peptide.

Expression of the _2A-adrenoreceptor in Rat 1 cells also allows agonist-mediated inhibition of forskolin-amplified adenylyl cyclase activity in membrane preparations (17). Following stable expression of _2AR-C351G G_i1 in these cells, UK14304 and adrenaline were able to mediate inhibition of forskolin-amplified adenylyl cyclase (Fig. 7). However, following pertussis toxin treatment, this inhibition was no longer observed (Fig. 7). The _2A-adrenoreceptor has been demonstrated to have the capacity to interact with the stimulatory G protein G_s as well as the G_i-like G proteins to regulate adenylyl cyclase activity (22-24). In most circumstances, the inhibitory effect is predominant, but following pertussis toxin treatment of cells the activation of G_s and thence adenylyl cyclase can often be unmasked (22-24). In Rat 1 cells stably expressing the isolated _2A-adrenoreceptor, neither adrenaline nor UK14304 produced marked effects on forskolin-amplified adenylyl cyclase activity in intact cells (Fig. 8). This may reflect the combination of capacity of the receptor to activate both G_i and G_s. In support of this concept, following pertussis toxin treatment to prevent interaction of the receptor with G_i, both agonists produced a strong activation of forskolin (50 µM)-amplified adenylyl cyclase activity (Fig. 8). By contrast, the agonist-mediated inhibition of forskolin-amplified adenylyl cyclase activity in intact, untreated clone RAGI 77 cells (Figs. 7 and 8) was not converted into a stimulation following pertussis toxin treatment (Fig. 8); indeed, under these conditions no effect of agonist was observed. As anticipated, forskolin-amplified adenylyl cyclase activity was not modified by adrenaline or UK14304 in either untreated or pertussis toxin-treated parental Rat 1 fibroblasts (Fig. 8). Such data are compatible with an inability of the agonist-occupied _2AR-C351G G_i1 fusion protein to interact effectively with endogenous G_s despite its clear activation of endogenous G_i.

View larger version (39K): [in this window] [in a new window]   Fig. 7.   Occupation of 2AR-C351G Gi1 by agonist results in inhibition of adenylyl cyclase via endogenous Gi proteins but not via 2AR-C351G Gi1. Clone RAGI 77 cells were prelabeled with [3H]adenine. Basal adenylyl cyclase activity (bars 1 and 5), its stimulation by forskolin (50 µM) (bars 2 and 6), and the capacity of UK14304 (100 µM) (bars 3 and 7) or adrenaline (100 µM) (bars 4 and 8) to inhibit forskolin-amplified activity were then measured in untreated (bars 1-4) and pertussis toxin-treated (25 ng/ml, 24 h) (bars 5-8) cells. Significant inhibition by the agonists was only observed in the untreated cells. Three independent experiments produced similar data.

View larger version (14K): [in this window] [in a new window]   Fig. 8.   Stimulation of adenylyl cyclase by the 2A-adrenoreceptor but not via 2AR-C351G Gi1 following pertussis toxin treatment. The regulation of forskolin (50 µM)-amplified adenylyl cyclase activity by adrenaline (A) and UK14304 (B) (both 100 µM) was measured in intact parental Rat 1 fibroblasts (bars 1 and 4), clone RAGI 77 (bars 3 and 6), and a clone of Rat 1 cells (bars 2 and 5) expressing the porcine 2A-adrenoreceptor to similar levels as 2AR-C351G Gi1 in clone RAGI 77, either without (bars 1-3) or following (bars 4-6) pertussis toxin (25 ng/ml, 24 h) treatment. Results are presented as a percentage of the activity produced by forskolin alone.

	DISCUSSION

Top Abstract Introduction Procedures Results Discussion References

Herein, we demonstrate for the first time the capacity of a GPCR constrained within a GPCR-G protein -subunit fusion protein to interact with and activate G proteins other than that physically linked to it. Furthermore, this interaction was selective between G protein families, indeed displaying greater selectivity than the isolated receptor. Using this system, the ability of the natural agonist adrenaline to stimulate high affinity GTPase activity, and the capacity of the GPCR-G protein fusion protein to be considered as an agonist-activated enzyme that contains and retains both GPCR and G protein function, we show that the porcine _2A-adrenoreceptor has the capacity to activate some 6 mol of G_i protein/mol at saturating concentrations of GTP. Such measurement could not have been obtained in native cell membranes without the use of the GPCR-G protein fusion protein. Given that the measured K_m for GTP was some 0.4 µM, with intracellular concentrations of GTP in the region of 10 µM and the measured stoichiometry of G protein activation being assessed at GTPase V_max, these estimates are likely to closely reflect the in vivo situation. We have also examined the capacity of the GPCR to activate downstream signal transduction via both the activated fusion protein-linked and endogenous G proteins.

We produced an _2A-adrenoreceptor-G_i1 fusion protein by simply ligating the 5'-end of the G protein cDNA to the 3'-end of the receptor cDNA. The open reading frame so produced is expressed as a single, intact polypeptide (3, 4). Following transient expression of this construct in COS-7 cells, we have demonstrated that the addition of an ₂-adrenoreceptor agonist to membranes of these cells results in guanine nucleotide exchange and its subsequent hydrolysis by the receptor-linked G protein (3, 4). To prove this latter point conclusively, we generated the fusion protein using a pertussis toxin-resistant form of G_i1 in which cysteine 351, which is the normal acceptor site for pertussis toxin-catalyzed ADP-ribosylation, was converted to glycine. Prior pertussis toxin treatment of the COS-7 cells resulted in modification of all of the endogenous G_i-like G proteins (6). Since this modification prevents functional interactions between GPCRs and G_i family G proteins (5), such treatment determined that the observed agonist regulation of high affinity GTPase activity could not reflect activation of the endogenous G_i proteins. Moreover, since agonist stimulation of high affinity GTPase activity in COS-7 cell membranes following expression of the isolated _2A-adrenoreceptor was completely blocked by pertussis toxin treatment (6), any measured activity could not reflect interactions of the receptor with other endogenously expressed G proteins, such as G_s, with which the _2A-adrenoreceptor has been reported to interact (22-24) (also see below). Pertussis toxin treatment of COS-7 cells expressing _2AR-C351G G_i1 resulted in little diminution of agonist signal (3). These data were consistent with interpretations centered on ideas that either the GPCR in the fusion protein could only contact its partner G protein or that the cellular distribution of the _2AR-C351G G_i1 was completely distinct from the bulk of the endogenously expressed G protein. It is certainly worth noting in this regard that a number of studies on GPCR expression on COS cells have indicated that much of the expressed protein is not targeted appropriately to the plasma membrane (e.g. see Ref. 25).

Simple attachment of another protein to the C-terminal tail of a GPCR is not inherently sufficient to prevent interaction of the GPCR with endogenous G proteins, as noted in studies in which GPCR-green fluorescent protein fusion proteins have the capacity to regulate effector function in an agonist-dependent manner (7, 8). Therefore, to directly assess possible interactions of the agonist-occupied GPCR within the _2AR-C351G G_i1 fusion protein with endogenously expressed G proteins, as well as the GPCR-associated G protein, it was necessary to express the fusion construct stably. The cell line selected for these studies was Rat 1 fibroblasts because we have previously stably expressed the porcine and human _2A-adrenoreceptors in these cells and shown them to be capable of allowing agonist-dependent stimulation of high affinity GTPase activity (17), inhibition of membrane-delineated forskolin-amplified adenylyl cyclase activity (17), and phosphorylation and activation of both the ERK MAP kinases (18-20) and p70 S6 kinase (15, 21).

Following stable transfection of _2AR-C351G G_i1, two clones were examined by RT-PCR to confirm expression. Using a primer pair selected to amplify sequence spanning the site of GPCR and G protein fusion we demonstrated expression of mRNA encoding the fusion protein in these clones but not in parental cells. Furthermore, ³H-antagonist binding studies demonstrated the fusion protein to be expressed at high levels. The addition of adrenaline to membranes of cells stably expressing _2AR-C351G G_i1 resulted in a robust stimulation of high affinity GTPase activity. This level of activity was greater per copy of the fusion protein than we had observed in transient transfection studies (3) (Fig. 4A), suggesting either that stable expression improved cellular targeting and function of the fusion protein or that G proteins other than the fusion partner were becoming activated. Following pertussis toxin treatment of the cells, the capacity of adrenaline to stimulate high affinity GTPase activity was reduced significantly. Since pertussis toxin treatment did not reduce levels of expression of _2AR-C351G G_i1, this confirmed that the GPCR within the fusion protein was capable of interacting with and activating endogenous G_i-like G proteins as well as the C351G G_i1 linked to the receptor.

This is the first demonstration of such a capacity; thus, to explore it in detail, we devised a series of approaches to examine similarities and differences in the regulation of these interactions. In the only other reported construct of a GPCR-G protein fusion protein, between the ₂-adrenoreceptor and G_s, its functionality was demonstrated by examining the ability of agonist to stimulate adenylyl cyclase activity following expression of the fusion protein in S49 cyc cells (26). Since S49 cyc cells lack endogenous G_s, this system was clearly not appropriate to address the types of questions posed herein.

To examine the relative contribution to the total agonist-stimulated GTPase activity of endogenous G_i compared with C351G G_i1 of the fusion protein, such assays were performed for 30 min, a period over which both enzymatic activities were proceeding in a maximal and linear fashion, in membranes of untreated and pertussis toxin-treated cells. 68.3 ± 2.6% of the total signal in response to adrenaline was produced via endogenous G_i proteins and the remainder from the fusion protein. These experiments were performed at a single (0.5 µM) subsaturating concentration of GTP, as is the norm for studies on agonist regulation of GTPase activity. Since the _2AR-C351G G_i1 fusion protein can be viewed as an agonist-regulated enzyme, the examination of GTPase activity with variation in GTP concentration allowed measurements of V_max for the GPCR-associated and endogenous G_i protein and direct assessment for the K_m for GTP as substrate. Interestingly, by resolving the contributions of the fusion protein-associated and endogenous G proteins to total agonist-induced signal by analyzing results in membranes of untreated and pertussis toxin-treated cells, we noted that the fusion protein partner G protein displayed higher affinity for GTP than the endogenous G proteins. Again, this is a unique set of observations. Since the levels of expression of the GPCR-linked C351G G_i1 are known from the ³H-ligand binding studies this allowed estimates of catalytic center activity (turnover number) of this protein. The measured V_max of the endogenous G protein population cannot be determined in this way, but assuming a similar catalytic center activity this would predict that the adrenaline-occupied _2AR-C351G G_i1 fusion protein can activate some 5 mol of endogenous G_i protein/mol. As noted above, this measurement of G protein activation stoichiometry could not have been obtained in native membranes without use of the GPCR-G protein fusion strategy.

A range of ligands with agonist activity at the _2A-adrenoreceptor are routinely considered as partial agonists compared with adrenaline. By examining the rate of GTPase activity produced by these ligands in membranes from untreated and pertussis toxin-treated clone RAGI 77 cells, this was observed to be true for activation of both the endogenous and GPCR-linked G proteins. The rank order of efficacy was the same for the various compounds at the two G protein pools, but, interestingly, in comparison with the effects of adrenaline and the other ligands that acted as full agonists, the partial agonists all displayed greater efficacy to activate the endogenous G_i-like G proteins. The basis for this difference is unclear and may relate to constraints imposed by the fusion or the C351G mutation that was necessary to distinguish agonist activation of these two G protein pools. Concentration-response curves for stimulation of high affinity GTPase of the endogenous and receptor-linked G_i demonstrated a 3-fold higher affinity for activation of the endogenous G protein pool. This difference, however, is likely to reflect differences in affinity of the agonist-occupied receptor to activate wild type and the C351G G protein. We have demonstrated previously that following independent co-expression of the _2A-adrenoreceptor with either wild type G_i1 or C351G G_i1, some 10-fold higher levels of the agonist UK14304 were required to stimulate the mutant G protein (6). Furthermore, in the present studies we have compared the capacity of agonist to stimulate the high affinity GTPase of _2AR-C351G G_i1 and a fusion protein in which the receptor is linked to the wild type G_i1 sequence (_2AR-C351 G_i1) following their transient expression in COS-7 cells. Some 5-fold higher levels of agonist were required to produce the same stimulation of the fusion protein containing the C351G mutation. This should not be considered inherently surprising, since the C-terminal tail of the G protein comprises a key contact site for a receptor, and the addition of ADP-ribose to the cysteine residue by pertussis toxin results in attenuation of receptor-G protein interactions (5).

₂-Adrenoreceptors are classical examples of GPCRs that mediate inhibition of adenylyl cyclase. Like many other G_i-linked GPCRs, when expressed in appropriate locations, they are also able to initiate cascades that result in the phosphorylation and activation of the ERK family of MAP kinases and of p70 S6 kinase. We have previously demonstrated that following expression in Rat 1 fibroblasts the _2A-adrenoreceptor is able to produce each of these effects in a pertussis toxin-sensitive manner (21). Following expression of the _2AR-C351G G_i1 fusion protein in these cells forskolin-amplified intact cell adenylyl cyclase activity was reduced by ₂-adrenoreceptor agonists. However, this was virtually abolished following treatment with pertussis toxin, demonstrating the effect to be due to activation of the endogenous G_i population (Fig. 7). As anticipated from studies with expressed isolated _2A-adrenoreceptors, in clone RAGI 77, agonists were able to cause the phosphorylation and activation of both the p44 MAP kinase and p70 S6 kinase. However, following pertussis toxin treatment, there was no ability of ₂-adrenoreceptor agonists to activate either of these kinase cascades, although, as anticipated, epidermal growth factor and PDGF acted as strong activators of both kinases in both untreated and pertussis toxin-treated cells (Figs. 5 and 6). It was further noteworthy that the stably expressed _2AR-C351G G_i1 fusion protein did not have the capacity to produce an enhancement of forskolin-stimulated adenylyl cyclase activity in pertussis toxin-treated intact clone RAGI 77 cells. A number of studies have noted the capacity of agonists at ₂-adrenoreceptors to stimulate adenylyl cyclase activity in defined circumstances (22-24). This is often effectively observed following pertussis toxin treatment of cells to eliminate interactions of the receptor with G_i-like G proteins (23, 24). Comparisons of the capacity in this regard of the isolated porcine _2A-adrenoreceptor and the _2AR-C351G G_i1 fusion protein were also illuminating (Fig. 8). Although _2AR-C351G G_i1 produced strong agonist-mediated inhibition of adenylyl cyclase in intact untreated clone RAGI 77 cells, the isolated receptor had little effect in these conditions. By contrast, following pertussis toxin treatment of the cells, although _2AR-C351G G_i1 now failed to modulate forskolin-amplified adenylyl cyclase activity, the addition of agonist to cells expressing the isolated receptor resulted in a substantial increase in activity. The most obvious interpretation of these results is that although _2AR-C351G G_i1 has the capacity to activate endogenous G_i in response to agonist, it does not interact effectively with endogenous G_s. This is in contrast to the isolated receptor, which in untreated cells presumably activates a combination of G_i and G_s to produce little net effect on forskolin-stimulated adenylyl cyclase and a strong stimulation in pertussis toxin-treated cells. These are again unique observations and provide rather unexpected differences between the isolated receptor and the fusion protein.

Previous studies with a ₂-adrenoreceptor-G_s fusion protein (26) demonstrated agonist-mediated stimulation of adenylyl cyclase following stable expression of the fusion protein in S49 cyc cells, which lack expression of endogenous G_s. It might be argued that the observed effect was due to activation of the endogenous population of ₂-adrenoreceptors present in S49 lymphoma cells now being able to utilize the provided fusion protein-attached G protein, as it is able to do when provided with exogenous G_s in cyc reconstitution assays. This appears unlikely, however, due to the high potency of agonist following expression of the fusion protein. Furthermore, even if it were an effect of the endogenous receptor, the stimulation of adenylyl cyclase would still reflect the capacity of the tethered G protein to access and activate adenylyl cyclase. The minimal capacity of the _2AR-C351G G_i1 fusion protein to mediate the inhibition of forskolin-amplified adenylyl cyclase via the physically linked G_i when the fusion protein-activated endogenous G_i can do so may then reflect limited spatial opportunities of the fusion protein-linked G protein or the relative G protein activation ratios that in the fusion protein must, by definition, be limited to mol/mol of agonist-occupied receptor. Certainly the _2A-adrenoreceptor has a short C-terminal tail compared with the ₂-adrenoreceptor, and little is known about the cellular distribution of the polypeptides of G protein-linked signaling cascades relative to one another (27, 28). It was perhaps even more surprising that the stably expressed