INTRODUCTION

Heterotrimeric G proteins transduce extracellular signals detected by transmembrane receptors into appropriate cellular responses (1, 2). The intensity and duration of these responses depend on the relative rates of biochemical reactions that turn G proteins on and off. The G protein switch turns on when receptors promote replacement of GTP for GDP bound by  subunits of trimers, leading to dissociation of active G·GTP from the dimer and consequent regulation of downstream effectors. A GTPase activity intrinsic to  subunits turns off signals by converting ·GTP to inactive G·GDP, which then binds to and inactivates . For pure G subunits in vitro the turnoff reaction is slow, 4 min¹ (2). In contrast, many G protein-mediated physiological responses must turn off much more rapidly, in fractions of a second.

Two classes of GTPase-activating protein (GAP)¹ have been reported to accelerate deactivation of trimeric G proteins. One class includes G protein effectors, such as phospholipase C (PLC) and the cGMP phosphodiesterase  subunit, which stimulate GTP hydrolysis by _q and _t, respectively (3, 4). Recent investigations have discovered and characterized a second class of G-GAPs, the RGS (regulators of G protein signaling) proteins. Pure recombinant RGS proteins display GAP activities for certain G protein  subunits (5-9). RGS proteins of mammals (8-12), yeast (13, 14), and Caenorhabditis elegans (11) share a conserved RGS domain and apparently share similar mechanisms of action. Indeed, a mammalian RGS can partially complement yeast mutations that inactivate Sst2p, the RGS of Saccharomyces cerevisiae (12).

Mammalian RGS proteins thus far examined appear to act selectively as GAPs for G proteins in the _i family (5-8), including _i, _o, _z, and most recently _t (9). Transient expression of RGS4 in HEK293 cells inhibits G_i-mediated activation of MAP kinase (MAPK) in response to stimulation of the interleukin-8 receptor (12). In the yeast two-hybrid system, in vitro binding, and co-immunoprecipitation assays, RGS4 interacts with _i family proteins but not with _s or ₁₂ (5-8, 10).

Recent experiments indicate that RGS4 can interact with _q/11 proteins albeit less efficiently than with _i. A high concentration of _q·GDP bound to AlF₄ can inhibit the GAP activity of RGS4 for _o·GTP, presumably because _q·GDP·AlF₄ competes against _o·GTP for binding the RGS protein (6). Moreover, RGS4 can stimulate the GTPase activity of _q in reconstituted vesicles (15). It is not known, however, whether RGS proteins can serve in intact cells as _q-GAPs and inhibitors of G_q-mediated cellular signals. Here we use expression of recombinant RGS4 in COS-7 cells to show that RGS4 can inhibit cellular signals mediated by G_q/11.

EXPERIMENTAL PROCEDURES

FLAG-tagged human RGS4 cDNA was a generous gift from John H. Kehrl at the Laboratory of Immunoregulation, NIAID, National Institutes of Health, Bethesda, MD. cDNA constructs for the bombesin receptor (BR), D₂ dopamine receptor (D₂R), and the ₂-adrenoreceptor were as described (16). Chinese hamster cDNA encoding an HA-tagged p44 MAPK was a gift from J. Pouysségur, Nice, France. pcDNAI and pCR3 were from Invitrogen, San Diego.

COS-7 cells were maintained in Dulbecco's modified Eagle's H21 medium with 10% calf serum. DNA was transfected with adenovirus and DEAE-dextran as described (17). Transfection efficiencies were determined by co-transfection of the plasmid pON249 encoding -galactosidase and assayed as described (17). Expression was consistently detected in over 90% of the cells. Expression of FLAG-tagged RGS4 and HA-tagged MAPK in total cell lysates was detected by immunoblotting with monoclonal antibodies M₂ (Eastman Kodak Co.) and 12CA5, respectively.

HA-MAPK activity was assayed by a procedure modified from that described by Faure et al. (18). COS-7 cells were transfected in 6-well plates at 0.8 × 10⁶ cells/well and placed in serum-free medium containing 0.1% bovine serum albumin after incubating for 24 h in medium containing 10% calf serum. MAPK activity was measured 48 h after transfection. After PTX pretreatment (100 ng/ml for 4 h), where indicated, cells were stimulated for 10 min with appropriate agonists. HA-MAPK immunoprecipitated from cell lysates was incubated with bovine myelin basic protein (MBP) (Sigma) as a substrate in the presence of [-³²P]ATP (DuPont NEN). ³²P-Phosphorylated MBP was quantitated with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) after resolution on a 14% polyacrylamide gel.

Total cellular inositol phosphates (IP) was measured according to Conklin et al. (19). 24 h after transfection cells were replated in a 24-well plate and labeled for 24 h with myo-[³H]inositol (4 µCi/ml, Amersham Corp.). After washing with medium containing 5 mM LiCl for 10 min, cells were incubated for 45 min at 37 °C with the appropriate agonist in the same medium containing LiCl. IP and total inositol fractions were resolved on a Dowex AG 1-X8 formate column (Bio-Rad) (12), and cellular IP content was expressed as the ratio of IP radioactivity to the sum of IP plus inositol radioactivity.

Intracellular [³H]cAMP accumulation was estimated by determining the ratio of cAMP to the cellular pool of ATP plus ADP as described (16). 24 h after transfection each 60-mm dish of 1 × 10⁶ cells was split into 9 wells in a 24-well plate and incubated in medium containing [³H]adenine (2 µCi/ml, Amersham). 18-24 h later, cells were washed once with 1 ml of assay medium and incubated in 1 ml of assay medium containing 1 mM 1-methyl-3-isobutylxanthine and agonist (isoproterenol) for 30 min as indicated.

RESULTS AND DISCUSSION

To assess effects of an RGS protein on cellular responses mediated by G proteins in the G_q/11 family, we compared MAPK activation by a G_q/11-coupled receptor, BR, and a G_i-coupled receptor, D₂R, that co-expressed with or without recombinant RGS4 in COS-7 cells (Figs. 1 and 2). Expression of RGS4 blocked MAPK activation by the G_q/11-coupled receptor agonist, bombesin (1 nM); PTX, which specifically blocks signaling by receptors that activate G_i proteins, did not inhibit the effect of bombesin (Fig. 1A). These results suggest that RGS4 inhibited bombesin signaling to MAPK by inhibiting the action of a G protein other than G_i, probably G_q/11. This inference was supported by additional experiments described below. Confirming a previous report (12) that recombinant RGS4 can inhibit G_i-mediated signals in an intact cultured cell, RGS4 markedly inhibited G_i-dependent MAPK activation by the D₂R agonist, quinpirole (10 nM; Fig. 1B); PTX also blocked the D₂R effect on MAPK (Fig. 1B). RGS4 did not alter the expression of HA-MAPK in these experiments (data not shown).

To further assess the effectiveness of RGS4 in blocking cellular responses mediated by G_q/11 and G_i, we measured MAPK activation by a range of concentrations of both agonists (Fig. 2). In both cases, a higher concentration of each agonist was required to produce equivalent MAPK activation in cells overexpressing RGS4, that is expression of RGS4 shifted the agonist concentration curve to the right. In RGS4-expressing cells, the apparent EC₅₀ of bombesin was increased ~5-fold (Fig. 2A). The quinpirole concentration-effect curve was shifted to the right ~10-fold (Fig. 2B) indicating a relatively greater effectiveness of RGS4 for inhibiting the G_i-mediated effect in comparison with that mediated by G_q/11. The apparent difference in potency could reflect different mechanisms by which RGS4 inhibits the two effects, but it is also consistent with a simpler interpretation that the RGS protein catalyzes GTP hydrolysis less efficiently with _q/11 than with _i proteins.

The RGS-induced rightward shift of signaling concentration-effect curves (Fig. 2) is predictable from the GAP mechanism of RGS action. When a GAP increases the rate of GTP hydrolysis, equivalent steady-state concentrations of G·GTP can only be achieved by an increased rate of receptor-catalyzed GTP-for-GDP exchange and thus by higher concentrations of agonist-occupied receptor. The extent of an RGS-induced rightward shift would be limited by the k_d of the agonist ligand for the relevant receptor. Although a GAP-induced shift in concentration-response curves has not been previously documented, it would be an attractive way to fine tune responsiveness of cells to extracellular stimuli. In phospholipid vesicles reconstituted with a M₁-muscarinic acetylcholine receptor and G_q, the EC₅₀ of carbachol for stimulating GTP hydrolysis was increased by addition of purified PLC1, an _q-GAP; in this case, addition of the GAP also markedly increased the maximal rate of GTP hydrolysis (3, 20).

To determine whether similar concentrations of cellular RGS4 are required to inhibit G_i- and G_q-dependent hormonal signals, we transfected cells with graded amounts of RGS4 plasmid (Fig. 3), which produced graded cellular amounts of RGS4 protein (Fig. 3C). RGS4 inhibited G_i- and G_q-mediated elevation of MAPK activity with similar dose-effect curves over a 16-fold range of transfected DNA (Fig. 3, A and B). Although the endogenous amounts of cellular RGS proteins are unknown, this result argues that similar amounts of RGS4 protein are required to produce both inhibitory effects. It is unlikely that all RGS proteins exhibit quantitatively similar abilities to inhibit G_i- and G_q-dependent hormonal signals. Indeed, another member of the RGS protein family, GAIP, inhibited G_i-mediated activation of MAPK much more effectively than that mediated by G_q/11.²

If RGS4 inhibits BR stimulation of MAPK activity by inactivating G_q/11, the RGS protein should also reduce BR-stimulated synthesis of IP by PLC, the principal effector of G_q/11. Indeed, expression of RGS4 reduced bombesin-induced IP accumulation by about 50% (Fig. 4A). The inhibitory effect of RGS4 was probably exerted on G_q/11 rather than on G_i because PTX failed to inhibit BR-induced IP accumulation (not shown). The BR is likely to stimulate IP accumulation via the  subunit of G_q/11 rather than via its subunit because PLC1 and PLC3, the G protein-responsive PLC isozymes of COS cells, are sensitive to _q/11 stimulation but relatively insensitive to stimulation by G; COS cells lack PLC2, the PLC isozyme that is most sensitive to (21).

RGS4 inhibits accumulation of second messengers in response to agonists for receptors coupled to G_q/11 and G_s.

RGS4 reduced maximal stimulation of IP accumulation by BR stimulation but did not alter the EC₅₀ for bombesin (Fig. 4A). In contrast, RGS4 expression did not affect maximal activation of MAPK by bombesin but did cause a rightward shift of the bombesin concentration-effect curve (Fig. 2A). How could the relations between agonist concentration and response be different, if as seems likely, both BR responses are mediated by G_q/11 and stimulation of PLC? Although we do not know the reason for this discrepancy, the two assays were performed under different conditions and reflect activation of G_q/11 and PLC in different ways. BR-mediated elevation of MAPK activity measured 10 min after addition of agonist probably results from some (undefined) combination of signals triggered by diacylglycerol activating protein kinase C isozymes and by inositol trisphosphate (InsP₃) elevating cytoplasmic Ca²⁺. The IP measurements, in contrast, assessed accumulation at 45 min of total radioactive inositol phosphates in cells labeled with radioactive inositol and exposed to LiCl, which inhibits IP degradation. InsP₃ constitutes only a fraction of the total IP pool, and LiCl may not alter concentrations of InsP₃ and total inositol phosphates in the same way. Consequently, the extent and time course of bombesin-induced changes in InsP₃ under conditions used in the MAPK experiments need not parallel bombesin-induced changes in total IP accumulation.

As expected from the reported (5-8) inability of RGS4 to stimulate GTP hydrolysis by the  subunit of G_s, the RGS protein had no effect on cAMP production stimulated by the ₂-adrenoreceptor agonist, isoproterenol (Fig. 4B) or on the cAMP-mediated activation of MAPK by isoproterenol (not shown).

RGS4 Interacts with _q·GDP·AlF₄ in Intact Cells

To support the idea that RGS4 inhibits PLC stimulation by an effect on _q/11, we took advantage of a recently discovered property of RGS proteins in vitro, their ability to bind G proteins whose nucleotide binding pockets contain GDP complexed with AlF₄ (6, 8, 9). This property is thought to reflect enhanced affinity of RGS proteins for a G conformation that mimics the transition state of GTP hydrolysis; it was useful for our purposes because the conformation of G·GDP·AlF₄ also allows it to regulate activity of the appropriate effector. Although RGS4 reportedly (6) binds G_q·GDP·AlF₄ less tightly than G_i·GDP·AlF₄, we imagined that an RGS4-_q interaction would inhibit stimulation of IP accumulation in cells transfected with recombinant G_q and exposed to AlF₄.

This turned out to be the case (Fig. 5). AlF₄ elevated cellular IP accumulation in cells expressing recombinant G_q but had no effect in untransfected cells; by itself, recombinant G_q produced a smaller but reproducible elevation of cellular IP content. These results suggest that increased abundance of G_q caused a modest elevation in the cellular concentration of its GTP-bound form, and that addition of AlF₄ activated PLC still further by binding to the GDP-bound form of transfected G_q, rendering it capable of activating the effector enzyme. Co-expression of RGS4 with G_q substantially inhibited IP accumulation in response to AlF₄ (Fig. 5). RGS4 presumably inhibited effector stimulation in this case not by accelerating GTP hydrolysis but by binding to and sequestering G_q·GDP·AlF₄. RGS4 also decreased the elevation of MAPK activity seen in untreated cells transfected with G_q (not shown), an effect that probably reflects acceleration of GTP hydrolysis by G_q.

In summary, we present two new sets of observations. While RGS proteins are known to inhibit signals mediated by G_i, we show for the first time that an RGS protein can interact with G_q/11 and inhibit signals transduced by G_q/11 in intact cells. RGS4 probably inhibits bombesin responses by acting as a GAP, that is, by stimulating the intrinsic GTPase activity of G_q/11. Our experiments also raise the possibility that RGS4 inhibits bombesin responses by sequestering the GTP-bound active conformation of G_q/11 (that is, by the mechanism that probably inhibits the AlF₄ response), in addition to stimulating GTP hydrolysis.

Second, we found that RGS4 induces rightward shifts in concentration-effect curves for agonists acting on receptors coupled to either G_i or G_q/11. It is likely that other RGS proteins modulate hormonal signals mediated by G_i and G_q/11 in much the same way. For each response, the extent of the rightward shift will depend on the local concentration of RGS protein and its relative affinity for the G protein involved. Thus different complements of RGS proteins could allow two cells to mount quantitatively different responses to the same concentration of a physiological agonist even when both cells use the same receptors and G proteins. If the relevant receptor couples to two distinct G proteins (for instance, to G_q and G_s or to G_i and G_q), differing cellular complements of RGS proteins with distinct G selectivities could even produce qualitatively different responses of two cells to the same agonist.