RGS4 Inhibits Gq-mediated Activation of Mitogen-activated Protein Kinase and Phosphoinositide Synthesis*

(Received for publication, December 23, 1996, and in revised form, February 21, 1997)

Yibing Yan , Patty P. Chi and Henry R. Bourne Dagger

From the Department of Cellular and Molecular Pharmacology, Programs in Cell Biology and Biomedical Sciences, and the Cardiovascular Research Institute, University of California, San Francisco, California 94143-0450

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Recombinant regulators of G protein-signaling (RGS) proteins stimulate hydrolysis of GTP by alpha  subunits of the Gi family but have not been reported to regulate other G protein alpha  subunits. Expression of recombinant RGS proteins in cultured cells inhibits Gi-mediated hormonal signals probably by acting as GTPase-activating proteins for Galpha i subunits. To ask whether an RGS protein can also regulate cellular responses mediated by G proteins in the Gq/11 family, we compared activation of mitogen-activated protein kinase (MAPK) by a Gq/11-coupled receptor, the bombesin receptor (BR), and a Gi-coupled receptor, the D2 dopamine receptor, transiently co-expressed with or without recombinant RGS4 in COS-7 cells. Pertussis toxin, which uncouples Gi from receptors, blocked MAPK activation by the D2 dopamine receptor but not by the BR. Co-expression of RGS4, however, inhibited activation of MAPK by both receptors causing a rightward shift of the concentration-effect curve for both receptor agonists. RGS4 also inhibited BR-stimulated synthesis of inositol phosphates by an effector target of Gq/11, phospholipase C. Moreover, RGS4 inhibited inositol phosphate synthesis activated by addition of AlF4- to cells overexpressing recombinant alpha q, probably by binding to alpha q·GDP·AlF4-. These results demonstrate that RGS4 can regulate Gq/11-mediated cellular signals by competing for effector binding as well as by acting as a GTPase-activating protein.


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 alpha  subunits of alpha beta gamma trimers, leading to dissociation of active Galpha ·GTP from the beta gamma dimer and consequent regulation of downstream effectors. A GTPase activity intrinsic to alpha  subunits turns off signals by converting alpha ·GTP to inactive Galpha ·GDP, which then binds to and inactivates beta gamma . For pure Galpha subunits in vitro the turnoff reaction is slow, <= 4 min-1 (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 gamma  subunit, which stimulate GTP hydrolysis by alpha q and alpha t, respectively (3, 4). Recent investigations have discovered and characterized a second class of Galpha -GAPs, the RGS (regulators of G protein signaling) proteins. Pure recombinant RGS proteins display GAP activities for certain G protein alpha  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 Galpha proteins in the alpha i family (5-8), including alpha i, alpha o, alpha z, and most recently alpha t (9). Transient expression of RGS4 in HEK293 cells inhibits Gi-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 alpha i family proteins but not with alpha s or alpha 12 (5-8, 10).

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


EXPERIMENTAL PROCEDURES

DNA Constructs and Transfection of COS-7 Cells

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 beta 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 beta -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.

Measurement of p44 HA-MAPK Activity

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 [gamma -32P]ATP (DuPont NEN). 32P-Phosphorylated MBP was quantitated with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) after resolution on a 14% polyacrylamide gel.

Inositol Phosphate Accumulation

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.

cAMP Assay

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

RGS4 Inhibits Gq-dependent MAPK Activation by Bombesin

To assess effects of an RGS protein on cellular responses mediated by G proteins in the Gq/11 family, we compared MAPK activation by a Gq/11-coupled receptor, BR, and a Gi-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 Gq/11-coupled receptor agonist, bombesin (1 nM); PTX, which specifically blocks signaling by receptors that activate Gi 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 Gi, probably Gq/11. This inference was supported by additional experiments described below. Confirming a previous report (12) that recombinant RGS4 can inhibit Gi-mediated signals in an intact cultured cell, RGS4 markedly inhibited Gi-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).


Fig. 1. RGS4 inhibits activation of MAPK by agonists for both Gi- and Gq/11-coupled receptors. Cells were transfected with plasmids encoding HA-MAPK (1 µg), BR (1 µg, panel A) or D2R (1 µg, panel B), and RGS4 (2 µg) or vector plasmid pCR3 (2 µg) and treated for 4 h with or without PTX as indicated. Cells were then exposed to 1 nM bombesin (A) or 10 nM quinpirole (B) for 10 min as indicated, and HA-MAPK activities were determined. HA-MAPK activities are expressed in arbitrary units of MBP phosphofluorescence (see "Experimental Procedures"). Data represent the mean ± S.D. of triplicate determinations; an additional experiment gave similar results.
[View Larger Version of this Image (31K GIF file)]


Fig. 2. RGS4 increases the agonist concentration required for activating MAPK. Cells were transfected with plasmids encoding HA-MAPK (1 µg), RGS4 (2 µg) or vector plasmid pCR3 (2 µg), and 1 µg of BR (A) or D2R (B). Cells were exposed for 10 min to the indicated concentration of bombesin (A) and quinpirole (B), and HA-MAPK activities were determined. HA-MAPK activities are expressed in arbitrary units of MBP phosphofluorescence (see "Experimental Procedures"). Data represent the mean ± S.D. of triplicate determinations; an additional experiment gave similar results.
[View Larger Version of this Image (20K GIF file)]

To further assess the effectiveness of RGS4 in blocking cellular responses mediated by Gq/11 and Gi, 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 Gi-mediated effect in comparison with that mediated by Gq/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 alpha q/11 than with alpha 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 Galpha ·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 kd 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 Gq, the EC50 of carbachol for stimulating GTP hydrolysis was increased by addition of purified PLCbeta 1, an alpha 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 Gi- and Gq-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 Gi- and Gq-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 Gi- and Gq-dependent hormonal signals. Indeed, another member of the RGS protein family, GAIP, inhibited Gi-mediated activation of MAPK much more effectively than that mediated by Gq/11.2


Fig. 3. RGS4 inhibits both Gi and Gq-mediated activation of MAPK in a dose-dependent manner. Panels A and B, cells were transfected with plasmids encoding HA-MAPK (1 µg), 1 µg each of BR and D2R, and the indicated amounts of RGS4. Vector plasmid pCR3 was added to keep the total amount of DNA constant. Cells were exposed for 10 min to 1 nM bombesin or 10 nM quinpirole, and HA-MAPK activities were determined. HA-MAPK activities are expressed in arbitrary units of MBP phosphofluorescence (see "Experimental Procedures"). Data represent the mean ± S.D. of triplicate determinations; two additional experiments gave similar results. Panel C, immunoblots of FLAG-tagged RGS4 and HA-tagged MAPK expressed in cells used in the MAPK assays shown in panels A and B. Total proteins from cell lysates were resolved in 14% polyacryamide gels and transferred to nitrocellulose membranes. After blotting with M2 FLAG antibody, the membranes were stripped and probed a second time with the 12CA5 antibody, directed against the HA tag. Blots were developed with an ECL kit (Amersham).
[View Larger Version of this Image (33K GIF file)]

RGS4 Reduces Accumulation of Inositol Phosphates Stimulated by Bombesin

If RGS4 inhibits BR stimulation of MAPK activity by inactivating Gq/11, the RGS protein should also reduce BR-stimulated synthesis of IP by PLC, the principal effector of Gq/11. Indeed, expression of RGS4 reduced bombesin-induced IP accumulation by about 50% (Fig. 4A). The inhibitory effect of RGS4 was probably exerted on Galpha q/11 rather than on Galpha i because PTX failed to inhibit BR-induced IP accumulation (not shown). The BR is likely to stimulate IP accumulation via the alpha  subunit of Gq/11 rather than via its beta gamma subunit because PLCbeta 1 and PLCbeta 3, the G protein-responsive PLC isozymes of COS cells, are sensitive to alpha q/11 stimulation but relatively insensitive to stimulation by Gbeta gamma ; COS cells lack PLCbeta 2, the PLC isozyme that is most sensitive to beta gamma (21).


Fig. 4. RGS4 inhibits accumulation of second messengers in response to agonists for receptors coupled to Galpha q/11 and Gs. A, cells were transfected with plasmids encoding BR (1 µg), plus RGS4 (2 µg), or vector plasmid pCR3 (2 µg) and labeled with myo-[3H]inositol. Cells were treated for 45 min with the indicated concentration of bombesin in medium containing 5 mM LiCl, and total cellular IP content was determined. B, cells were transfected with plasmids encoding the beta 2-adrenoreceptor (1 µg), plus RGS4 (2 µg), or vector plasmid pCR3 (2 µg) and labeled with [3H]adenine. Cells were treated for 30 min with the indicated concentration of isoproterenol, and cAMP accumulation was determined. Data represent the mean ± S.D. of triplicate determinations; two additional experiments gave similar results.
[View Larger Version of this Image (22K GIF file)]

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 Gq/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 Gq/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 alpha  subunit of Gs, the RGS protein had no effect on cAMP production stimulated by the beta 2-adrenoreceptor agonist, isoproterenol (Fig. 4B) or on the cAMP-mediated activation of MAPK by isoproterenol (not shown).

RGS4 Interacts with alpha q·GDP·AlF4- in Intact Cells

To support the idea that RGS4 inhibits PLC stimulation by an effect on alpha q/11, we took advantage of a recently discovered property of RGS proteins in vitro, their ability to bind Galpha 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 Galpha conformation that mimics the transition state of GTP hydrolysis; it was useful for our purposes because the conformation of Galpha ·GDP·AlF4- also allows it to regulate activity of the appropriate effector. Although RGS4 reportedly (6) binds Galpha q·GDP·AlF4- less tightly than Galpha i·GDP·AlF4-, we imagined that an RGS4-alpha q interaction would inhibit stimulation of IP accumulation in cells transfected with recombinant Galpha q and exposed to AlF4-.

This turned out to be the case (Fig. 5). AlF4- elevated cellular IP accumulation in cells expressing recombinant Galpha q but had no effect in untransfected cells; by itself, recombinant Galpha q produced a smaller but reproducible elevation of cellular IP content. These results suggest that increased abundance of Galpha 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 Galpha q, rendering it capable of activating the effector enzyme. Co-expression of RGS4 with Galpha 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 Galpha q·GDP·AlF4-. RGS4 also decreased the elevation of MAPK activity seen in untreated cells transfected with Galpha q (not shown), an effect that probably reflects acceleration of GTP hydrolysis by Galpha q.


Fig. 5. RGS4 inhibits IP accumulation induced by AlF4- acting on Galpha q/11. Cells were transfected with 1 µg of Galpha q DNA (+) or pcDNAI (-) and 2 µg of RGS4 (+) or pCR3 (-) and labeled with myo-[3H]inositol. 48 h after transfection, cells were incubated in medium with (+) or without (-) 30 µM AlCl3 and 10 mM NaF for 30 min before determination of cellular IP content. Data represent the mean ± S.D. of triplicate determinations; two additional experiments gave similar results.
[View Larger Version of this Image (33K GIF file)]

In summary, we present two new sets of observations. While RGS proteins are known to inhibit signals mediated by Gi, we show for the first time that an RGS protein can interact with Galpha q/11 and inhibit signals transduced by Galpha q/11 in intact cells. RGS4 probably inhibits bombesin responses by acting as a GAP, that is, by stimulating the intrinsic GTPase activity of Galpha q/11. Our experiments also raise the possibility that RGS4 inhibits bombesin responses by sequestering the GTP-bound active conformation of Galpha 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 Gi or Gq/11. It is likely that other RGS proteins modulate hormonal signals mediated by Gi and Gq/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 Gq and Gs or to Gi and Gq), differing cellular complements of RGS proteins with distinct Galpha selectivities could even produce qualitatively different responses of two cells to the same agonist.


FOOTNOTES

*   This work was supported by National Institutes of Health Grants CA54427 and GM27800 (to H. R. B.), NIH National Research Service Award Postdoctoral Fellowship GM17533 (to Y. Y.), and HHMI Research Training Fellowship for Medical Students (to P. P. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed. Tel.: 415-476-8161; Fax: 415-476-5292; E-mail: h_bourne{at}quickmail.ucsf.edu.
1   The abbreviations used are: GAP, GTPase-activating protein; PLC, phospholipase C; G protein, heterotrimeric guanine nucleotide-binding protein; RGS, regulator of G protein signaling; MAPK, mitogen-activated protein kinase; BR, bombesin receptor; D2R, D2 dopamine receptor; PTX, pertussis toxin; MBP, myelin basic protein; IP, inositol phosphates; InsP3, inositol 1,4,5-trisphosphate; HA, hemagglutinin.
2   P. P. Chi, unpublished result.

ACKNOWLEDGEMENTS

We thank Tom Baranski, Simon Fishburn, Pablo Garcia, Paul Herzmark, Taroh Iiri, Janine Morales, Enid Neptune, and Soren Sheikh for valuable discussions.


REFERENCES

  1. Bourne, H. R., Sanders, D. A., and McCormick, F. (1990) Nature 348, 125-132 [Medline] [Order article via Infotrieve]
  2. Gilman, A. G. (1987) Annu. Rev. Biochem. 56, 615-649 [CrossRef][Medline] [Order article via Infotrieve]
  3. Berstein, G., Blank, J. L., Jhon, D. Y., Exton, J. H., Rhee, S. G., and Ross, E. M. (1992) Cell 70, 411-418 [Medline] [Order article via Infotrieve]
  4. Arshavsky, V. Y., and Bownds, M. D. (1992) Nature 357, 416-417 [Medline] [Order article via Infotrieve]
  5. Berman, D. M., Wilkie, T. M., and Gilman, A. G. (1996) Cell 86, 445-452 [Medline] [Order article via Infotrieve]
  6. Berman, D. M., Kozasa, T., and Gilman, A. G. (1996) J. Biol. Chem. 271, 27209-27212 [Abstract/Free Full Text]
  7. Watson, N., Linder, M. E., Druey, K. M., Kehrl, J. H., and Blumer, K. J. (1996) Nature 383, 172-175 [Medline] [Order article via Infotrieve]
  8. Hunt, T. W., Fields, T. A., Casey, P. J., and Peralta, E. G. (1996) Nature 383, 175-177 [Medline] [Order article via Infotrieve]
  9. Chen, C.-K., Wieland, T., and Simon, M. I. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 12885-12889 [Abstract/Free Full Text]
  10. De Vries, L., Mousli, M., Wurmser, A., and Farquhar, M. G. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 11916-11920 [Abstract]
  11. Koelle, M. R., and Horvitz, H. R. (1996) Cell 84, 115-125 [Medline] [Order article via Infotrieve]
  12. Druey, K. M., Blumer, K. J., Kang, V. H., and Kehrl, J. H. (1996) Nature 379, 742-746 [Medline] [Order article via Infotrieve]
  13. Dohlman, H. G., Apaniesk, D., Chen, Y., Song, J., and Nusskern, D. (1995) Mol. Cell. Biol. 15, 3635-3643 [Abstract]
  14. Dohlman, H. G., Song, J., Ma, D., Courchesne, W. E., and Thorner, J. (1996) Mol. Cell. Biol. 16, 5194-5209 [Abstract]
  15. Hepler, J. R., Berman, D. M., Gilman, A. G., and Kozasa, T. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 428-432 [Abstract/Free Full Text]
  16. Lustig, K. D., Conklin, B. R., Herzmark, P., Taussig, R., and Bourne, H. R. (1993) J. Biol. Chem. 268, 13900-13905 [Abstract/Free Full Text]
  17. Forsayeth, J. R., and Garcia, P. D. (1994) BioTechniques 17, 354-358 [Medline] [Order article via Infotrieve]
  18. Faure, M., Voyno-Yasenetskaya, T. A., and Bourne, H. R. (1994) J. Biol. Chem. 269, 7851-7854 [Abstract/Free Full Text]
  19. Conklin, B. R., Chabre, O., Wong, Y. H., Federman, A. D., and Bourne, H. R. (1992) J. Biol. Chem. 267, 31-34 [Abstract/Free Full Text]
  20. Biddlecome, G. H., Berstein, G., and Ross, E. M. (1996) J. Biol. Chem. 271, 7999-8007 [Abstract/Free Full Text]
  21. Jiang, H., Kuang, Y., Wu, Y., Smrcka, A., Simon, M. I., and Wu, D. (1996) J. Biol. Chem. 271, 13430-13434 [Abstract/Free Full Text]

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.