©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Distinct Pathways of G- and G-mediated Mitogen-activated Protein Kinase Activation (*)

(Received for publication, April 3, 1995)

Brian E. Hawes (§) , Tim van Biesen(§)(¶) , Walter J. Koch (**) , Louis M. Luttrell , Robert J. Lefkowitz (§§)

From the Howard Hughes Medical Institute, Departments of Medicine (Cardiology) and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Receptors that couple to the heterotrimeric G proteins, G or G, can stimulate phosphoinositide (PI) hydrolysis and mitogen-activated protein kinase (MAPK) activation. PI hydrolysis produces inositol 1,4,5-trisphosphate and diacylglycerol, leading to activation of protein kinase C (PKC), which can stimulate increased MAPK activity. However, the relationship between PI hydrolysis and MAPK activation in G and G signaling has not been clearly defined and is the subject of this study. The effects of several signaling inhibitors are assessed including expression of a peptide derived from the carboxyl terminus of the adrenergic receptor kinase 1 (ARKct), which specifically blocks signaling mediated by the subunits of G proteins (G), expression of dominant negative mutants of p21 (RasN17) and p74 (NRaf), protein-tyrosine kinase (PTK) inhibitors and cellular depletion of PKC. The G-coupled 2A adrenergic receptor (AR) stimulates MAPK activation which is blocked by expression of ARKct, RasN17, or NRaf, or by PTK inhibitors, but unaffected by cellular depletion of PKC. In contrast, MAPK activation stimulated by the G-coupled 1B AR or M1 muscarinic cholinergic receptor is unaffected by expression of ARKct or RasN17 expression or by PTK inhibitors, but is blocked by expression of NRaf or by PKC depletion. These data demonstrate that G- and G-coupled receptors stimulate MAPK activation via distinct signaling pathways. G is responsible for mediating G-coupled receptor-stimulated MAPK activation through a mechanism utilizing p21 and p74 independent of PKC. In contrast, G mediates G-coupled receptor-stimulated MAPK activation using a p21-independent mechanism employing PKC and p74. To define the role of G in G-coupled receptor-mediated PI hydrolysis and MAPK activation, direct stimulation with G was used. Expression of G resulted in MAPK activation that was sensitive to inhibition by expression of ARKct, RasN17, or NRaf or by PTK inhibitors, but insensitive to PKC depletion. By comparison, G-mediated PI hydrolysis was not affected by ARKct, RasN17, or NRaf expression or by PTK inhibitors. Together, these results demonstrate that G mediates MAPK activation and PI hydrolysis via independent signaling pathways.


INTRODUCTION

The ubiquitous mitogen-activated protein kinases (MAPK)()represent a point of convergence for mitogenic signals originating from several distinct classes of cell surface receptor. Activation of these kinases may be achieved via several pathways. Tyrosine phosphorylation of growth factor receptors (e.g. the epidermal growth factor receptor) leads to the formation of a membrane-associated multi-protein complex(1, 2, 3) , which catalyzes GTP exchange on and activation of the low molecular weight GTP-binding protein, p21(4) . Activation of p21results in the membrane association and activation of the serine/threonine protein kinase p74, which initiates a protein phosphorylation cascade culminating in the phosphorylation and activation of MAPK(5, 6, 7, 8, 9) . Alternatively, activators of PKC (e.g. phorbol esters) have been reported to stimulate MAPK activation (10, 11, 12) through both p21-dependent and independent mechanisms(13, 14, 15) .

Recently, a number of receptors that couple to heterotrimeric G proteins have been shown to stimulate MAPK activation(16, 17, 18) , including both receptors that couple to G and to G. Activation of G protein-coupled receptors catalyzes exchange of GTP for GDP on the G protein subunit (G) leading to complex dissociation and release of free G-GTP and G subunits (19, 20) , each of which may then interact with effector molecules. Once released, G subunits modulate numerous effectors, including adenylate cyclases, phospholipase C (PLC) isoforms, and ion channels (21) . G subunits activate isoforms of adenylate cyclase(22, 23) and PLC(24, 25, 26, 27, 28, 29) , phospholipase A2(30) , potassium channels(31) , and phosphatidylinositol 3`-kinase(32, 33) . In addition, G subunits mediate translocation of -adrenergic receptor kinases (ARK) from the cytosol to their membrane receptor substrates(34) .

Given the diversity of effector molecules regulated by G protein-coupled receptors, it is possible that MAPK activation by this class of receptor can proceed by multiple pathways. Such diversity is suggested by the observation that cellular expression of G subunits or constitutively activated G subunits, but not constitutively activated G subunits, leads to MAPK activation(18) . The G-mediated pathway may utilize PLC-dependent PKC activation, whereas the mechanism of G-mediated p21 and MAPK activation (16, 17, 18) has not been clearly defined.

Activation of either G- or G-coupled receptors releases both free G and G subunits, either of which might initiate a sequence of events leading to MAPK activation. The relative contributions of G subunit-mediated p21activation versus G- or G-mediated PLC activation to the stimulation of MAPK by these receptors remains in question. This study compares the mechanisms of MAPK activation employed by G- and G-coupled receptors using inhibitors of cellular signal transduction (e.g. a G subunit-sequestrant peptide, dominant negative mutants of p21 and p74, protein-tyrosine kinase inhibitors, and cellular depletion of PKC) in a transfected cell system. We find that G-coupled receptor-mediated MAPK activation is predominantly G-mediated, PKC-dependent, and p21-independent and cannot be dissociated from the ability of these receptors to activate PLC, whereas the G-coupled receptor pathway is G subunit-mediated, p21-dependent, PKC-independent, and dissociable from PLC activation.


EXPERIMENTAL PROCEDURES

Materials

COS-7 and CHO cells were from the American Type Culture Collection. Culture media and LipofectAMINE® were from Gibco-BRL. Fetal bovine serum and gentamicin were from Life Technologies, Inc. Monoclonal antibody 12CA5 was from Boehringer Mannheim. Protein A-agarose was from Pharmacia Biotech Inc. Dowex AG1-X8 was from Bio-Rad. Econofluor, myo-[H]inositol, and [-P]ATP were from DuPont NEN. Herbimycin A, genistein, myelin basic protein (MBP), carbachol,(-)-epinephrine, and phorbol 12-myristate 13-acetate (PMA) were from Sigma. UK-14304 was from Pfizer. Dowex AG1-X8 was from Bio-Rad. Pertussis toxin was from List Biologicals.

DNA Constructs

The cDNAs for the human 2-C10 AR and ARK1 were cloned in our laboratory(35) . Human M1 AChR cDNA was provided by E. Peralta(36) ; cDNAs encoding G1, G2, G3, G4, G1, G2, and G3 were provided by M. Simon; DNA encoding hemagglutinin (HA)-tagged p44 (Erk1) was from J. Pouysségur; DNA encoding the p21dominant negative mutant was from D. Altschuler and M. Ostrowski; and DNA encoding the p74 dominant negative mutant (NRaf) was from L. T. Williams. The ARKct peptide-encoding minigene, containing cDNA encoding the carboxyl-terminal 195 amino acids of ARK1, was prepared as described previously(28) .

Cell Culture and Transfection

COS-7 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS) and 50 µg/ml gentamicin. CHO cells were maintained in F-12 medium supplemented with 10% FBS and 50 µg/ml gentamicin. For transient transfection using LipofectAMINE, cells at 80% confluence in six-well culture plates were washed once with serum-free medium and incubated for 3-5 h at 37 °C in 1.0 ml of serum-free medium containing the indicated amounts of plasmid DNA and 6.0 µl of LipofectAMINE/µg of DNA. The transfection mixture was then replaced with 2.0 ml of growth medium and the cells maintained for 24 h. Prior to assay, transfected cells were incubated overnight in medium containing 0.5% FBS or medium containing 10% FBS plus 2 µCi/ml [H]inositol for experiments measuring MAPK activity or IP production, respectively.

Measurement of MAPK Activity

Activity of epitope-tagged p44 (Erk1) was determined following immunoprecipitation, using MBP as substrate(37) . Briefly, serum-starved transfected cells were stimulated for 5 min with the indicated agonist, lysed in 200 µl of cold lysis buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 1% (v/v) Nonidet P-40, 0.5% (w/v) sodium deoxycholate, 0.1% SDS, 10 mM NaF, 10 mM sodium pyrophosphate, 0.1 mM phenylmethylsulfonylfluoride) and clarified by centrifugation (15 min, 16000 g). Supernatants were transferred to tubes containing 6.5 µg of anti-HA 12CA5 antibody and 25 µl of a 50% slurry of protein A-agarose beads to immunoprecipitate p44 (rotated 1 h at 4C). Immune complexes were washed twice with cold lysis buffer and twice with kinase buffer (20 mM HEPES, pH 7.4, 10 mM MgCl, 1 mM dithiothreitol). Phosphorylation of MBP was performed in 40 µl of kinase buffer containing 250 µg/ml MBP, 20 µM ATP, and 4 µCi/ml [-P]ATP at room temperature for 30 min. Reactions were terminated by the addition of 2 Laemmli sample buffer and P-labeled MBP resolved by SDS-polyacrylamide gel electrophoresis. Quantitation of labeled MBP was performed using a Molecular Dynamics PhosphorImager.

Measurement of IP Production

Transfected cells, labeled overnight with [H]inositol, were washed once with phosphate-buffered saline, incubated in phosphate-buffered saline containing 10 mM LiCl and 1.0 mM CaCl, and stimulated as indicated for 45-60 min. Cells were lysed by addition of 0.8 ml of 0.4 M perchloric acid (1.0 ml/well), and lysates were neutralized by addition of 0.4 ml of 0.72 M KOH, 0.6 M KHCO. Total IP accumulation was determined by Dowex anion exchange chromatography and liquid scintillation spectroscopy.


RESULTS

Stimulation of IP Production and MAPK Activity by G- and G-coupled Receptors

To determine whether diversity exists in the mechanisms of activation of MAPK employed by G- and G-coupled receptors, we studied the effects of the G subunit-sequestrant ARKct peptide, dominant negative mutants of p21 and p74, cellular depletion of PKC, and inhibitors of protein-tyrosine kinases on MAPK activation mediated by each class of receptor. As shown in Fig. 1, both PI hydrolysis and MAPK activation stimulated by the G-coupled 2A AR are sensitive to pertussis toxin (PTX), while PI hydrolysis and MAPK activity stimulated by the G-coupled 1B AR and M1AChR are PTX-insensitive. Since expression of ARKct selectively inhibits G-mediated signal transduction, this peptide can be utilized to distinguish G and G signaling pathways(28) . Coexpression of ARKct blocks 2A AR-stimulated PI hydrolysis and MAPK activation but does not affect 1B AR- or M1AChR-mediated signaling (Fig. 1). These results suggest that G mediates both G-coupled receptor-stimulated PI hydrolysis and MAPK activation. In contrast, G-coupled receptor-mediated PI hydrolysis and MAPK activation are ARKct-insensitive, suggesting that these effects are mediated by the G subunits.


Figure 1: Effect of ARKct peptide expression and PTX treatment on G- and G-coupled receptor-stimulated PI hydrolysis and MAPK activation. COS-7 cells were transiently transfected with plasmid DNA encoding 2A AR (0.2 µg/well), 1B AR (0.3 µg/well), or M1AChR (0.3 µg/well) as described. In experiments in which MAPK activation was measured, cells were cotransfected with plasmid DNA encoding p44 (0.1 µg/well). Where indicated, cells were preincubated overnight with PTX (100 ng/ml) prior to assay. Cells were stimulated with 1.0 µM UK-14304 (2A AR), 100 µM epinephrine (1B AR), or 1.0 mM carbachol (M1AChR) for 45 min prior to determination of IP production (A) or for 5 min prior to determination of MAPK activity (B). Data are expressed as a percent of total inositol phosphates or MAPK activity produced by agonist stimulation in control (vehicle pretreated) cells. -Fold IP production stimulated by agonist for each receptor under control conditions was 3.0-fold for 2A AR, 3.3-fold for 1B AR, and 9.6-fold for M1AChR. -Fold MAPK activity stimulated by agonist for each receptor under control conditions was 7.4-fold for 2A AR, 3.2-fold for 1B AR, and 3.0-fold for M1AChR. Values shown represent the mean ± S.E. from at least three separate experiments. Openbar, control; filledbar, PTX;shadedbar, ARKct.



G-coupled receptor-mediated MAPK activation requires p21 and p74 activity(38, 39, 40) . In order to compare the roles of p21and p74 in G- and G-coupled receptor mediated MAPK activation, the effects of the dominant negative mutants RasN17 and NRaf on receptor-stimulated MAPK activation were assessed. As shown in Fig. 2A, expression of RasN17 or NRaf inhibits 2A AR-stimulated MAPK activation. In contrast, MAPK activation via the G-coupled M1AChR is not affected by RasN17 expression but is sensitive to inhibition by NRaf. Results similar to the M1AChR were obtained with the G-coupled 1B AR (data not shown).


Figure 2: Effect of RasN17 and NRaf expression or PKC depletion on G- and G-coupled receptor-mediated MAPK activation. COS-7 cells (A) were cotransfected with plasmid DNA encoding p44 (0.1 µg/well), 2A AR (0.2 µg/well), or M1AChR (0.3 µg/well) plus either vector plasmid alone (openbars) or plasmid DNA encoding RasN17 (darkshadedbars) or NRaf (2.0 µg/well; lightshadedbars). Cells were stimulated with or without agonist for 5 min and MAPK activity determined as described. CHO cells (B) were cotransfected with plasmid DNA encoding p44 (0.1 µg/well) plus either 2A AR (0.2 µg/well), 1B AR (0.3 µg/well), G1 and G2 subunits (0.5 µg each/well), or empty plasmid vector. Cells were pretreated with (filledbar) or without (openbar) 1.0 µM PMA for 24 h prior to stimulation for 5 min with or without agonist or 1.0 µM PMA and MAPK activity was measured. Data are expressed as -fold MAPK activation in which the MAPK activity produced in unstimulated cells was defined as 1.0. Values shown represent mean ± S.E. from four separate experiments.



G- and G-mediated MAPK activation are also distinguished by differences in dependence upon PKC. Fig. 2B depicts the effects of cellular PKC depletion in transiently transfected CHO cells. Following prolonged exposure to PMA, further stimulation with phorbol ester fails to provoke MAPK activation, demonstrating that the cells are functionally depleted of PKC activity. The G-coupled 2A AR fully activates MAPK in PKC-depleted cells, but the G-coupled 1B AR response is abolished, as is M1AChR-stimulated MAPK activation (data not shown). These data suggest that the signaling pathway utilized by G-coupled receptors involves G, p21, and p74 and is independent of PKC, whereas the G-coupled receptor pathway involves G and PKC but is independent of p21. The two pathways apparently converge downstream of p21, since both are inhibited by expression of NRaf.

A role for PTKs in G-coupled receptor-mediated MAPK activation has been proposed(41, 42) . As shown in Fig. 3A, MAPK activation mediated by the 2A AR, but not the M1 AChR, is inhibited by pretreatment with the PTK inhibitors genistein or herbimycin A. By comparison, PI hydrolysis stimulated by either G- or G-coupled receptors is unaffected by pretreatment with PTK inhibitors. These data suggest a role for protein-tyrosine phosphorylation in MAPK activation mediated by G-coupled receptors, but not by G-coupled receptors. These results also suggest that G-mediated MAPK activation can be dissociated from PI hydrolysis.


Figure 3: Effect of protein-tyrosine kinase inhibitors on G and G-stimulated PI hydrolysis and MAPK activation. COS-7 cells were transfected with plasmid DNA encoding 2A AR (0.2 µg/well), or M1AChR (0.3 µg/well). In experiments in which MAPK activation was measured, cells were cotransfected with plasmid DNA encoding p44 (0.1 µg/well). Cells were pretreated for 20 min with vehicle, genistein (100 µM), or herbimycin A (1.0 µg/ml) and stimulated for 5 min prior to determination of MAPK activity (A) or for 45 min prior to measurement of IP production (B). Data are expressed as the percent of IP production or MAPK activity produced by agonist stimulation in control (vehicle-pretreated) cells. -Fold MAPK activity stimulated by agonist for each receptor under control conditions was 8.5-fold for 2A AR and 3.8-fold for M1AChR. -Fold IP production stimulated by agonist for each receptor under control conditions was 3.0-fold for 2A AR and 9.5-fold for M1AChR. Values shown represent mean ± S.E. from at least three separate experiments. Openbar, control; filledbar, genistein; shadedbar, herbimycin A.



Stimulation of PI Hydrolysis and MAPK Activity by Cellular Expression of G Subunits

In order to clarify the role of G subunits in G-coupled receptor-mediated PI hydrolysis and MAPK activation, we studied the effects of direct G expression on both processes. As shown in Fig. 4, expression of G1 alone in COS-7 cells provokes a small increase in MAPK activity, presumably due to an association with endogenous G subunits. Coexpression of combinations of G11, G12, G13, G22, and G23 subunits each cause a 6-10-fold increase in basal MAPK activation compared to control cells. Transfection with plasmids encoding G2 and G1 subunits, and all the combinations using G3 or G4 subunits, fails to activate MAPK beyond the basal level exhibited in cells transfected with G1 alone. The combinations of G and G that stimulate MAPK activity also promote a 3-4-fold increase in PI hydrolysis compared to control cells. Thus, G and G subunit combinations known to form G complexes when coexpressed in cellular systems (43, 44, 45, 46) (G11, G12, G13, G22, and G23) all stimulate both MAPK activation and PI hydrolysis, while those combinations which are incapable of forming complexes (G21, G31, and G32) do not produce activity greater than that observed by transfection of G1 alone.


Figure 4: Stimulation of MAPK activation and IP production by coexpression of various combinations of G and G subunits. COS-7 cells were transfected with pRK5 vector (Control), or plasmid DNA encoding the indicated combinations of G and G subunits (1.0 µg each/well). For determination of MAPK activity, cells were cotransfected with plasmid DNA encoding p44 (0.1 µg/well). The ability of each combination of G subunit to stimulate MAPK activation (A) and IP production (B) was determined as described. Data are presented as -fold stimulation of MAPK activity or IP production normalized to the value obtained in cells transfected with vector alone. Values shown represent the mean ± S.E. from three separate experiments.



As observed with G-coupled receptor-mediated signaling, G-stimulated MAPK activation and PI hydrolysis appear to be independent processes. Although ARKct expression blocks both MAPK activation and IP accumulation stimulated by G12, coexpression of either RasN17 or NRaf abolishes G-mediated MAPK activation, without affecting G-mediated IP production (Fig. 5). In addition, the PTK inhibitors genistein and herbimycin A attenuate G-stimulated MAPK activation in a dose-dependent manner, with no effect on G-stimulated IP production (Fig. 6). Moreover, as shown in Fig. 2B, G12-mediated MAPK activation is unaffected by PKC depletion of CHO cells.


Figure 5: Differential effects of ARKct, RasN17, and NRaf expression on G-stimulated MAPK activation and IP production. COS-7 cells were transfected with plasmid DNA encoding G1 and G2 subunits (1.0 µg/well each), plus either pRK5 vector, ARKct, RasN17, or NRaf (2.0 µg/well). Where indicated, p44 (0.1 µg/well) was cotransfected for determination of MAPK activity. The effect of ARKct, RasN17, or NRaf expression on G-mediated MAPK activation (A) and IP production (B) were determined as described. Data are presented as -fold stimulation of MAPK activity or IP production normalized to the value obtained in cells transfected with vector alone. Values shown represent the mean ± S.E. from four separate experiments.




Figure 6: Effect of the protein-tyrosine kinase inhibitors genistein and herbimycin A on G-stimulated MAPK activation and IP production. COS-7 cells were cotransfected with pRK5 vector or plasmid DNA encoding G1 and G2 (1.0 µg each/well). Where indicated, p44 (0.1 µg/well) was cotransfected for determination of MAPK activity. Cells were pretreated for 2 h with the indicated concentration of genistein or herbimycin A, and MAPK activity (left panel) and IP production (right panel) were determined as described. The data are presented as percent of G subunit-stimulated MAPK activity or IP production in cells not exposed to genistein or herbimycin A. Absolute values for G-stimulated signaling under control conditions were 4.8-fold for MAPK activation and 4.8-fold for IP production. The values represent the mean ± S.E. from at least four separate experiments. Open bar, control; bar with thin diagonal stripes, 10 µM genistein; lightly shaded bar, 50 µM genistein; dark shaded bar, 100 µM genistein; bar with thick diagonal stripes, 200 µM genistein; solid bar, 1.0 µg/ml herbimycin.




DISCUSSION

Collectively, these results suggest that the release of G subunits is responsible for mediation of G-coupled receptor-stimulated PI hydrolysis and MAPK activation. G likely stimulates increased IP accumulation by directly activating a G-sensitive PLC(24, 25, 26, 27, 28, 29) . Although G stimulates both MAPK activation and PI hydrolysis, the sensitivity of G-mediated MAPK activation to RasN17, NRaf, and PTK inhibitors, as well as its insensitivity to PKC depletion, indicate that G-mediated PI hydrolysis and MAPK activation represent independent signaling pathways.

Previous studies of the G protein subunit responsible for mediating G-coupled receptor-stimulated MAPK activation have produced contradictory results(16, 17, 18) . One study found that expression of the G subunit of transducin (T), which like the ARKct peptide sequesters free G subunits, inhibited M1AChR-stimulated MAPK activation(17) , suggesting that G-coupled receptor-mediated MAPK activation was mediated by G subunits. Another study reported that neither M1AChR- nor bombesin receptor-stimulated MAPK activation was sensitive to inhibition by T subunits(18) . The present results strongly support the hypothesis that multiple mechanisms exist for G protein-coupled receptor-mediated MAPK activation. The data demonstrate that G- and G-coupled receptor-mediated MAPK activation can be distinguished by several characteristics. (i) Expression of the G subunit-sequestrant ARKct peptide blocks MAPK activation stimulated by receptors coupled to G but not to G; (ii) G-coupled receptor mediated MAPK activation is abolished by PKC depletion, while G-coupled receptor mediated MAPK activation is unaffected; (iii) expression of the dominant negative mutant RasN17 inhibits G- but not G-mediated MAPK activation; and (iv) the PTK inhibitors genistein and herbimycin A attenuate G-mediated, but not G-mediated MAPK activation.

The effects of PTK inhibitors on G-coupled receptor- and G subunit-mediated MAPK activation suggest a role for a protein-tyrosine kinase in the G-mediated signaling pathway. We have found that MAPK activation resulting from expression of a constitutively activated p21 mutant, p21(47) , is insensitive to PTK inhibitors, indicating that the inhibitor-sensitive step lies upstream of p21.() Genistein has been reported to inhibit several classes of protein-tyrosine kinases including epidermal growth factor receptor, pp60, and pp110(48) , while herbimycin A demonstrates more selective inhibition of Src family kinases (e.g.Src, Yes, Fbs, Ros, Abl, and ErbB)(49) . Since both genistein and herbimycin A inhibit G subunit-mediated MAPK activation, we speculate that a tyrosine phosphorylation, possibly mediated by a member of the Src family, is required for G-mediated p21 activation. If so, the pathways of MAPK activation utilized by classical tyrosine kinase growth factor receptors and G may converge at a very early point.

It is likely that other signaling pathways capable of mediating MAPK activation and mitogenesis via G protein-coupled receptors exist. A distinct signaling mechanism may be mediated by the G protein-coupled receptor for platelet-activating factor (PAF). PAF stimulates both PI hydrolysis and MAPK activation, but does not activate p21 in CHO cells(50) . PAF-stimulated MAPK activation, but not PI hydrolysis, is inhibited by PTX, suggesting that the PAF receptor activates MAPK via a signaling pathway that utilizes a PTX-sensitive G protein and is independent of both PI hydrolysis and p21. It has also been reported that PKC, which is activated in response to numerous G protein-coupled receptors, provokes MAPK activation via both p21-dependent and independent mechanisms (13, 14, 15) . These data suggest that considerable heterogeneity exists in the signaling pathways employed by G proteincoupled receptors leading to MAPK activation. The mechanism utilized by a given receptor to stimulate MAPK activation is likely dependent on the class of G protein to which the receptor can couple and on the signaling machinery available in a given cell type.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant HL 16037. 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.

§
These authors contributed equally to this work.

Recipient of a postdoctoral award from the Alberta Heritage Foundation for Medical Research.

**
Current address: Dept. of Surgery, Duke University Medical Center, Durham, NC 27710.

§§
To whom correspondence and requests for reprints should be addressed: Howard Hughes Medical Institute, Depts. of Medicine (Cardiology) and Biochemistry, Duke University Medical Center, Box 3821, Durham, NC 27710. Tel.: 919-684-2974; Fax: 919-684-8875.

The abbreviations used are: MAPK, mitogen-activated protein kinase; PI, phosphoinositide; AR, adrenergic receptor; AChR, cholinergic receptor; ARK, the adrenergic receptor kinase; ARKct, the carboxyl-terminal peptide of ARK; G proteins, GTP-binding proteins; G and G, the and the subunits, respectively, of G proteins; PKC, Ca-dependent protein kinase; PLC, phospholipase C; PTK, protein-tyrosine kinase; RasN17, a dominant negative mutant of p21; NRaf, a dominant negative mutant of p74; PAF, platelet-activating factor; PMA, phorbol 12-myristate 13-acetate; MBP, myelin basic protein; CHO, Chinese hamster ovary; IP, inositol phosphate; PTX, pertussis toxin; FBS, fetal bovine serum.

B. E. Hawes, T. van Biesen, and R. J. Lefkowitz, unpublished observations.


ACKNOWLEDGEMENTS

We thank Dr. Kazushige Touhara for helpful discussion, S. Exum for technical assistance, and D. Addison and M. Holben for excellent secretarial services.


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