©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Effect of Cellular Expression of Pleckstrin Homology Domains on G-coupled Receptor Signaling(*)

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

From the (1) Howard Hughes Medical Institute and the Departments of Medicine and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Pleckstrin homology (PH) domains are 90-110 amino acid regions of protein sequence homology that are found in a variety of proteins involved in signal transduction and growth control. We have previously reported that the PH domains of several proteins, including ARK1, PLC, IRS-1, Ras-GRF, and Ras-GAP, expressed as glutathione S-transferase fusion proteins, can reversibly bind purified bovine brain G subunits in vitro with varying affinity. To determine whether PH domain peptides would behave as antagonists of G subunit-mediated signal transduction in intact cells, plasmid minigene constructs encoding these PH domains were prepared, which permit transient cellular expression of the peptides. Pertussis toxin-sensitive, G subunit-mediated inositol phosphate (IP) production was significantly inhibited in COS-7 cells transiently coexpressing the 2-C10 adrenergic receptor (AR) and each of the PH domain peptides. Pertussis toxin-insensitive, G subunit-mediated IP production via coexpressed M1 muscarinic acetylcholine receptor (M1 AChR) was attenuated only by the PLC PH domain peptide, suggesting that the inhibitory effect of most of the PH domain peptides was G subunit-specific. Stimulation of the mitogen-activated protein (MAP) kinase pathway by G-coupled receptors in COS-7 cells has been reported to require activation of p21 and to be independent of protein kinase C. Since several proteins involved in activation contain PH domains, the effect of PH domain peptide expression on 2-C10 AR-mediated p21 -GTP exchange and MAP kinase activation as well as direct G subunit-mediated activation of MAP kinase was determined. In each assay, coexpression of the PH domain peptides resulted in significant inhibition. Increasing G subunit expression surmounted PH domain peptide-mediated inhibition of MAP kinase activation. These data suggest that the PH domain peptides behave as specific antagonists of G-mediated signaling in intact cells and that interactions between PH domains and G subunits or structurally related proteins may play a role in the activation of mitogenic signaling pathways by G protein-coupled receptors.


INTRODUCTION

Pleckstrin homology (PH)() domains, so named because of their original description as internal repeats in pleckstrin, the major protein kinase C substrate in platelets (1, 2) , are 90-110 amino acid regions of protein sequence homology that are found in a variety of proteins involved in signal transduction and growth control. Included in the list of approximately 70 known PH domain-containing proteins are several that participate in the function of Ras including Ras-GRF, Sos1, and Ras-GAP, the growth factor-binding protein Grb7, the insulin receptor substrate IRS-1, serine/threonine kinases including Rac-, Rac-, and the -adrenergic receptor kinases ARK1 and ARK2, tyrosine kinases including the Bruton tyrosine kinase (Btk), Tec, and TSK, phospholipase C species including PLC and PLC, the cytoskeletal protein spectrin, the microtubule-binding GTPase dynamin, and oxysterol binding protein (3-5). The three-dimensional structures of the pleckstrin (6) , spectrin (7) , and dynamin (8, 9) PH domains demonstrate that they consist of seven antiparallel -sheets and a carboxyl-terminal amphiphilic -helix. The three structures are essentially superimposable, indicating that despite relatively low primary sequence homology, PH domains apparently represent a protein domain of defined tertiary structure.

The function of PH domains is unknown. A possible clue to their function comes from the localization of a PH domain to the carboxyl terminus of ARK1 and ARK2, where it corresponds approximately to that region of the protein which functions as a specific recognition domain for the G subunits of heterotrimeric G proteins. With ARK, this region is responsible for agonist-induced translocation of the kinase from the cytosol to the plasma membrane, where it initiates the process of homologous desensitization (10, 11, 12) . We have previously shown that the PH domains of several other proteins, expressed as glutathione S-transferase fusion proteins, can reversibly bind bovine brain G subunits in vitro with varying affinity (13) . This observation, coupled with recent reports that Ras-dependent activation of MAP kinase by those G protein-coupled receptors that utilize pertussis toxin-sensitive G proteins is mediated by G subunits (14, 15, 16) , suggests that the PH domains of other proteins may function as recognition domains for G subunits in the intact cell.

To determine whether PH domains can function as G subunit-binding domains in the intact cell, we have prepared a series of plasmid minigene constructs that permit transient cellular expression of PH domain-containing peptides. We tested whether expression of peptides comprising the PH domains of ARK1, IRS-1, PLC, Ras-GAP and Ras-GRF could specifically antagonize G-coupled receptor-mediated PLC activation in transiently transfected COS-7 cells, i.e. whether the peptides could act as free G subunit scavengers within the cell. We also assessed whether PH domain peptide expression could antagonize G-coupled receptor-mediated Ras-GTP exchange and MAP kinase activation as well as the activation of MAP kinase that resulted directly from overexpression of G subunits. Our data suggest that the PH domains of several proteins can function as G subunit-binding domains in the intact cell and that PH domain-directed association of these proteins with G subunits or structurally related proteins may play an important role in the regulation of cellular mitogenesis.


EXPERIMENTAL PROCEDURES

Materials

The cDNA for the human 2-C10 AR was cloned in our laboratory (17) . The cDNA for the human M1 AChR (18) was provided by Dr. Ernest Peralta. The cDNAs encoding G1 (19) and G2 (20) were provided by Dr. Mel Simon. The cDNA encoding hemagglutinin-tagged p44 (erk1) (p44 ) (21) was provided by Dr. Jacques Pouyssegur. The sources of cDNA fragments encoding the PH domains of bovine ARK1, rat IRS-1, rat PLC, bovine Ras-GAP, and rat Ras-GRF were as described previously (13) . LipofectAMINE was obtained from Life Technologies, Inc. Rabbit anti-ARK carboxyl terminus was prepared in our laboratory (11) . Rabbit polyclonal anti-IRS-1 was from Transduction Laboratories (Lexington, KY). Mixed monoclonal anti-GAP and anti-PLC were from Upstate Biotechnology Incorporated (Lake Placid, NY). Rabbit polyclonal anti-erk2 (C14) agarose conjugate was from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal antibody 12CA5 was from Boehringer Mannheim. Monoclonal anti-p21 antibody Y13-259, Protein A-agarose, and Protein G plus-agarose were from Oncogene Science (Cambridge, MA). Protein G Sepharose 4 Fast Flow was from Pharmacia Biotech Inc. Sources of other reagents were as described previously (14, 22) .

DNA Constructs

The peptide-encoding cDNA fragments spanning the previously described (13) G subunit-binding regions of the PH domains of bovine ARK1 (Gly-Leu), rat IRS-1 (Phe-Glu), rat PLC (Asn-Lys), bovine Ras-GAP (Asp-Asn) and rat Ras-GRF (Phe-Ser), shown schematically in Fig. 1 , were amplified using the polymerase chain reaction to create minigene peptide coding inserts. Expression constructs were prepared by subcloning the amplified peptide coding cassette into the minigene peptide coding region as described previously (22) . The full-length minigene DNA fragments were subcloned into the pRK5 vector for transient eukaryotic cell expression. Receptor cDNAs and cDNAs encoding G1, G2, and p44 were subcloned into pRK5 or pcDNA eukaryotic expression vectors.


Figure 1: Construction of PH domain peptide minigenes. A, the cDNA sequences encoding the indicated regions of ARK1, PLC, IRS-1, Ras-GRF, and Ras-GAP were employed in the construction of PH domain peptide minigenes. Boxedareas represent the relative location of PH domain sequences within the peptide minigene product and the approximate position of the G subunit-binding domains of ARK1, Ras-GRF, and PLC as previously determined (13). PH domain sequences are aligned on the conserved tryptophan residue (W) in subdomain 6 as shown. B, schematic representation of the peptide minigene construct employed for PH domain peptide expression. aa, amino acids.



Cell Culture and Transfection

COS-7 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 100 µg/ml gentamicin at 37 °C in a humidified, 5% CO atmosphere. For transfection using DEAE-dextran, cells were seeded in 6-well tissue culture plates and transfected using a total of 1-2 µg of DNA/well as described previously (22, 23) . For transfection using the LipofectAMINE reagent, cells in 6-well tissue culture plates were incubated at 37 °C with a transfection mixture composed of 1 ml of serum-free DMEM containing 2 µg of DNA/well and 12 µl of LipofectAMINE. After 5 h, 1 ml of DMEM containing 20% fetal bovine serum was added, and the cells were incubated overnight. LipofectAMINE transfection consistently resulted in COS-7 cell transfection efficiencies of greater than 80% (data not shown). Assays were performed 48 h after transfection. Empty pRK5 vector was added to transfections as needed to keep the total mass of DNA added per well constant within an experiment.

Expression of the ARK1 PH domain was determined by protein immunoblotting of whole cell detergent lysates using rabbit anti-ARK1 carboxyl terminus antiserum as described (24) . Cellular expression of the IRS-1, PLC, and Ras-GAP PH domain peptides was documented following metabolic labeling with [S]methionine. Transfected COS-7 cells in 100-mm dishes were incubated overnight in 3 ml of methionine-free medium containing [S]methionine (100 µCi/ml), lysed in 1 ml of RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.25% deoxycholate), and clarified by centrifugation. Supernatants were incubated with anti-IRS-1 (4 µg), anti-PLC (8 µg), or anti-Ras-GAP (8 µg); immune complexes were collected on Protein A-agarose (anti-IRS-1) or Protein G plus-agarose (anti-PLC and anti-Ras-GAP) and resolved by 16% Tricine SDS-polyacrylamide gel electrophoresis. Labeled peptides were visualized by autoradiography. Receptor expression was quantitated by saturation binding analysis as described (22) .

Inositol Phosphate Production

For measurement of total inositol phosphates (IPs), transfected COS-7 cells were labeled for 18-24 h with [H]myo-inositol (2 mCi/ml) in DMEM supplemented with 3% fetal bovine serum. After labeling, cells were washed for 20 min at 37 °C with calcium-free Dulbecco's phosphate-buffered saline, preincubated for 20 min in Dulbecco's phosphate-buffered saline supplemented with 20 mM LiCl and 1 mM CaCl and stimulated for 45 min with or without agonist (10 µM UK-14304 for the 2-C10 AR and 100 µM carbachol for the M1 AChR). Following stimulation, IPs were extracted in 0.4 M perchloric acid (1 ml/well); 0.8 ml of each sample was neutralized with 0.4 ml of 0.72 M KOH, 0.6 M KHCO3; and 1.0 ml of the neutralized supernatant was analyzed for total IPs as described (22) .

Measurement of MAP Kinase Activity

Activation of endogenous p42 (erk2) and epitope-tagged p44 (erk1) was measured using a modification of the procedure of Meloche et al.(21) . Briefly, transfected COS-7 cells in 6-well plates, preincubated overnight in low serum medium (DMEM, 0.5% fetal bovine serum), were stimulated for 5 min with the indicated agonist, washed with ice-cold calcium- and magnesium-free phosphate-buffered-saline, lysed in 200 µl of ice-cold lysis buffer (50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mM NaF, 10 mM sodium pyrophosphate, 0.1 mM phenylmethylsulfonyl fluoride) and clarified by centrifugation. Endogenous p42 was immunoprecipitated using anti-erk2 agarose conjugate (8 µg) plus 25 µl of a 50% slurry of protein A-agarose agitated for 1 h at 4 °C. Coexpressed p44 was immunoprecipitated using 12CA5 antibody (6.5 µg) and 30 µl of a 50% slurry of protein A-agarose. Immune complexes were washed twice with ice-cold lysis buffer and twice more with kinase buffer (20 mM HEPES, pH 7.4, 10 mM MgCl, 1 mM dithiothreitol). The beads were resuspended in 40 µl of kinase buffer containing 250 µg/ml myelin basic protein (MBP), 20 µM ATP, 2.5 µCi of [-P]ATP and incubated for 20 min at 30 °C. The reaction was terminated by the addition of 40 µl of 2 Laemmli sample buffer and P-labeled MBP resolved by SDS-polyacrylamide gel electrophoresis. Phosphorylation was quantitated using a Molecular Dynamics PhosphorImager.

Activation of p21

Transfected COS-7 cells in 6-well plates were serum-starved overnight as described, labeled for 2 h in phosphate-free DMEM containing [P]orthophosphate (200 µCi/ml), and stimulated for 2 min with or without UK-14304. Following agonist stimulation, monolayers were lysed in detergent buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 25 mM MgCl, 1% Nonidet P-40), and p21 was immunoprecipitated using monoclonal antibody Y13-259 with Protein G-Sepharose 4 Fast Flow (75 µl of a 20% slurry in lysis buffer). p21 -bound GDP and GTP were resolved by thin-layer chromatography on polyethylenimine-cellulose plates in 0.75 M KH2PO4 (pH 3.4) as described (14, 25) . Labeled GDP and GTP were quantitated using a Molecular Dynamics PhosphorImager, and data were expressed as the percentage of GTP over total labeled guanine nucleotides.


RESULTS

Effects of PH Domain Peptide Expression on G Subunit-mediated IP Production

In COS-7 cells, transiently expressed 2-C10 AR mediates a Bordetella pertussis toxin-sensitive increase in IP production, which apparently reflects G subunit-mediated activation of a isoform of PLC (24) . In contrast, transiently expressed M1 AChR mediates a pertussis toxin-insensitive increase in IP production that is dependent upon G subunits (26) . We have previously shown that the G-binding ARK1 PH domain (10-12) specifically antagonizes 2-C10 AR-mediated IP production with no effect on that mediated by the M1 AChR (24) in COS-7 cells. G subunit-mediated conditional stimulation of type II adenyl cyclase in vitro(27) as well as in intact (24) and permeabilized (27) transfected cells is also blocked. Thus, it appears that in these cells, the ability to selectively antagonize these G-coupled receptor-mediated events correlates with the ability to sequester free G subunits.

To determine whether PH domain peptides would behave as G-binding peptides in the intact cell, we studied the effects of PH domain minigene expression on both 2-C10 AR- and M1 AChR-mediated IP production in COS-7 cells transiently cotransfected with each minigene and receptor. Fig. 1depicts schematically the PH domain-containing regions of ARK1, Ras-GRF, Ras-GAP, PLC, and IRS-1 employed in the construction of minigenes. Expression of PH domain minigene products was confirmed by immunoprecipitation following [S]methionine labeling of transfected cells (data not shown). Fig. 2depicts the effect of coexpression of each PH domain-containing peptide on 2-C10 AR-mediated IP production in transiently cotransfected COS-7 cells. In control (vector cotransfected) cells, agonist-stimulated IP production increased directly as a function of the level of receptor expression. Coexpression of a minigene encoding the third intracellular (3i) domain of the 1B AR, which antagonizes G-coupled receptor-mediated IP production (22) , had no effect on the 2-C10 AR-mediated response (Fig. 2A). In contrast, coexpression of each of the five PH domain-containing peptides inhibited the 2-C10 AR-mediated response (Fig. 2, B-F), suggesting that each could antagonize G subunit-mediated PLC activation.


Figure 2: Effect of PH domain peptides on 2-C10 AR-mediated IP production. COS-7 cells were transiently cotransfected with plasmid DNA encoding the 2-C10 AR (0.02-2 µg/well) plus empty pRK5 vector () or the indicated peptide minigene (2 µg/well) () by the DEAE-dextran method. Receptor expression and basal and maximal agonist-stimulated IP production were determined as described. Each data point represents the mean of triplicate binding and IP determinations performed on an independent transfection. Vector cotransfected cell data are shown in each panel for comparison with 1B 3i domain (A), ARK1 PH domain (B), PLC PH domain (C), IRS-1 PH domain (D), Ras-GRF PH domain (E), and Ras-GAP PH domain (F) cotransfected cells.



To determine whether the inhibition was specific for G subunit-mediated PLC activation, the effects of coexpression of the 1B 3i domain peptide and the PH domain-containing peptides on 2-C10 AR- and M1 AChR-mediated IP production were compared, as shown in Fig. 3. At equal levels of receptor expression, coexpression of each of the PH domain peptides resulted in a 50-80% attenuation of 2-C10 AR-mediated IP production (Fig. 3A). The 1B 3i domain peptide had no significant effect. When assayed for the ability to antagonize M1 AChR-mediated IP production, only the 1B 3i domain peptide and PLC PH domain exhibited significant activity (Fig. 3B). Thus, the 1B 3i domain peptide selectively antagonized the G subunit-mediated M1 AChR signal (22) ; the ARK1, IRS-1, Ras-GAP, and Ras-GRF PH domains selectively antagonized the G subunit-dependent 2-C10 AR signal; and the PLC PH domain was inhibitory to both pathways.


Figure 3: Comparison of the effects of PH domain peptides on IP production mediated by 2-C10 AR and M1 AChR. COS-7 cells were transiently cotransfected with plasmid DNA encoding the 2-C10 AR (A) or M1 AChR (B) (0.2 µg/well) plus empty pRK5 vector or the indicated peptide minigene (2 µg/well) by the DEAE-dextran method. Receptor expression and basal and maximal agonist-stimulated IP production were determined as described. Mean levels of receptor expression were not significantly different between vector cotransfected and minigene cotransfected cells. IP production is presented as percentage of the maximal agonist-induced stimulation observed in vector cotransfected cells in each experiment. Each bar represents mean ± S.E. values for six to eight separate experiments performed with the 2-C10 AR and three separate experiments performed with the M1 AChR. *, value less than control, p < 0.05.



Effects of PH Domain Peptide Expression on G Subunit-mediated Activation of MAP Kinase and p21

Several receptors that couple to pertussis toxin-sensitive G proteins, including the 2 AR, the M2 AChR, and the receptors for lysophosphatidic acid and -thrombin, have been shown to activate MAP kinase in various cell types (16, 28, 29, 30, 31, 32, 33) . In COS-7 cells (16, 29) and Rat 1 fibroblasts (30-33) this pathway is dependent upon the activation of p21 and is independent of protein kinase C. Recently, G subunits have been implicated in this direct Ras-dependent activation of MAP kinase. Coexpression of G12 subunits in COS-7 cells leads directly to MAP kinase activation (15, 16) . Furthermore, the G subunit-binding ARK1 PH domain specifically antagonizes 2-C10 AR-, M2 AChR- and lysophosphatidic acid receptor-stimulated Ras-GTP exchange and MAP kinase activation, with no effect on G subunit-mediated, protein kinase C-dependent, 1B AR-mediated signaling (14) .

If PH domains function as G subunit-binding domains in the intact cell, then expression of PH domain peptide minigenes should result in selective inhibition of G-coupled receptor-mediated activation of MAP kinase. To test this hypothesis, COS-7 cells were cotransfected with the G-coupled 2-C10 AR or the G-coupled M1 AChR, plus each of the PH domain peptide minigenes. Receptor-stimulated MAP kinase activity was determined following immunoprecipitation of p42. As shown in Fig. 4, stimulation of both the G-coupled 2-C10 AR and the G-coupled M1 AChR produced a severalfold increase in MAP kinase activity. Coexpression of each of the PH domain peptides resulted in a 55-75% attenuation of 2-C10 AR-mediated MAP kinase activation (Fig. 4A). The 1B 3i domain peptide had no effect (data not shown). When assayed for the ability to antagonize M1 AChR-mediated MAP kinase activation, which proceeds via a p21 -independent pathway (14, 16) , only the PLC PH domain peptide exhibited significant activity (Fig. 4B). This probably reflects inhibition of G-mediated, PLC-dependent activation of protein kinase C (Fig. 3B). As we have previously shown for the ARK1 PH domain (14) , none of the PH domain peptides inhibited epidermal growth factor-stimulated MAP kinase activation (data not shown). The effect of PH domain peptide expression on 2-C10 AR-mediated activation of p21 -GTP exchange, an event known to be involved upstream of MAP kinase in the pathway of G subunit-mediated mitogenic signaling, was also determined. As shown in Fig. 4C, COS-7 cells coexpressing 2-C10 AR and the ARK1, IRS-1 and Ras-GRF PH domain peptides showed impaired agonist-stimulated p21 -GTP exchange compared with controls. Differences in the apparent activity of the PH domain constructs were similar to, but more striking than, that observed for inhibition of MAP kinase activation, with the ARK1 and Ras-GRF PH domains exhibiting the greatest effects.


Figure 4: Effect of PH domain peptides on G protein-coupled receptor-mediated MAP kinase activation and p21-GTP exchange. A, COS-7 cells were transiently cotransfected with plasmid DNA encoding the 2-C10 AR (0.2 µg/well) plus empty pRK5 vector or the indicated PH domain peptide minigene (2 µg/well) by the LipofectAMINE method. Basal and maximal agonist-stimulated MAP kinase activity were determined as described. MAP kinase activity is presented as the -fold increase in p42-catalyzed [P]phosphate incorporation into MBP. Bars represent mean ± S.E. values for five separate experiments performed in duplicate. B, COS-7 cells were transiently cotransfected with plasmid DNA encoding the M1 AChR (0.2 µg/well) plus empty pRK5 vector or the indicated PH domain peptide minigene (2 µg/well) and assayed as described. MAP kinase activity is presented as the -fold increase in p42-catalyzed [P]phosphate incorporation into MBP. Bars represent mean ± S.E. values for three separate experiments performed in duplicate. C, COS-7 cells were transiently cotransfected with plasmid DNA encoding the 2-C10 AR (0.2 µg/well) plus empty pRK5 vector or the indicated PH domain peptide minigene (2 µg/well). Following agonist stimulation, guanine nucleotides bound to p21 were quantitated as described. Data are expressed as the -fold increase in the ratio of GTP to total guanine nucleotides following agonist stimulation. Bars represent mean ± S.E. values for three separate experiments performed in duplicate. In each panel, * signifies value less than control, p < 0.05.



To confirm that the PH domain peptide effects on 2-C10 AR-mediated MAP kinase activation resulted from the sequestration of G subunits, the effect of PH domain peptide expression on direct G12 subunit-mediated MAP kinase activation was determined. COS-7 cells were cotransfected with plasmid DNA encoding hemagglutinin-tagged p44 and G1 and G2 subunits, plus each of the PH domain peptide minigenes. Activity of immunoprecipitated p44 was determined to assess MAP kinase activation in the transfected cell pool. As shown in Fig. 5A, p44 activity increased as a function of the amount of G12 subunit expression. Coexpression of increasing amounts of the ARK1 PH domain peptide resulted in progressive inhibition of this G12 subunit-mediated effect. Conversely, as shown in Fig. 5B, increasing G12 subunit expression diminished the ARK1 PH domain peptide-mediated inhibition, suggesting that the observed inhibition resulted from the competitive interaction of G subunits and the PH domain peptide. As shown in Fig. 5C, coexpression of the PLC, IRS-1, Ras-GAP, and Ras-GRF PH domain peptides also resulted in significant attenuation of p44 kinase activity, with the greatest inhibition observed in cells expressing the ARK1, PLC, and Ras-GRF PH domains.


Figure 5: Effect of PH domain peptides on direct G subunit-mediated MAP kinase activation. A, COS-7 cells were transiently cotransfected with plasmid DNA encoding p44 (0.1 µg/well) plus increasing amounts of plasmid DNA encoding G1 and G2 subunits (0.01-2.0 µg of each/well) by the LipofectAMINE method, and basal MAP kinase activity was determined as described. MAP kinase activity is presented as the -fold increase in p44-catalyzed [P]phosphate incorporation into MBP. Data shown represent mean values for duplicate determinations in one of two separate experiments. B, COS-7 cells were transiently cotransfected with the ARK1 PH domain minigene (0.1 or 0.5 µg/well) and plasmid DNA encoding p44 (0.1 µg/well) plus increasing amounts of plasmid DNA encoding G1 and G2 subunits (0.01-2.0 µg of each/well), and basal MAP kinase activity was determined as described. Data are presented as the percent inhibition of MAP kinase activation in ARK1 PH domain-expressing cells compared with control cells expressing only p44 plus each amount of G12 subunit plasmid DNA. Data shown represent mean values for duplicate determinations in one of two separate experiments. C, COS-7 cells were transiently cotransfected with plasmid DNA encoding G1 and G2 subunits (1.0 µg of each/well) and plasmid DNA encoding p44 (0.1 µg/well) plus empty pRK5 vector or the indicated PH domain peptide minigene (2 µg/well), and basal MAP kinase activity was determined as described. MAP kinase activity is presented as the -fold increase in p44-catalyzed [P]phosphate incorporation into MBP. Data shown represent mean ± S.E. values for three separate experiments performed in duplicate. *, value less than control, p < 0.05.




DISCUSSION

The data presented here provide evidence that PH domain-containing peptides derived from five distinct proteins behave as G subunit-binding domains when expressed in intact cells. Each peptide, when expressed in COS-7 cells, impaired 2-C10 AR-mediated stimulation of IP production, a G subunit-dependent process. Four of the peptides (the ARK1, IRS-1, Ras-GAP, and Ras-GRF PH domains) were specific for G subunit-mediated PLC activation in that G subunit-mediated PLC activation via the M1 AChR was not significantly attenuated. When these peptides were assayed for the ability to antagonize 2-C10 AR-mediated p21 -GTP exchange and MAP kinase activation, two steps in a pathway of G-coupled receptor-mediated mitogenic signaling suspected to involve G subunits, similar effects were observed. Furthermore, direct G subunit-mediated activation of MAP kinase was attenuated by the peptide minigenes, providing the most direct evidence that these PH domain peptides act as G sequestrants in the intact cell.

Recent observations have expanded the role of G subunits in cellular signal transduction by G protein-coupled receptors (34, 35) . In addition to activating ARK1 and ARK2, G subunits mediate the conditional stimulation of types II and IV adenyl cyclase, the PLC 2 and 3 isozymes, phospholipase A2, a phosphatidylinositol-3`-kinase (36) , and the atrial K channel as well as the inhibition of type I adenyl cyclase and the PLC 1 isozyme. Through as yet undefined pathways, G subunits apparently mediate the p21 -dependent activation of MAP kinase, independent of any effects on PLC and protein kinase C (14, 16).

Appreciation of the broad importance of G subunits in cellular signaling has focused attention on other proteins that might interact with G subunits. We have previously shown that glutathione S-transferase fusion proteins containing the PH domains of nine proteins, ARK1, Ras-GRF, Ras-GAP, PLC, IRS-1, Rac, Atk, spectrin, and oxysterol binding protein, reversibly bound bovine brain G subunits in vitro to varying extents (13) . G subunit binding to the fusion proteins was inhibited by G subunits, indicating that the binding of G subunits to the G subunits and the PH domains was mutually exclusive. Deletion mapping of the G subunit-binding regions of ARK1 and Ras-GRF (13) and Btk (37) has indicated that only sequences in and just distal to the carboxyl-terminal half of the PH domain were required for G subunit binding. In each case, the most highly conserved region of the PH domain, subdomain 6, which contains the carboxyl-terminal -helix, was required for in vitro G subunit binding.

The solution structures of the PH domains of pleckstrin (6) , spectrin (7), and dynamin (8, 9) bear considerable similarity to proteins that bind small hydrophobic molecules such as FK506 binding protein and retinal binding protein, which belong to a structural superfamily termed the calycins. Indeed, some PH domains can apparently bind phosphatidylinositol 4,5-bisphosphate or phosphatidylinositol 4-phosphate in the cleft of the -barrel (38) . Other molecules may associate with PH domains under physiologic conditions as well. The Btk PH domain has been reported to bind protein kinase C (39) as well as G subunits (37) , and the PH domain of -spectrin has been reported to be the site of membrane interaction (40) . Since G subunit-binding by PH domains requires only the carboxyl-terminal subdomains, it is tempting to speculate that the binding of G subunits or structurally similar proteins to PH domains may be regulated in complex ways by other molecules, such as phosphatidylinositol phosphates, or by protein phosphorylation. Thus, PH domains that exhibit weak or absent G-binding as glutathione S-transferase fusion proteins in vitro, e.g. glutathione S-transferase-spectrin (13) and glutathione S-transferase-dynamin() may be modulated in vivo.

The previously characterized glutathione S-transferase-PH domain fusion proteins exhibited a relative order of potency for inhibition of bovine brain G subunit-mediated translocation of ARK1 in vitro; ARK1 > PLC oxysterol binding protein > GRF > spectrin (13) , suggesting differences in the apparent affinity of these peptides for a mixed population of G subunits. It is difficult to make such direct comparisons of the relative potency of the PH domain peptides for inhibition of G subunit-mediated signaling in transfected cell systems, since differences in metabolic labeling efficiency and antibody affinity make accurate comparisons of cellular expression impossible. It is noteworthy that those PH domain constructs most effective at inhibiting 2-C10 AR-mediated IP production in the intact cell were not necessarily as effective at inhibiting 2-C10 AR- or direct G subunit-mediated MAP kinase activation. Whereas the ARK1 PH domain was about equally effective in both assays, the PH domain of IRS-1 was apparently a better inhibitor of G subunit-mediated PLC activation, while the Ras-GRF PH domain was a better inhibitor of G subunit-mediated MAP kinase activation. Such differences suggest that while the ability to bind G subunits may be a property shared by each of the PH domains studied, these PH domains may recognize different endogenous ``pools'' of G subunits with different affinity, thereby imparting additional specificity. PH domains may also recognize other members of the G-transducin superfamily of WD40 motif-containing proteins, which includes more than 11 nonorthologous proteins (41, 42, 43) , among them the receptor for activated C kinase, RACK1, and phospholipase A2 activating protein (44) .

Our previous work with PH domain-glutathione S-transferase fusion proteins in vitro(13) as well as the data presented here on the behavior of PH domain peptides expressed in intact cells provide strong evidence that some PH domains can function as G subunit-binding domains. The presence of PH domains in several proteins involved in signal transduction and growth control along with the growing appreciation that G subunits are involved in modulating the activity of several of these pathways, from the conditional stimulation of PLC species to the direct activation of p21, suggests that PH domains may play a major role in the assembly of membrane-associated protein complexes that trigger signal transduction cascades in a manner analogous to the well characterized G subunit-mediated translocation of ARK through its PH domain.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant HL16037. 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 the work presented here.

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

**
To whom correspondence and reprint requests should be addressed: Howard Hughes Medical Inst., Room 468 Carl Bldg., Box 3821, Duke University Medical Center, Durham, NC 27710. Tel.: 919-684-2974; Fax: 919-684-8875.

The abbreviations used are: PH, pleckstrin homology; AChR, acetylcholine receptor; AR, adrenergic receptor; ARK, -adrenergic receptor kinase; G protein, heterotrimeric guanine nucleotide-binding regulatory protein; G, inhibitory G protein regulator of adenyl cyclase; G, stimulatory G protein regulator of phospholipase C; G, complex formed by the and subunits of G proteins; IRS-1, insulin receptor substrate 1; MAP, mitogen-activated protein; MBP, myelin basic protein; PLC, phospholipase C; Ras-GAP, GTPase-activating protein regulator of p21; Ras-GRF, guanine nucleotide-releasing factor of p21; DMEM, Dulbecco's modified Eagle's medium; IP, inositol phosphate; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.

K. Touhara and R. J. Lefkowitz, unpublished observation.


ACKNOWLEDGEMENTS

We thank S. T. Exum for excellent technical assistance and D. Addison and M. Holben for secretarial assistance.


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