COMMUNICATION
Receptor Docking Sites for G-protein beta gamma Subunits
IMPLICATIONS FOR SIGNAL REGULATION*

Guangyu Wu, Jeffrey L. BenovicDagger §, John D. Hildebrandt, and Stephen M. Lanier

From the Department of Pharmacology, Medical University of South Carolina, Charleston, South Carolina 29425 and the Dagger  Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

    ABSTRACT
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Abstract
Introduction
Procedures
Results & Discussion
References

We report the direct interaction of Gbeta gamma with the third intracellular (i3) loop of the M2- and M3-muscarinic receptors (MR) and the importance of this interaction relative to effective phosphorylation of the receptor subdomain. The i3 loop of the M2- and the M3-MR were expressed in bacteria and purified as glutathione S-transferase fusion proteins for utilization as an affinity matrix and to generate substrate for receptor subdomain phosphorylation. In its inactive heterotrimeric state stabilized by GDP, brain G-protein did not associate with the i3 peptide affinity matrix. However, stimulation of subunit dissociation by GTPgamma S/Mg2+ resulted in the retention of Gbeta gamma , but not the Galpha subunit, by the M2- and M3-MR i3 peptide resin. Purified Gbeta gamma bound to the M3-MR i3 peptide with an apparent affinity similar to that observed for the Gbeta gamma binding domain of the receptor kinase GRK2 and Bruton tyrosine kinase, whereas transducin beta gamma was not recognized by the M3-MR i3 peptide. Effective phosphorylation of the M3-MR peptide by GRK2 required both Gbeta gamma and lipid as is the case for the intact receptor. Incubation of purified GRK2 with the i3 peptide in the presence of Gbeta gamma resulted in the formation of a functional ternary complex in which Gbeta gamma served as an adapter protein. Such a complex provides a mechanism for specific spatial translocation of GRK2 within the cell positioning the enzyme on its substrate, the activated receptor. The apparent ability of Gbeta gamma to act as a docking protein may also serve to provide an interface for this class of membrane-bound receptors to an expanded array of signaling pathways.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results & Discussion
References

The precise functional roles of the intracellular domains of the family of receptors coupled to heterotrimeric G-proteins remain unclear. The seven-membrane span core motif for such receptors is fairly compact presenting a relatively small surface area at the cytoplasmic membrane face. Thus, the intracellular domains assume a significant structural presence within the receptor's microenvironment and likely play a role in receptor trafficking, signaling efficiency, receptor phosphorylation, and signal termination. The juxtamembrane segments of the third intracellular (i3)1 loops of most such receptors are critical for G-protein activation by the agonist-occupied receptor, whereas other segments of the i3 loop or other cytoplasmic domains of the receptor may interact with various accessory proteins that regulate signal propagation and contribute to the formation of a signal transduction complex on the inner face of the membrane (1-3). As part of a continuing effort to define such a signal transduction complex we used modular receptor domains to identify interacting proteins and the subdomains participating in such interactions. We initially focused on the i3 loop of muscarinic receptor (MR) subtypes.

The M2- and M4-MR primarily couple to the pertussis toxin-sensitive G-proteins Gi/Go (4). The M1-, M3-, and M5-MR generally couple to the Gq family of G-proteins but are also capable of activating Go and Gi (4-6). As is the case with most members of this receptor superfamily, there is considerable flexibility in the type of G-protein activated by a given receptor. Both the M2-MR and the M3-MR are phosphorylated by the receptor kinase GRK2 in an agonist-dependent manner (7-9). Receptor phosphorylation is a key regulatory event in the processing of external stimuli by the cell. There are three components required for effective receptor phosphorylation: 1) lipid, 2) Gbeta gamma , and 3) an activated conformation of the receptor (10-12). Gbeta gamma likely plays a key role in mediating receptor phosphorylation in the intact cell. Gbeta gamma binds directly to the pleckstrin homology domain of GRK2 and is postulated to mediate translocation of GRK2 from the cytosol to the inner face of the plasma membrane. Mechanistically, how this translocation occurs and results in receptor phosphorylation is unclear (13-16). In the present manuscript, we report the direct interaction of Gbeta gamma with the i3 loop of the M2-MR and M3-MR. The interaction of Gbeta gamma with the i3 loop was dependent upon G-protein activation and resulted in the formation of a ternary complex consisting of the i3 peptide, Gbeta gamma , and GRK2 essentially positioning the enzyme on its substrate. In addition to its role in receptor regulation, the interaction of Gbeta gamma with the i3 loop may also facilitate the interface of G-protein-coupled receptors to diverse signaling pathways via complex formation with additional Gbeta gamma -binding proteins.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results & Discussion
References

Materials-- Heterotrimer G-protein and Gbeta gamma dimer were purified from bovine brain as described previously (17). Transducin was kindly provided by Dr. Heidi E. Hamm (Dept. of Molecular Pharmacology, Northwestern University). Antisera to the amino-terminal 16 amino acids of Goalpha was kindly provided by Dr. Graeme Milligan (Dept. of Biochemistry and Pharmacology, University of Glasgow). Antisera to the carboxyl-terminal 10 amino acids of Gbeta 1, which recognizes Gbeta 1-4, was generated as described (18). Monoclonal antibody 3A10 recognizes an epitope within residues 500-531 of the carboxyl terminus of bovine GRK2 (19). GRK2 was purified from Sf9 insect cells infected with recombinant virus as described (20). The AC2Q peptide corresponding to amino acids Gln956 to Lys982 of adenylyl cyclase II was synthesized and purified by Biosynthesis Inc. (Lewisville, TX). All other materials were obtained as described elsewhere (1, 2).

Plasmid Constructions and Protein Expression-- The M2-MR i3 peptide (His208-Arg387; MW = 19,603 plus GST) and the GRK2-ct (Tyr466-Leu689; MW = 26,073 plus GST) constructs were generated as described previously (1, 13). The construct encoding the PH-domain (Met1-Gln196; MW = 22,965 plus GST) of Bruton tyrosine kinase (Btk-PH) was kindly provided by Dr. Owen N. Witte (Dept. of Microbiology and Molecular Genetics, Howard Hughes Medical Institute, UCLA). The full-length rat M3-MR cDNA was kindly provided by Dr. Tom Bonner (Laboratory of Cell Biology, NIMH). The M3-MR i3 peptide (Arg252-Gln490; MW = 26,471 plus GST) and the M3-MR carboxyl terminus (Asn547-Leu589; MW = 5,351 plus GST) constructs were generated by DNA amplification using the polymerase chain reaction and inserted into the BamHI and EcoRI restriction sites of the pGEX-4T-1 vector. To generate the M3-MR i1 peptide (Lys93-Tyr104; MW = 1,449 plus GST) and the M3-MR i2 peptide (Asp164-Lys182; MW = 2,374 plus GST) constructs, complementary oligonucleotides from these regions were synthesized and annealed prior to ligation into the BamHI and EcoRI restriction sites of the pGEX-4T-1 vector. The structure of each construct used in the present study was verified by restriction mapping and nucleotide sequence analysis. GST fusion proteins were expressed in bacteria and purified on a glutathione-Sepharose matrix as described previously (1). GST and GST fusion proteins were eluted from the resin with 10 mM glutathione and subsequently concentrated/desalted by ultrafiltration (Centricon 3).

Protein Interaction Assays-- Purified receptor subdomains (~5 µg) immobilized on a glutathione resin (2-10 µl of packed resin) or eluted from the resin were incubated with G-protein or GRK2 in a total volume of 250 µl of buffer A (20 mM Tris-HCl, pH 7.5, 0.6 mM EDTA, 1 mM dithiothreitol, 70 mM NaCl, 0.01% Thesit) at 4 °C for 90 min with gentle rotation. In experiments using receptor subdomains not tethered to resin, samples were subsequently incubated with 10 µl of packed resin at 4 °C for 20 min with gentle rotation. The resin was washed three times with 0.5 ml of buffer A at 4 °C, and the retained proteins were solubilized in Laemmli sample buffer and applied to a denaturing 10% polyacrylamide gel. Polyvinylidene difluoride membrane transfers were evaluated by immunoblotting (1).

Phosphorylation of the M3-MR i3 Peptide by GRK2-- The incubation conditions for phosphorylation reactions were essentially as described previously for the intact M2-MR, M3-MR, and beta -adrenergic receptor (AR) (10, 21, 22). Briefly, the reaction was carried out in a total volume of 50 µl of buffer (20 mM Tris-HCl, pH 7.2, 2 mM EDTA, 7 mM MgCl2) containing 2-4 pmol of the GST-M3-MR i3 peptide fusion protein and 50 nM GRK2 with or without various additions as described in the figure legends. Unless indicated otherwise, all phosphorylation reactions contained 300 µM PI. The reactions were initiated by addition of 0.1 mM [gamma -32P]ATP (500-1000 cpm/pmol) and incubated at 30 °C for 40 min. The reactions were stopped by addition of 50 µl of 2 × Laemmli sample buffer and electrophoresed on 10% SDS-polyacrylamide gels. The gels were dried and subsequently exposed to Kodak XAR-5 film for 1 to 12 h. The amount of peptide phosphorylation was determined by cutting the phosphorylated bands from the gel and quantitation by liquid scintillation spectrometry.

    RESULTS AND DISCUSSION
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Abstract
Introduction
Procedures
Results & Discussion
References

The interaction of G-protein subunits with the i3 peptides (M2-MR i3 = His208-Arg387, M3-MR i3 = Arg252-Gln490) was first addressed using heterotrimeric G-protein or dissociated G-protein subunits. The i3 peptides were bound to a glutathione-Sepharose matrix and incubated with bovine brain G-protein in the presence of GDP/EDTA or GTPgamma S/Mg2+, which would either stabilize the heterotrimer or promote subunit dissociation, respectively. The resin was washed, and retained proteins were determined by SDS/PAGE and subsequent immunoblotting with either a Goalpha or Gbeta common antisera. In its heterotrimeric form, immunoblotting with either antisera indicated that G-protein did not effectively interact with the i3 peptide resin (Fig. 1A). However, following activation and subunit dissociation, Gbeta gamma , but not Goalpha , bound to the i3 peptides (Fig. 1A). Similar experiments with transducin resulted in undetectable Gbeta gamma binding to the M2- or M3-MR i3 peptide (Fig. 1A) consistent with the distinct functional properties of transducin and brain G-protein (~80% of which is Go) (23). These data suggest that the i3 loop presents a motif for brain Gbeta gamma binding and that the Gbeta gamma domain recognized by this motif is masked by the Galpha subunit.


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Fig. 1.   Interaction of receptor subdomains with brain G-proteins. A, heterotrimeric brain G-protein or transducin (30 nM) were incubated (4 °C for 90 min) with the control GST, M2-MR i3, or M3-MR i3 affinity matrix (~5 µg of protein) in a total volume of 250 µl of buffer containing either 10 µM GDP or 10 µM GTPgamma S plus 5 mM Mg2+. The resins were washed, and bound proteins were visualized by immunoblotting of membrane transfers following SDS-PAGE. The membrane transfers were first incubated with Gbeta antisera (upper panel) and then stripped for reprobing with Goalpha antisera (lower panel). The numbers to the left of the gel indicate the migration of standards of known molecular weight × 10-3. Std, 200 ng of brain G-protein or transducin without resin incubation. B and C, GST fusion proteins (~5 µg of protein) were incubated with Gbeta gamma or transducin beta gamma (30 nM), and samples were processed as described under "Experimental Procedures" using Gbeta antisera. PTP1C (amino acids 1-296), Na+/H+ exchanger (amino acids 168-345), c-Jun (amino acids 1-79). Std, 100 ng of brain Gbeta gamma or transducin beta gamma without resin incubation. Similar results were obtained in three to four experiments for A-C using different preparations of fusion protein and brain G-protein.

The interaction of the i3 peptides with Gbeta gamma was also observed using purified bovine brain Gbeta gamma that was physically separated from brain Galpha (Fig. 1, B and C). Purified Gbeta gamma did not bind to GST control, protein-tyrosine phosphatase 1C (PTP1C), Na+/H+ exchanger or c-Jun fusion proteins (Fig. 1B) and also did not interact with the M3-MR i1, M3-MR i2, or M3-MR carboxyl terminus peptides (Fig. 1C). Although the M3-MR i3 peptide interacted with brain Gbeta gamma , it did not recognize transducin beta gamma (Fig. 1C). As brain Gbeta gamma and transducin beta gamma differ in their gamma  subunits (23), these data indicate that the gamma  subunit plays an important role in the recognition of Gbeta gamma by the receptor subdomain.

To provide additional insight as to the relative properties of Gbeta gamma binding to the i3 peptides, the M3-MR i3 was compared with Gbeta gamma binding domains of the proteins, GRK2 and Btk. GRK2 phosphorylates selected G-protein-coupled receptors following their activation by agonist, whereas Btk is a tyrosine kinase involved in human X chromosome-linked immunodeficiency. The binding of Gbeta gamma to the M3-MR i3, GRK2-ct, and Btk-PH was dependent upon Gbeta gamma concentration and exhibited similar affinities although maximum binding varied (Fig. 2). The direct association of Gbeta gamma with the receptor subdomain may relate to earlier reports indicating that Gbeta gamma plays diverse roles in signaling by G-protein-coupled receptors (23-28).


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Fig. 2.   Binding of Gbeta gamma to M3-MR i3, GRK2-ct, or Btk-PH fusion proteins. Increasing concentrations of purified brain Gbeta gamma (4-71 nM) were incubated with the M3-MR i3, GRK2-ct, or Btk-PH affinity matrix (~5 µg of protein). Right panel, the immunoblots were analyzed by densitometry, and the results are expressed as a percent of the signal observed at a Gbeta gamma concentration of 71 nM. Similar results were obtained in three experiments using different preparations of fusion protein.

Gbeta gamma is postulated to mediate translocation of GRK2 from the cytosol to the membrane leading to phosphorylation of activated adrenergic, muscarinic, neurokinin, and other receptors of this structural class. Mechanistic aspects of this translocation are unclear. Gbeta gamma may increase the amount of membrane-associated GRK2 by using the acylated gamma  subunit of Gbeta gamma to anchor to hydrophobic sites in the membrane bilayer. A second possibility is that the binding of Gbeta gamma to GRK2 initiates a conformational change in the enzyme that reveals a "membrane binding domain" of GRK2. Another possibility is that Gbeta gamma may provide an anchor to the membrane by interacting with the i3 loop of the activated receptor. In such a situation, the kinase would be positioned adjacent to its substrate suggesting that one role of Gbeta gamma is to "position" enzyme and substrate facilitating the phosphorylation reaction. If such a hypothesis is true, then Gbeta gamma should promote the formation of a ternary complex consisting of the activated receptor, Gbeta gamma , and GRK2. We addressed these issues experimentally using the M3-MR i3 peptide.

We first determined if the M3-MR i3 peptide was a suitable substrate for phosphorylation by GRK2 and if so, how this phosphorylation compared with that observed with the intact agonist-activated receptor itself. As is the case for the intact M2-MR, M3-MR, and beta 2-AR, phosphorylation of the M3-MR i3 peptide required both Gbeta gamma and the acidic lipid PI when assayed under ionic conditions similar to cytosol (Fig. 3A). Gbeta gamma and PI stimulated phosphorylation of the i3 peptide in a synergistic manner (Fig. 3A). The M3-MR i3 peptide was phosphorylated by GRK2 to a stoichiometry of ~1-3 mol of phosphate/mol of peptide, which is comparable to the results obtained with the intact, agonist-activated M2-MR, M3-MR, and beta 2-AR. The ability of Gbeta gamma to stimulate GRK2-mediated phosphorylation of the M3-MR i3 peptide was concentration-dependent, and it was blocked by the AC2Q peptide, a 27-residue peptide derived from adenylyl cyclase type II that blocks Gbeta gamma regulation of various effector proteins (Fig. 3, B and C) (29). The interaction of Gbeta gamma with the M3-MR i3 peptide described earlier was also blocked by the AC2Q peptide (data not shown).


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Fig. 3.   Phosphorylation of the M3-MR i3 peptide by GRK2. Purified GST-M3-MR i3 peptide fusion protein (2-4 pmol) was incubated with GRK2 (50 nM) and 0.1 mM [gamma -32P]ATP under various conditions, and samples were processed for SDS-PAGE/autoradiography. GST itself was not phosphorylated by GRK2 under any incubation conditions. A, phosphorylation of the M3-MR i3 peptide by GRK2 in the presence and absence of Gbeta gamma (120 nM) and/or PI (300 µM). The arrow indicates the migration of the M3-MR i3 peptide. B, phosphorylation of the M3-MR i3 peptide by GRK2 in the presence of increasing concentrations of Gbeta gamma (0-600 nM). C, phosphorylation of the M3-MR i3 peptide by GRK2 in the presence of Gbeta gamma (120 nM) and increasing concentrations of the AC2Q peptide (0-100 µM). Similar results were obtained in four experiments. All incubation buffers contained 140 mM KCl.

As observed for the intact agonist-activated beta 2-AR, M2-MR, and M3-MR (9, 21, 22), the phosphorylation of the M3-MR i3 peptide was sensitive to increased ionic strength of the incubation buffer (Fig. 4A), and this apparently reflects the loss of a direct interaction between GRK2 and the i3 peptide (Fig. 4B, upper panel). Addition of Gbeta gamma to the incubation buffer reversed the inhibition of M3-MR i3 peptide phosphorylation by increased salt concentrations, suggesting that Gbeta gamma facilitated the interaction of GRK2 with its substrate. The interaction of Gbeta gamma with the M3-MR i3 peptide was actually augmented by increasing the concentration of KCl or NaCl (Fig. 4B, lower panel), an effect that was directly opposite to the influence of increased ionic strength on i3 peptide/receptor phosphorylation and the association of GRK2 with the M3-MR i3 peptide (Fig. 4, A and B, upper panel). Thus, under ionic conditions similar to those found in cytosol, GRK2 did not effectively phosphorylate the intact agonist-activated receptor or the i3 peptide unless Gbeta gamma was present. Indeed, addition of Gbeta gamma resulted in the association of GRK2 with the M3-MR i3 peptide affinity matrix in the presence of cytosolic concentrations of potassium (Fig. 4C). This association is likely mediated by a ternary complex consisting of the receptor subdomain, Gbeta gamma , and GRK2. These data indicate that Gbeta gamma served as a docking protein to allow formation of this ternary complex, which is apparently required for effective phosphorylation by GRK2.


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Fig. 4.   A, phosphorylation of the M3-MR i3 peptide by GRK2 in the presence of increasing concentrations of KCl (0-140 mM) with or without Gbeta gamma (120 nM). B, effect of ionic strength on the interaction of GRK2 (upper panel) or Gbeta gamma (lower panel) with the M3-MR i3 peptide. The M3-MR i3 or GST-substituted resin were incubated with brain Gbeta gamma (30 nM) or GRK2 (25 nM) in the presence of increasing concentrations of sodium chloride, potassium chloride, or potassium acetate and processed as described in the legend to Fig. 1. C, interaction of GRK2 with the M3-MR i3 peptide in the presence and absence of Gbeta gamma . GRK2 (50 nM) was incubated with the M3-MR i3 peptide affinity matrix (~5 µg of protein) in the absence (lane 2) or presence of Gbeta gamma (50 nM, lanes 3-5), and samples were processed as described in the legend to Fig. 1. Each incubation contained 140 mM KCl. Lanes 3-5 involved experiments using three different preincubation conditions (4 °C, 30 min) in which the three interacting entities were added in a different order: lane 3, preincubation of the M3-MR i3 peptide with GRK2; lane 4, preincubation of Gbeta gamma with GRK2; lane 5, preincubation of the M3-MR i3 peptide with Gbeta gamma . Std (lane 1), 100 ng of GRK2 and 100 ng of Gbeta gamma without resin incubation. Similar results were obtained in three to five experiments using different preparations of GRK2, fusion protein, and Gbeta gamma .

There are three important points concerning the interaction of Gbeta gamma with the M3-MR i3 peptide. First, the interaction of the M3-MR i3 peptide with Gbeta gamma requires dissociation of heterotrimeric G-protein. Second, the inability of the M3-MR i3 peptide to recognize transducin beta gamma indicates that the interaction with Gbeta gamma is isoform-selective. Third, it is hypothesized that when the M3-MR i3 peptide is expressed free of the conformational constraints imposed by the receptor's membrane spans, it assumes an activated conformation (see "Discussion" in Ref. 1). This hypothesis is supported by structural analysis of the cytoplasmic domains of rhodopsin where key atomic distances were identical with those observed by chemical cross-linking of the intact activated receptor (30, 31). Such an interpretation is also supported by the interaction of arrestin with the i3 loop of the M2-MR, M3-MR, and alpha 2-AR (1) and the ability of GRK2 to phosphorylate the M3-MR i3 peptide in a manner similar to that observed with intact, agonist-activated G-protein-coupled receptors.

The dependence of Gbeta gamma binding to the i3 peptide upon G-protein dissociation is of particular note relative to both potential downstream signaling events and various aspects of receptor regulation. Upon receptor activation, Gbeta gamma may be "trapped" by the M3-MR i3 loop and present a motif that is recognized by GRK2 positioning the kinase in close proximity to its substrate, the activated receptor. Although such a scenario may not be operative for all GRKs or G-protein-coupled receptors, it is of particular interest for several reasons. Gbeta gamma is suggested to interact with several molecules involved in signal propagation. The regulation of structurally diverse molecules by Gbeta gamma suggests that its structure has unique properties that perhaps facilitate simultaneous interaction with more than one protein. These structural properties might reside in the repetitive elements of the WD-40 motifs and would be conducive to the function of Gbeta gamma as an adaptor protein. The apparent ability of Gbeta gamma to act as a docking protein suggests that Gbeta gamma may anchor the formation of a signal transduction complex. Such an anchor may also allow the interface of selected G-protein-coupled receptors to signaling pathways involving soluble tyrosine kinases and/or low molecular weight G-proteins leading to activation of mitogenic signaling pathways and/or changes in cellular architecture.

    ACKNOWLEDGEMENTS

We thank Dr. Owen N. Witte, Dr. Gen-Shen Feng (Dept. of Biochemistry and Molecular Biology, Indiana University School of Medicine), Dr. Larry Fliegel (Dept. of Biochemistry and Pediatrics, University of Alberta, Alberta, Canada), and Dr. Steven Rosenzweig (Dept. of Pharmacology, Medical University of South Carolina) for providing the Btk-PH, PTP1C, Na+/H+ exchanger, and c-Jun fusion protein constructs, respectively. We thank Dr. Tom Bonner for the rat M3-muscarinic receptor cDNA and Dr. Heidi E. Hamm for transducin and transducin beta gamma . The expression vector containing the peptide derived from the third intracellular loop of the M3-muscarinic receptor was generated by Dr. Pablo Escriba in the laboratory of Dr. Lanier. We appreciate the technical assistance of Lisa Kless in the laboratory of S. M. L. and the contributions of Dr. Jane Dingus and Bronwyn Tatum for G-protein purification in the laboratory of J. D. H.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants NS24821 (to S. M. L.), DK37219 (to J. D. H.), and GM47417 (to J. L. B.) and Council for Tobacco Research Grant 2235 (to S. M. L.).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.

§ Established Investigator of the American Heart Association.

To whom correspondence should be addressed: Dept. of Pharmacology, Medical University of South Carolina, 171 Ashley Ave., Charleston, SC 29425. Tel.: 803-792-2574; Fax: 803-792-2475; E-mail: laniersm{at}musc.edu.

1 The abbreviations used are: i3, third intracellular loop; MR, muscarinic receptor; AR, adrenergic receptor; MW, calculated molecular weight; GST, glutathione S-transferase; PI, phosphatidylinositol; PAGE, polyacrylamide gel electrophoresis; GTPgamma S, guanosine 5'-O-(3-thiotriphosphate).

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Abstract
Introduction
Procedures
Results & Discussion
References

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