COMMUNICATION:
Interaction of Phosducin-like Protein with G Protein beta gamma Subunits*

(Received for publication, January 21, 1997, and in revised form, March 18, 1997)

Christelle Thibault , Michael W. Sganga Dagger and Michael F. Miles §

From the Ernest Gallo Clinic and Research Center, Department of Neurology, University of California at San Francisco, San Francisco, California 94110

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Phosducin-like protein (PhLP), a widely expressed ethanol-responsive gene (Miles, M. F., Barhite, S., Sganga, M., and Elliott, M. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10831-10835), is a homologue of phosducin, a known major regulator of Gbeta gamma signaling in retina and pineal gland. However, although phosducin has a well characterized role in retinal phototransduction, function of the PhLP remains unclear. In this study we examine the ability of PhLP to bind Gbeta gamma dimer in vitro and in vivo. Using PhLP glutathione S-transferase fusion proteins, we show that PhLP directly binds Gbeta gamma in vitro. Studies with a series of truncated PhLP fusion proteins indicate independent binding of Gbeta gamma to both the amino- and C-terminal halves of PhLP. Protein-protein interactions between Gbeta gamma and PhLP are inhibited by the alpha  subunit of Go and Gi3, suggesting that PhLP can bind only free Gbeta gamma . Finally, we show that PhLP complexes, at least partially, with Gbeta gamma in vivo. Following overexpression of epitope-tagged PhLP together with Gbeta 1gamma 2 proteins in COS-7 cells, a PhLP-Gbeta gamma complex is co-immunoprecipitated by monoclonal antibody directed against the epitope tag. Similarly, polyclonal anti-PhLP antibody co-precipitates endogenous PhLP and Gbeta gamma proteins from NG108-15 cell lysates. These data are consistent with the hypothesis that PhLP is a widely expressed modulator of Gbeta gamma function. Furthermore, because alternate forms of the PhLP transcript are expressed, there may be functional implications for the existence of two Gbeta gamma -binding domains on PhLP.


INTRODUCTION

Heterotrimeric guanine nucleotide-binding proteins (G proteins) play a major role in transmembrane signaling processes by transducing extracellular signals from the superfamily of heptahelical cell surface receptors to their appropriate intracellular effectors (1, 2). In its trimeric form, Galpha beta gamma is inactive, and the Galpha subunit binds a molecule of GDP. Upon ligand binding, the receptor catalyzes the exchange of GDP for GTP on Galpha that causes its activation and dissociation from the tightly bound Gbeta gamma complex.1 Inactivation and reassociation of the heterotrimer is initiated by the hydrolysis of bound GTP into GDP by an intrinsic GTPase activity of the Galpha subunit. It is now known that both the free GTP-bound Galpha and the Gbeta gamma dimer can bind and regulate downstream effectors including adenylyl cyclases, phospholipases, and ion channels, and thereby modulate second messenger levels and ion flux (3).

The discovery of several specific Gbeta gamma binding proteins has recently shed light on new roles for Gbeta gamma in the propagation and termination of cellular signaling. The dimer has been shown to recruit beta -adrenergic receptor kinase (beta ARK)2 to its membrane-associated receptor substrate and thus initiate receptor desensitization (4, 5). This process occurs via direct binding of Gbeta gamma to the C terminus of a putative pleckstrin homology domain on beta ARK (6). Furthermore, the responsiveness of G protein-regulated signaling systems may be directly modulated through the interaction of Gbeta gamma subunits with intracellular regulatory proteins. For instance, phosducin, a phosphoprotein mainly expressed in the retina and pineal gland, inhibits the phototransduction cascade by scavenging beta gamma subunits of the G protein transducin (Gt), thus preventing their reassociation with the Gtalpha subunit (7, 8). Because phosducin has a higher affinity for Gtbeta gamma than does Gtalpha , it has been suggested that the formation of the phosducin/Gtbeta gamma complex is a major factor regulating photoreceptor responsiveness (9). From in vitro binding and co-transfection assays, it was proposed that phosducin may also compete with other targets for Gbeta gamma binding, such as beta ARK and phospholipase C type beta 2 (10, 11).

We recently isolated a rat brain cDNA encoding a phosducin-like protein (PhLP), which has 65% amino acid homology to phosducin (12). We also described several 5'-end splice variants that generate two predicted isoforms of the protein: PhLP long (PhLP) of 301 amino acids containing the entire coding sequence and PhLP short (PhLPS) of 218 amino acids missing the first 83 N-terminal residues of PhLP (12, 13). Based on sequence homology with phosducin, we have suggested that PhLP proteins regulate Gbeta gamma signaling in nonretinal tissues. In favor of this hypothesis, a recent report showed that recombinant PhLPS inhibits several Gbeta gamma functions in vitro (14). Interestingly, these authors suggested that unlike phosducin (11, 15), the N terminus of PhLP was unlikely to contain a Gbeta gamma -binding domain.

To more directly characterize the interaction of PhLP with Gbeta gamma , we studied PhLP binding to Gbeta gamma both in vivo and in vitro. Our results here, using in vitro binding studies with a series of truncated PhLP/glutathione S-transferase (GST) fusion proteins, show that PhLP binds beta gamma through a bipartite binding domain. The Gbeta gamma -PhLP interaction was confirmed by co-immunoprecipitation of the complex from cell lysates. Our findings support the hypothesis that PhLP can modulate Gbeta gamma function (14) in many tissues through direct protein-protein interactions. Regulation of PhLP/Gbeta gamma interactions could be an important factor in controlling G protein signaling.


EXPERIMENTAL PROCEDURES

Materials

PhLP and PhLPS cDNAs were cloned in our laboratory (12). The GST-phosducin construct was obtained from Dr. Cheryl Craft (University of Southern California). Gbeta gamma and Goalpha proteins purified from bovine brain were kindly provided by Dr. Eva Neer (Brighman and Woman's Hospital, Boston, MA). Gbeta 1 and Ggamma 2 expression vectors were kind gifts from Dr. H. Bourne (University of California at San Francisco). Purified recombinant Gbeta 1gamma 2 was generously provided by Dr. Rene Onrust in the Bourne laboratory. Recombinant Gialpha 3 protein was from Calbiochem.

DNA Constructs

The full-length PhLP (amino acids 1-301) as well as different regions of the protein corresponding to amino acid residues 84-301 (referred to as PhLPS), 1-167, 1-115, 1-70, 50-167, 84-167, 161-301, and 200-301 were expressed as GST fusion proteins. DNA fragments encoding PhLP and its derivatives were amplified by polymerase chain reaction using rat PhLP cDNA as template and 5'- and 3'-primers containing BamHI and EcoRI sites, respectively. The amplified fragments were ligated in frame with the 3'-end of the coding region of GST into BamHI and EcoRI sites of the pGEX-2T vector (Pharmacia Biotech Inc.). The resultant constructs were verified by DNA sequencing using the chain termination method (Sequenase version 2.0, U. S. Biochemical Corp.) and used to transform Escherichia coli strain BL21.

An epitope-tagged PhLP expression vector was generated by fusing an 8 amino acid peptide from the hemagglutinin (HA) of influenza virus to the C terminus of PhLP. Sense and antisense oligonucleotides corresponding to the HA epitope (YDVPDYAS), flanked by a 5' EcoRI site and a 3' NotI site, were synthesized, annealed, and inserted into pcDNA3 (Invitrogen) between EcoRI and NotI sites. The full-length PhLP coding sequence, amplified as described above, was then ligated between the BamHI and EcoRI sites of the modified pcDNA3 vector, so as to fuse the PhLP C terminus in frame with the 5'-end of the HA tag.

Expression of GST Fusion Proteins and Gbeta gamma Binding Assay

Fusion protein expression was induced with 0.1 mM isopropyl-1-thiol-beta -D-galactopyranoside for 90 min, and the proteins were solubilized and purified on glutathione-Sepharose 4B resin (Pharmacia) by the Sarkosyl method (16). In a typical binding assay, following immobilization on glutathione-agarose beads, fusion proteins at a final concentration of 0.5-1.0 µM were incubated with 50-100 nM Gbeta gamma purified from bovine brain in 50 µl of phosphate-buffered saline (PBS) containing 0.01% Lubrol for 2 h at 4 °C. Following six washes in 200 µl of PBS containing 0.01% Lubrol, the beads were resuspended in SDS sample buffer and boiled for 10 min. The eluted proteins were separated with 10% SDS-polyacrylamide gel electrophoresis (PAGE) and electrotransferred onto nitrocellulose membranes using standard methods. The blots were probed with a polyclonal anti-beta antiserum (1:1000; DuPont NEN) and processed using the enhanced chemiluminescense detection system (Amersham Corp.). Occasionally, blots were stripped and reprobed with a polyclonal anti-GST antiserum (1:4000; Santa Cruz).

Anti-PhLP Antiserum

The entire coding region of PhLPS cDNA was amplified by polymerase chain reaction and fused in frame with the maltose binding protein coding region in the vector pMAL-c2 (New England Biolabs). The maltose binding protein-PhLPS fusion protein migrated at approximately 72 kDa on SDS-polyacrylamide gels as expected. The fusion protein was purified to apparent homogeneity by amylose resin chromatography exactly as described by the manufacturer (New England Biolabs) and was injected into rabbits by a commercial source (CalTag) for generation of a polyclonal antiserum. This antiserum was affinity-purified over a column of GST-PhLPS coupled to CnBr-activated Sepharose 4B (Pharmacia).

Cell Culture and Transient DNA Transfection

NG108-15 cells were grown as described (12) in Dulbecco's modified Eagle's medium (DMEM) containing 10% serum+ (JRH Biosciences). COS-7 cells (3 × 105 cells/well) were seeded 48 h before transfection in 6-well plates in DMEM supplemented with 10% fetal bovine serum. Cells were incubated for 5 h in serum-free DMEM with DNA plasmids premixed with lipofectAmine (Life Technologies, Inc.) and were then incubated overnight at 37 °C in DMEM containing 10% fetal bovine serum. The total amount of DNA in all transfections was 2 µg/well. When required, the empty pcDNA3 vector was used to maintain a constant amount of DNA.

Immunoprecipitation

2 × 106 transfected COS-7 cells or 4 × 106 NG108-15 cells were washed twice with ice-cold PBS and lysed in Nonidet P-40 lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 2 µg/ml aprotinin and leupeptin, 20 µg/ml soybean trypsin inhibitor). After 15 min of incubation on ice, insoluble material was removed by centrifugation at 10,000 × g at 4 °C for 10 min, and the lysate was precleared in the presence of protein A-agarose (Santa Cruz) for 30 min. COS-7 cell lysate was then incubated with 5 µg of monoclonal antibody 12CA5 (Boehringer Mannheim) to the HA tag, whereas NG108-15 cell lysate was incubated with 5 µg of affinity-purified polyclonal anti-PhLP or an equivalent amount of preimmune serum. The incubations were conducted overnight at 4 °C before precipitation in the presence of protein A-agarose. The immunoprecipitates were washed four times with PBS containing 0.01% Lubrol, and protein complexes were eluted in SDS sample buffer and analyzed by Western blot using the 12CA5 antibody and/or a monoclonal antibody to the Gbeta 1 subunit (Transduction Laboratories).


RESULTS AND DISCUSSION

To determine whether PhLP directly interacts with Gbeta gamma , we generated PhLP and PhLPS GST fusion proteins and examined their ability to bind Gbeta gamma purified from bovine brain. Following immobilization of the fusion proteins on glutathione-agarose beads, Gbeta gamma binding was detected by Western blot analysis using an anti-beta antiserum. As controls, we tested Gbeta gamma binding by the GST protein itself and GST fused to phosducin (GST-phosducin), a known Gbeta gamma -binding protein. The fusion proteins migrate at their expected molecular weight as visualized by Coomassie Blue-staining (Fig. 1A, lower panel) and can be detected with an anti-GST antibody on Western blot analysis (Fig. 1B, lower panel). In addition, GST-PhLP proteins are recognized by an affinity-purified polyclonal antiserum directed against PhLPS (data not shown).


Fig. 1. Binding of bovine brain Gbeta gamma by GST-phosducin and GST-PhLP. A, Western blot (upper panel) analysis of Gbeta gamma binding to GST fusion proteins. GST fusion proteins (500 nM) or GST (1.5 µM) were incubated with 70 nM of purified bovine brain Gbeta gamma . Bound Gbeta gamma was fractionated by SDS-PAGE and visualized by Western blot using a polyclonal anti-beta antiserum as described under "Experimental Procedures." Lane 1, GST; lane 2, GST-PhLPS; lane 3, GST-PhLP; lane 4, GST-phosducin. Also shown is 500 ng of purified Gbeta gamma alone. The lower panel shows Coomassie Blue staining of expressed GST fusion proteins following purification on glutathione-Sepharose resin. Approximate molecular masses (in kDa) are indicated at the right. B, titration of Gbeta gamma binding to GST-PhLP (left) and GST-phosducin (right). GST fusion proteins were present at 1 µM in all reactions, and increasing concentrations of Gbeta gamma were added as indicated. The amounts of GST fusion proteins and bound Gbeta gamma were monitored by Western blot using anti-GST (lower panels) and anti-beta (upper panels) antisera, respectively. The results are representative of experiments repeated at least three times.
[View Larger Version of this Image (40K GIF file)]

As previously reported by other investigators (11), GST-phosducin bound detectable amounts of Gbeta gamma at a molar ratio of beta gamma :GST-phosducin of approximately 1:7 (Fig. 1A, upper panel). Under similar conditions, GST-PhLPS and GST-PhLP also retained Gbeta gamma (Fig. 1A, upper panel). By contrast, GST protein did not bind Gbeta gamma subunits even when present at a 3-fold higher concentration than the GST-PhLP proteins (Fig. 1A, upper panel), indicating that Gbeta gamma binding by the fusion proteins is specified by the PhLP protein sequence. The affinity of phosducin for Gbeta gamma was previously reported to be in the nanomolar range (11, 14). Under our experimental conditions, GST-PhLP appeared to have a slightly lower affinity for Gbeta gamma than GST-phosducin but of the same order of magnitude because titration with varying amounts of Gbeta gamma protein showed that GST-PhLP retained only slightly lower amounts of Gbeta gamma than did GST-phosducin (Fig. 1B, upper panel).

To map the Gbeta gamma binding domain of PhLP, we examined recombinant Gbeta 1gamma 2 interaction in vitro with GST fusion proteins containing various regions of PhLP. Fig. 2 shows an alignment of PhLP deletion constructs with the full-length PhLP. Each construct produced a protein that migrated at the expected molecular weight on SDS-PAGE (data not shown). Surprisingly, we found Gbeta gamma binding activity of PhLP at two areas in the N- and C-terminal regions. In the N-terminal half of PhLP (PhLP1-167), amino acids 50-115 appeared sufficient for Gbeta gamma binding, because PhLP1-115 and PhLP50-167 retained Gbeta gamma , whereas PhLP1-70 and PhLP84-167 did not (Fig. 2). The 50-115 region contains an 11-amino acid stretch (57-67: TGPKGVINDWR) that is perfectly conserved between PhLP and phosducin and is known to be essential for Gbeta gamma binding by phosducin (11). Furthermore, the crystal structure of the Gt beta gamma -phosducin complex, reported while this manuscript was in preparation, showed that this highly conserved sequence has extensive and tight interactions with the center of the Gbeta propellar (17). This conserved sequence region of PhLP (amino acids 57-67) may be important for binding of Gbeta gamma because PhLP84-167 totally lacked beta gamma binding, whereas PhLP50-167 had essentially full binding activity. However, although the 57-67 region may be important for Gbeta gamma binding by PhLP, additional elements seem required because PhLP1-70 did not bind Gbeta gamma .


Fig. 2. Analysis of the Gbeta gamma binding regions of PhLP. Western blot analysis of Gbeta gamma binding to GST-PhLP and different deletion constructs using an anti-beta antiserum (upper panel). GST fusion proteins (1 µM) were incubated with 50 nM Gbeta 1gamma 2 recombinant proteins. The lower portion shows schematized diagram of PhLP and deletion constructs. The predicted Gbeta gamma interacting residues in PhLP (17) are shaded. The deletion constructs are illustrated together with their relative binding. The results are representative of experiments repeated at least three times.
[View Larger Version of this Image (21K GIF file)]

Additional deletions revealed that the C-terminal half of PhLP (PhLP161-301) also binds Gbeta gamma . This binding activity was further localized to the C-terminal 101 residues of PhLP (PhLP200-301) (Fig. 2). This C-terminal binding domain may explain why PhLPS (PhLP84-301), which does not contain the 57-67 conserved sequence, still retained significant Gbeta gamma binding activity.

The crystal structure of Gt beta gamma -phosducin also showed two spatially and possibly functionally distinct domains in phosducin (17). These two domains roughly correspond to the N-terminal and C-terminal halves of the molecule and do not interact with each other but both contact Gbeta gamma . The N-terminal domain may compete with Galpha , whereas the C-terminal, thioredoxin-like domain was suggested to be responsible for Gbeta gamma translocation away from the membrane (17). Based on sequence homology, Gaudet et al. proposed a similar structure for PhLP and predicted the Gbeta gamma interacting residues in this protein (17). The regions occupied by these amino acids, depicted in Fig. 2, correspond to residues 54-69 and 114-152 in the N-terminal domain and to residues 240-247 and 270-277 in the C-terminal domain. Our results are in perfect agreement with these predictions and also suggest that the N-terminal and C-terminal domains can interact with Gbeta gamma independently. Because these domains may affect different functions of Gbeta gamma , they might be useful tools to study different aspects of Gbeta gamma regulation as suggested by Gaudet et al. (17).

Previous studies have demonstrated that the binding of Gbeta gamma to Galpha subunit inhibits its interaction with phosducin or beta ARK (15, 18). In contrast, the N-terminal domain of the G protein-gated K+ channel as well as the small GTPase, ADP-ribosylation factor were shown to interact with either Gbeta gamma alone or trimeric Galpha beta gamma (19, 20). We found that recombinant Gialpha 3 inhibited Gbeta gamma binding to GST-PhLP (Fig. 3). Similarly, Goalpha -GDPbeta S but not Goalpha -GTPgamma S partially abolished the interaction of Gbeta gamma to GST-PhLP (data not shown). Together, these results suggest that only free Gbeta gamma can interact with PhLP. Western blot analysis with a common anti-alpha antiserum (DuPont NEN) indicated that neither Gialpha 3 or Goalpha was retained on GST-PhLP along with the Gbeta gamma dimer (Fig. 3 and data not shown). In addition, Gialpha 3 by itself did not bind to GST-PhLP (Fig. 3). It should be noted that a relatively high concentration of recombinant Gialpha 3 was required to totally eliminate Gbeta gamma binding to GST-PhLP (Fig. 3). This may reflect the fact that Gbeta gamma interacts more tightly with PhLP than with Galpha subunit, as was previously found for phosducin and Gtalpha interaction with Gbeta gamma (17).


Fig. 3. Galpha inhibits binding of Gbeta gamma to GST-PhLP. Western blot analysis showing Gbeta gamma binding to GST-PhLP in the presence of Gialpha 3 recombinant protein. GST-PhLP (1 µM) immobilized on glutathione-Sepharose beads was incubated in the presence of bovine brain Gbeta gamma (35 nM) and/or in the presence of various concentrations of recombinant protein Gialpha 3, as indicated. Following 2 h of incubation, the Gbeta gamma and the Gialpha 3 retained on the beads (top and middle panels) as well as the unbound Gialpha 3 (bottom panel) were detected by Western blot analysis using anti-beta or anti-alpha antiserum (DuPont NEN). The results are representative of experiments repeated twice.
[View Larger Version of this Image (33K GIF file)]

To demonstrate Gbeta gamma -PhLP interaction in vivo, the complex was immunoprecipitated following overexpression of the proteins in COS-7 cells. For these experiments, the C terminus of PhLP was tagged with a HA epitope. COS-7 cells were transiently transfected with plasmids encoding PhLP-HA, Gbeta 1 and Ggamma 2 subunits, or a combination of these proteins. Expression of the proteins in COS-7 cells was monitored by Western blot analysis using monoclonal anti-HA and anti-beta 1 antibodies (Fig. 4A, lower panels). PhLP-HA was specifically precipitated by anti-HA antibody (Fig. 4A, upper panel). In addition, this antibody co-precipitated overexpressed Gbeta 1gamma 2 subunits, only in cells co-expressing PhLP-HA (Fig. 4A, upper panel). We also detected Gbeta in immunoprecipitates from cells transfected only with PhLP-HA plasmid (Fig. 4A, upper panel), suggesting that PhLP-HA interacts with both overexpressed and endogenous Gbeta gamma subunits.


Fig. 4. Co-immunoprecipitation of Gbeta gamma with PhLP. A, COS-7 cells were transiently transfected either alone or in combination with PhLP-HA expression vector, Gbeta 1 and Ggamma 2 expression vectors, or with empty vector as indicated. Anti-HA immunoprecipitates (upper panel) from COS-7 cells transfected as shown were analyzed for the presence of Gbeta 1 and PhLP-HA proteins by immunoblotting with anti-Gbeta 1 and anti-HA antibodies. Aliquots of the precleared whole cell lysates (lower panels) were also monitored for expression levels of PhLP-HA and Gbeta 1. <FR><NU>1</NU><DE>30</DE></FR> volume of each lysate was loaded on the gel. B, endogenous PhLP protein was immunoprecipitated from NG108-15 cell lysates using preimmune serum (PI) or a polyclonal anti-PhLP antibody. The precipitated proteins were then analyzed by Western blot for the presence of Gbeta subunits using a monoclonal anti-Gbeta 1 antibody. The position of immunoglobulin heavy and light chains is indicated (*). Experiments were repeated at least twice with similar results.
[View Larger Version of this Image (27K GIF file)]

Interaction of endogenous PhLP and Gbeta gamma was examined in NG108-15 neuroblastoma × glioma cells because this cell line expresses high basal levels of PhLP.3 Endogenous PhLP protein from NG108-15 cell lysates was immunoprecipitated by an affinity-purified polyclonal antiserum directed against PhLPS. On Western blot analysis of NG108-15 cell lysates, this antiserum recognized a single band migrating at 46 kDa, the expected molecular mass for full-length PhLP protein (data not shown) (12). The antiserum also immunoprecipitated a 46-kDa protein from [35S]methionine-labeled NG108-15 cells (data not shown). Anti-PhLP immunoprecipitates contained Gbeta as detected by Western blot analysis (Fig. 4B). Preimmune serum did not precipitate Gbeta (Fig. 4B). These results confirm the overexpression studies (Fig. 4A) and suggest that PhLP might exist, at least partially, as a complex with Gbeta gamma subunits in vivo.

In conclusion, these studies have documented the direct interaction of PhLP with Gbeta gamma through both in vivo and in vitro analyses. Our deletion analysis, together with the recent crystal structure of the phosducin-Gtbeta gamma complex, suggests that two potentially independent domains on PhLP interact with Gbeta gamma . This complements prior studies on PhLP that suggested that regions beyond the N terminus were involved in inhibition of Gbeta gamma function in vitro (14). Because the two domains of PhLP are predicted to interact with functionally different regions of Gbeta (17) and we have previously shown the existence of multiple forms of the PhLP transcript, it is tempting to speculate that alternate forms of PhLP might produce distinct changes in Gbeta gamma signaling. For example, PhLPS contains predominantly the C-terminal Gbeta gamma -binding domain and thus might produce different kinetics or extent of changes in Gbeta gamma function than the full-length PhLP protein. It remains to be determined which of the diverse Gbeta gamma cellular effects are functionally modified by PhLP-Gbeta gamma interactions.


FOOTNOTES

*   This work was supported by grants from the National Institute on Alcohol Abuse and Alcoholism (to M. F. M.) and by intramural funding from the Ernest Gallo Clinic and Research Center.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    Present address: Veterans Administration Medical Center, Box 0114, 4150 Clement St., San Francisco, CA 94121.
§   Recipient of career development award AA00118 (K02) from the National Institute on Alcohol Abuse and Alcoholism. To whom correspondence should be addressed: Ernest Gallo Clinic and Research Center, San Francisco General Hospital, Bldg. 1, Rm. 101, 1001 Potrero Ave., San Francisco, CA 94110. Tel.: 415-648-7111; Fax: 415-648-7116; E-mail: miles{at}itsa.ucsf.edu.
1   Gbeta and Ggamma are thought to exist as an obligate complex and are thus referred to here as Gbeta gamma even in instances where direct interaction may only involve Gbeta .
2   The abbreviations used are: beta ARK, beta -adrenergic receptor kinase; DMEM, Dulbecco's modified Eagle's medium; GST, glutathione S-transferase; HA, hemagglutinin; PhLP, phosducin-like protein; PhLPS, phosducin-like protein, short form (PhLP84-301); PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis.
3   C. Thibault and M. F. Miles, unpublished results.

ACKNOWLEDGEMENTS

We thank Steven Barhite, Ivan Diamond, Ulrike Heberlein, and Robert Messing at the Ernest Gallo Clinic and Research Center for many helpful discussions during the course of these studies. We also thank Henry Bourne, Cheryl Craft, Eva Neer, and Rene Onrust for gifts of proteins and plasmids as noted under "Experimental Procedures."


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