Interaction of Phosducin-like Protein with G Protein βγ Subunits

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 Gβγ 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 Gβγ dimer in vitro and in vivo. Using PhLP glutathione S-transferase fusion proteins, we show that PhLP directly binds Gβγ in vitro. Studies with a series of truncated PhLP fusion proteins indicate independent binding of Gβγ to both the amino- and C-terminal halves of PhLP. Protein-protein interactions between Gβγ and PhLP are inhibited by the α subunit of Go and Gi3, suggesting that PhLP can bind only free Gβγ. Finally, we show that PhLP complexes, at least partially, with Gβγin vivo. Following overexpression of epitope-tagged PhLP together with Gβ1γ2 proteins in COS-7 cells, a PhLP-Gβγ complex is co-immunoprecipitated by monoclonal antibody directed against the epitope tag. Similarly, polyclonal anti-PhLP antibody co-precipitates endogenous PhLP and Gβγ proteins from NG108-15 cell lysates. These data are consistent with the hypothesis that PhLP is a widely expressed modulator of Gβγ function. Furthermore, because alternate forms of the PhLP transcript are expressed, there may be functional implications for the existence of two Gβγ-binding domains on PhLP. 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 Gβγ 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 Gβγ dimer in vitro and in vivo. Using PhLP glutathione S-transferase fusion proteins, we show that PhLP directly binds Gβγ in vitro. Studies with a series of truncated PhLP fusion proteins indicate independent binding of Gβγ to both the amino- and C-terminal halves of PhLP. Protein-protein interactions between Gβγ and PhLP are inhibited by the α subunit of Go and Gi3, suggesting that PhLP can bind only free Gβγ. Finally, we show that PhLP complexes, at least partially, with Gβγin vivo. Following overexpression of epitope-tagged PhLP together with Gβ1γ2 proteins in COS-7 cells, a PhLP-Gβγ complex is co-immunoprecipitated by monoclonal antibody directed against the epitope tag. Similarly, polyclonal anti-PhLP antibody co-precipitates endogenous PhLP and Gβγ proteins from NG108-15 cell lysates. These data are consistent with the hypothesis that PhLP is a widely expressed modulator of Gβγ function. Furthermore, because alternate forms of the PhLP transcript are expressed, there may be functional implications for the existence of two Gβγ-binding domains on PhLP. 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 (1Gilman A.G. Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Scopus (4714) Google Scholar, 2Simon M.I. Strathmann M.P. Gautam N. Science. 1991; 252: 802-808Crossref PubMed Scopus (1585) Google Scholar). In its trimeric form, Gαβγ is inactive, and the Gα subunit binds a molecule of GDP. Upon ligand binding, the receptor catalyzes the exchange of GDP for GTP on Gα that causes its activation and dissociation from the tightly bound Gβγ complex. 1Gβ and Gγ are thought to exist as an obligate complex and are thus referred to here as Gβγ even in instances where direct interaction may only involve Gβ. Inactivation and reassociation of the heterotrimer is initiated by the hydrolysis of bound GTP into GDP by an intrinsic GTPase activity of the Gα subunit. It is now known that both the free GTP-bound Gα and the Gβγ dimer can bind and regulate downstream effectors including adenylyl cyclases, phospholipases, and ion channels, and thereby modulate second messenger levels and ion flux (3Neer E.J. Cell. 1995; 80: 249-257Abstract Full Text PDF PubMed Scopus (1287) Google Scholar). The discovery of several specific Gβγ binding proteins has recently shed light on new roles for Gβγ in the propagation and termination of cellular signaling. The dimer has been shown to recruit β-adrenergic receptor kinase (βARK) 2The abbreviations used are: βARK, β-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. to its membrane-associated receptor substrate and thus initiate receptor desensitization (4Haga K. Haga T. J. Biol. Chem. 1992; 267: 2222-2227Abstract Full Text PDF PubMed Google Scholar, 5Pitcher J.A. Inglese J. Higgins J.B. Arriza J.L. Casey P.J. Kim C. Benovic J.L. Kwatra M.M. Caron M.G. Lefkowitz R.J. Science. 1992; 257: 1264-1267Crossref PubMed Scopus (573) Google Scholar). This process occurs via direct binding of Gβγ to the C terminus of a putative pleckstrin homology domain on βARK (6Pitcher J.A. Touhara K. Payne E.S. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 11707-11710Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar). Furthermore, the responsiveness of G protein-regulated signaling systems may be directly modulated through the interaction of Gβγ subunits with intracellular regulatory proteins. For instance, phosducin, a phosphoprotein mainly expressed in the retina and pineal gland, inhibits the phototransduction cascade by scavenging βγ subunits of the G protein transducin (Gt), thus preventing their reassociation with the Gtα subunit (7Lee R.H. Lieberman B.S. Lolley R.N. Biochemistry. 1987; 26: 3983-3990Crossref PubMed Scopus (154) Google Scholar, 8Lee R.H. Ting T.D. Lieberman B.S. Tobias D.E. Lolley R.N. Ho Y.-K. J. Biol. Chem. 1992; 267: 25104-25112Abstract Full Text PDF PubMed Google Scholar). Because phosducin has a higher affinity for Gtβγ than does Gtα, it has been suggested that the formation of the phosducin/Gtβγ complex is a major factor regulating photoreceptor responsiveness (9Yoshida T. Willardson B.M. Wilkins J.F. Jensen G.J. Thornton B.D. Bitensky M.M. J. Biol. Chem. 1994; 269: 24050-24057Abstract Full Text PDF PubMed Google Scholar). From in vitro binding and co-transfection assays, it was proposed that phosducin may also compete with other targets for Gβγ binding, such as βARK and phospholipase C type β2 (10Hekman M. Bauer P.H. Söhlemann P. Lohse M.J. FEBS Lett. 1994; 343: 120-124Crossref PubMed Scopus (44) Google Scholar, 11Xu J. Wu D. Slepak V.Z. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2086-2090Crossref PubMed Scopus (66) Google Scholar). We recently isolated a rat brain cDNA encoding a phosducin-like protein (PhLP), which has 65% amino acid homology to phosducin (12Miles M.F. Barhite S. Sganga M. Elliott M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10831-10835Crossref PubMed Scopus (87) Google Scholar). 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 (12Miles M.F. Barhite S. Sganga M. Elliott M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10831-10835Crossref PubMed Scopus (87) Google Scholar, 13Wang J.-F. Barhite S. Thibault C. Miles M.F. Alcohol. Clin. Exp. Res. 1995; 19 (abstr.): 34Crossref Scopus (66) Google Scholar). Based on sequence homology with phosducin, we have suggested that PhLP proteins regulate Gβγ signaling in nonretinal tissues. In favor of this hypothesis, a recent report showed that recombinant PhLPS inhibits several Gβγ functionsin vitro (14Schröder S. Lohse M.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2100-2104Crossref PubMed Scopus (63) Google Scholar). Interestingly, these authors suggested that unlike phosducin (11Xu J. Wu D. Slepak V.Z. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2086-2090Crossref PubMed Scopus (66) Google Scholar, 15Hawes B.E. Touhara K. Kurose H. Lefkowitz R.J. Inglese J. J. Biol. Chem. 1994; 269: 29825-29830Abstract Full Text PDF PubMed Google Scholar), the N terminus of PhLP was unlikely to contain a Gβγ-binding domain. To more directly characterize the interaction of PhLP with Gβγ, we studied PhLP binding to Gβγ 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 βγ through a bipartite binding domain. The Gβγ-PhLP interaction was confirmed by co-immunoprecipitation of the complex from cell lysates. Our findings support the hypothesis that PhLP can modulate Gβγ function (14Schröder S. Lohse M.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2100-2104Crossref PubMed Scopus (63) Google Scholar) in many tissues through direct protein-protein interactions. Regulation of PhLP/Gβγ interactions could be an important factor in controlling G protein signaling. PhLP and PhLPS cDNAs were cloned in our laboratory (12Miles M.F. Barhite S. Sganga M. Elliott M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10831-10835Crossref PubMed Scopus (87) Google Scholar). The GST-phosducin construct was obtained from Dr. Cheryl Craft (University of Southern California). Gβγ and Goα proteins purified from bovine brain were kindly provided by Dr. Eva Neer (Brighman and Woman's Hospital, Boston, MA). Gβ1 and Gγ2 expression vectors were kind gifts from Dr. H. Bourne (University of California at San Francisco). Purified recombinant Gβ1γ2 was generously provided by Dr. Rene Onrust in the Bourne laboratory. Recombinant Giα3 protein was from Calbiochem. 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 andEcoRI sites, respectively. The amplified fragments were ligated in frame with the 3′-end of the coding region of GST intoBamHI 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) betweenEcoRI and NotI sites. The full-length PhLP coding sequence, amplified as described above, was then ligated between theBamHI 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. Fusion protein expression was induced with 0.1 mm isopropyl-1-thiol-β-d-galactopyranoside for 90 min, and the proteins were solubilized and purified on glutathione-Sepharose 4B resin (Pharmacia) by the Sarkosyl method (16Frangioni J.V. Neel B.G. Ann. Biochem. 1993; 210: 179-187Crossref Scopus (832) Google Scholar). 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 nmGβγ 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-β 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). 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). NG108-15 cells were grown as described (12Miles M.F. Barhite S. Sganga M. Elliott M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10831-10835Crossref PubMed Scopus (87) Google Scholar) 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. 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 Gβ1 subunit (Transduction Laboratories). To determine whether PhLP directly interacts with Gβγ, we generated PhLP and PhLPS GST fusion proteins and examined their ability to bind Gβγ purified from bovine brain. Following immobilization of the fusion proteins on glutathione-agarose beads, Gβγ binding was detected by Western blot analysis using an anti-β antiserum. As controls, we tested Gβγ binding by the GST protein itself and GST fused to phosducin (GST-phosducin), a known Gβγ-binding protein. The fusion proteins migrate at their expected as by and can be detected with an anti-GST antibody on Western blot analysis In proteins are by an affinity-purified polyclonal antiserum directed against PhLPS As by other (11Xu J. Wu D. Slepak V.Z. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2086-2090Crossref PubMed Scopus (66) Google Scholar), GST-phosducin bound of Gβγ at a of of approximately GST-PhLPS and also Gβγ GST protein bind Gβγ subunits even at a higher concentration than the proteins that Gβγ binding by the fusion proteins is by the PhLP protein The affinity of phosducin for Gβγ was to be in the (11Xu J. Wu D. Slepak V.Z. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2086-2090Crossref PubMed Scopus (66) Google Scholar, S. Lohse M.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2100-2104Crossref PubMed Scopus (63) Google Scholar). our to have a affinity for Gβγ than GST-phosducin of the of because with of Gβγ protein showed that only of Gβγ than GST-phosducin To the Gβγ binding domain of PhLP, we examined recombinant Gβ1γ2 interaction in vitro with GST fusion proteins containing regions of PhLP. an of PhLP constructs with the full-length PhLP. construct a protein that migrated at the expected on we Gβγ binding activity of PhLP at two in the and C-terminal In the N-terminal of PhLP amino acids for Gβγ binding, because and Gβγ, whereas The region an acid that is between PhLP and phosducin and is known to be for Gβγ binding by phosducin (11Xu J. Wu D. Slepak V.Z. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2086-2090Crossref PubMed Scopus (66) Google Scholar). Furthermore, the of the this was in showed that this sequence has and interactions with the of the A. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). This sequence region of PhLP (amino acids may be important for binding of Gβγ because βγ binding, whereas binding However, although the region may be important for Gβγ binding by PhLP, because bind Gβγ. that the C-terminal of PhLP also binds Gβγ. This binding activity was to the C-terminal residues of PhLP This C-terminal binding domain may PhLPS which does contain the sequence, Gβγ binding The of also showed two and domains in phosducin A. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). These two domains to the N-terminal and C-terminal halves of the molecule and with other both Gβγ. The N-terminal domain may compete with whereas the domain was suggested to be for Gβγ from the A. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). Based on sequence proposed a for PhLP and predicted the Gβγ residues in this protein A. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). The regions by these amino in to residues and in the N-terminal domain and to residues and in the C-terminal domain. Our results are in with these and also that the N-terminal and C-terminal domains can with Gβγ Because these domains may different of Gβγ, be to study different of Gβγ as suggested by A. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). studies have that the binding of Gβγ to Gα subunit inhibits its interaction with phosducin or βARK B.E. Touhara K. Kurose H. Lefkowitz R.J. Inglese J. J. Biol. Chem. 1994; 269: 29825-29830Abstract Full Text PDF PubMed Google Scholar, K. Inglese J. J.A. Lefkowitz R.J. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). In the N-terminal domain of the G as well as the factor were shown to with Gβγ or trimeric Gαβγ Casey P.J. 1995; Full Text PDF PubMed Scopus Google Scholar, M.I. Inglese J. C. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). We that recombinant Giα3 inhibited Gβγ binding to Similarly, the interaction of Gβγ to these results that only free Gβγ can with PhLP. Western blot analysis with a antiserum NEN) that Giα3 or Goα was on with the Gβγ dimer and data In Giα3 by itself bind to It be that a concentration of recombinant Giα3 was to Gβγ binding to This may the that Gβγ interacts more tightly with PhLP than with Gα as was for phosducin and Gtα interaction with Gβγ A. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). To Gβγ-PhLP interaction in the complex was following overexpression of the proteins in COS-7 For these the C terminus of PhLP was with a HA COS-7 cells were transfected with plasmids encoding Gβ1 and Gγ2 or a of these proteins. of the proteins in COS-7 cells was by Western blot analysis using monoclonal and 4 was by antibody 4 In this antibody Gβ1γ2 only in cells 4 We also detected in immunoprecipitates from cells transfected only with 4 suggesting that interacts with both and endogenous Gβγ of endogenous PhLP and Gβγ was examined in NG108-15 × cells because this cell levels of PhLP. Thibault and M. PhLP protein from NG108-15 cell was by an affinity-purified polyclonal antiserum directed against Western blot analysis of NG108-15 cell this antiserum a at the expected for full-length PhLP protein (12Miles M.F. Barhite S. Sganga M. Elliott M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10831-10835Crossref PubMed Scopus (87) Google Scholar). The antiserum also a protein from NG108-15 cells immunoprecipitates as detected by Western blot analysis 4 4 These results the overexpression studies 4 and that PhLP at least partially, as a complex with Gβγ subunits in vivo. In these studies have the direct interaction of PhLP with Gβγ through both in vivo and in vitro Our together with the recent of the that two independent domains on PhLP with Gβγ. This studies on PhLP that suggested that regions the N terminus were in of Gβγ function in vitro (14Schröder S. Lohse M.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2100-2104Crossref PubMed Scopus (63) Google Scholar). Because the two domains of PhLP are predicted to with different regions of A. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) and we have shown the existence of forms of the PhLP it is to that alternate forms of PhLP in Gβγ signaling. For PhLPS the C-terminal Gβγ-binding domain and thus different or of in Gβγ function than the full-length PhLP protein. It remains to be which of the Gβγ cellular are modified by PhLP-Gβγ interactions. We Barhite, and at the and for many the of these We also Cheryl Eva and Rene Onrust for gifts of proteins and plasmids as