RGS4 Binds to Membranes through an Amphipathic α-Helix
2000; Elsevier BV; Volume: 275; Issue: 24 Linguagem: Inglês
10.1074/jbc.m000618200
ISSN1083-351X
AutoresLeah S. Bernstein, Andrew A. Grillo, Stephanie S. Loranger, Maurine E. Linder,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoRGS4, a mammalian GTPase-activating protein for G protein α subunits, requires its N-terminal 33 amino acids for plasma membrane localization and biological activity (Srinivasa, S. P., Bernstein, L. S., Blumer, K. J., and Linder, M. E. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 5584–5589). In this study, we tested the hypothesis that the N-terminal domain mediates membrane binding by forming an amphipathic α-helix. RGS4 bound to liposomes containing anionic phospholipids in a manner dependent on the first 33 amino acids. Circular dichroism spectroscopy of a peptide corresponding to amino acids 1–31 of RGS4 revealed that the peptide adopted an α-helical conformation in the presence of anionic phospholipids. Point mutations that either neutralized positive charges on the hydrophilic face or substituted polar residues on the hydrophobic face of the model helix disrupted plasma membrane targeting and biological activity of RGS4 expressed in yeast. Recombinant mutant proteins were active as GTPase-activating proteins in solution but exhibited diminished binding to anionic liposomes. Peptides corresponding to mutants with the most pronounced phenotypes were also defective in forming an α-helix as measured by circular dichroism spectroscopy. These results support a model for direct interaction of RGS4 with membranes through hydrophobic and electrostatic interactions of an N-terminal α-helix. RGS4, a mammalian GTPase-activating protein for G protein α subunits, requires its N-terminal 33 amino acids for plasma membrane localization and biological activity (Srinivasa, S. P., Bernstein, L. S., Blumer, K. J., and Linder, M. E. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 5584–5589). In this study, we tested the hypothesis that the N-terminal domain mediates membrane binding by forming an amphipathic α-helix. RGS4 bound to liposomes containing anionic phospholipids in a manner dependent on the first 33 amino acids. Circular dichroism spectroscopy of a peptide corresponding to amino acids 1–31 of RGS4 revealed that the peptide adopted an α-helical conformation in the presence of anionic phospholipids. Point mutations that either neutralized positive charges on the hydrophilic face or substituted polar residues on the hydrophobic face of the model helix disrupted plasma membrane targeting and biological activity of RGS4 expressed in yeast. Recombinant mutant proteins were active as GTPase-activating proteins in solution but exhibited diminished binding to anionic liposomes. Peptides corresponding to mutants with the most pronounced phenotypes were also defective in forming an α-helix as measured by circular dichroism spectroscopy. These results support a model for direct interaction of RGS4 with membranes through hydrophobic and electrostatic interactions of an N-terminal α-helix. regulator of G protein signaling 1,2-dipalmitoyl-sn-glycero-3-phosphocholine 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] liver phosphatidylcholine brain phosphatidylserine green fluorescent protein dithiothreitol 3-[N-morpholino]propanesulfonic acid wild type GTPase-activating protein Regulators of G protein signaling (RGS proteins)1 are a recently appreciated family of proteins that participate as negative regulators or effectors in G protein pathways (reviewed in Refs. 1.Hepler J.R. Trends Pharm. Sci. 1999; 20: 376-382Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar and 2.Diverse-Pierluissi M.A. Fischer T. Jordan J.D. Schiff M. Ortiz D.F. Farquhar M.G. De Vries L. J. Biol. Chem. 1999; 274: 14490-14494Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). RGS proteins catalytically accelerate GTP hydrolysis on α subunits, resulting in faster termination of G protein signaling. The GAP activity of RGS proteins may account for discrepancies between the measured intrinsic rates of GTP hydrolysis of the α subunit and the deactivation rate of physiological effectors. In addition to their functions as GAPs, some RGS proteins may also regulate G protein pathways by serving as effector antagonists (3.Hepler J.R. Berman D.M. Gilman A.G. Kozasa T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 428-432Crossref PubMed Scopus (338) Google Scholar, 4.Wieland T. Chen C.-K. Simon M.I. J. Biol. Chem. 1997; 272: 8853-8856Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). As new RGS family members are identified and characterized, it has become clear that RGS proteins can act as effectors, as well as inhibitors, of G protein pathways (5.Hart M.J. Jiang X. Kozasa T. Roscoe W. Singer W.D. Gilman A.G. Sternweis P.C. Bollag G. Science. 1998; 280: 2112-2114Crossref PubMed Scopus (677) Google Scholar). More than 20 mammalian RGS proteins have been identified to date (1.Hepler J.R. Trends Pharm. Sci. 1999; 20: 376-382Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). All RGS family members share sequence similarity that extends over approximately 120 amino acids. In many RGS proteins, this so-called "RGS box" or core domain is sufficient to bind G protein α subunits and catalyze GTPase activity in vitro (6.Faurobert E. Hurley J.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2945-2950Crossref PubMed Scopus (94) Google Scholar, 7.Popov S., Yu, K. Kozasa T. Wilkie T.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7216-7220Crossref PubMed Scopus (149) Google Scholar, 8.Chen C. Lin S.-C. FEBS Lett. 1998; 422: 359-362Crossref PubMed Scopus (36) Google Scholar, 9.Srinivasa S.P. Watson N. Overton M.C. Blumer K.J. J. Biol. Chem. 1998; 273: 1529-1533Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). However in a cellular context, regions outside the RGS domain are necessary for biological activity of the protein (8.Chen C. Lin S.-C. FEBS Lett. 1998; 422: 359-362Crossref PubMed Scopus (36) Google Scholar, 10.Srinivasa S.P. Bernstein L.S. Blumer K.J. Linder M.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5584-5589Crossref PubMed Scopus (131) Google Scholar). Thus, important regulatory information is likely to be contained within these highly divergent flanking regions of RGS proteins. One way in which these RGS flanking regions can modulate protein activity is by determining subcellular localization. For several RGS proteins, regions near the N terminus are responsible for targeting the proteins to particular cellular locations. RGS-GAIP and RGSZ1 contain cysteine string motifs near their N termini. These domains contain multiple sites for palmitoylation, which is presumed to promote association with membranes (11.DeVries L. Elenko E. Hubler L. Jones T.L.Z. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15203-15208Crossref PubMed Scopus (157) Google Scholar, 12.Wang J. Ducret A. Tu Y. Kozasa T. Aebersold R. Ross E.M. J. Biol. Chem. 1998; 273: 26014-26025Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Many other RGS proteins contain protein-protein interaction domains that may determine their localization (1.Hepler J.R. Trends Pharm. Sci. 1999; 20: 376-382Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). We demonstrated previously that a domain in RGS4 consisting of the first 33 amino acids of the protein is necessary for biological activity and is responsible for targeting RGS4 to the plasma membrane (10.Srinivasa S.P. Bernstein L.S. Blumer K.J. Linder M.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5584-5589Crossref PubMed Scopus (131) Google Scholar). RGS5 and RGS16 share significant sequence homology in this region, suggesting that this region codes for a functionally important domain. Indeed, a recent study has revealed that the corresponding region of RGS16 is also required for membrane association and biological activity (13.Chen C. Seow K.T. Guo K. Yaw L.P. Lin S.-C. J. Biol. Chem. 1999; 274: 19799-19806Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Wilkie and co-workers (14.Zeng W. Xu X. Popov S. Mukhopadhyay S. Chidiac P. Swistok J. Danho W. Yagaloff K.A. Fisher S.L. Ross E.M. Muallem S. Wilkie T.M. J. Biol. Chem. 1998; 273: 34687-34690Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar) have shown that the N-terminal domain of RGS4 mediates receptor selectivity, affirming its functional importance. RGS4 and RGS16 are palmitoylated at cysteine residues within the conserved N-terminal domain. However, mutation of the N-terminal cysteines in RGS4 or RGS16 does not interfere with membrane association of the proteins (10.Srinivasa S.P. Bernstein L.S. Blumer K.J. Linder M.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5584-5589Crossref PubMed Scopus (131) Google Scholar, 13.Chen C. Seow K.T. Guo K. Yaw L.P. Lin S.-C. J. Biol. Chem. 1999; 274: 19799-19806Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 15.Druey K.M. Ugur O. Caron J.M. Chen C.-K. Backlund P.S. Jones T.L.Z. J. Biol. Chem. 1999; 274: 18836-18842Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). This suggests that other structural features of the N-terminal domain are mediating membrane attachment. We proposed a model based on secondary structure predictions that formation of an amphipathic α-helix within the N-terminal domain is responsible for membrane targeting (10.Srinivasa S.P. Bernstein L.S. Blumer K.J. Linder M.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5584-5589Crossref PubMed Scopus (131) Google Scholar). When modeled as an α-helical wheel, this domain is an amphipathic α-helix with hydrophobic residues, including the two palmitoylated cysteines, lying on one face of the helix and positively charged hydrophilic residues on the opposing face (Fig. 1). Basic residues aligned on the hydrophilic face of the helix could associate with the head groups of anionic phospholipids of the membrane. Additionally, the nonpolar residues and the palmitate molecules on the cysteine residues may insert partially into the lipid bilayer of the membrane, yielding hydrophobic interactions with the lipid tails in the inner surface of the membrane. The helix is not necessarily continuous throughout the domain. At least two other proteins, CTP:phosphocholine cytidylyltransferase and prostaglandin endoperoxide H synthases 1 and 2, rely on amphipathic α-helices for mediating their associations with membranes (16.Dunne S.J. Cornell R.B. Johnson J.E. Glover N.R. Tracey A.S. Biochemistry. 1996; 35: 11975-11984Crossref PubMed Scopus (91) Google Scholar, 17.Picot D. Loll P.J. Garavito M. Nature. 1994; 367: 243-249Crossref PubMed Scopus (1154) Google Scholar, 18.Li Y. Smith T. Grabski S. DeWitt D.L. J. Biol. Chem. 1998; 273: 29830-29837Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Lin and co-workers (13.Chen C. Seow K.T. Guo K. Yaw L.P. Lin S.-C. J. Biol. Chem. 1999; 274: 19799-19806Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) have modeled residues 12–30 of RGS16 as an amphipathic helix. Mutations that disrupt hydrophobic residues of the nonpolar face of the helix and positively charged side chains along the polar/nonpolar interface of the helix prevent plasma membrane localization of RGS16, suggesting that amphipathic features of RGS16 are required for membrane association. In this study, we demonstrate using model membranes that RGS4 has an intrinsic affinity for anionic lipids that is dependent upon its N-terminal 33-amino acid domain. We show that a peptide corresponding to this domain adopts an α-helical conformation in the presence of anionic phospholipids. We further demonstrate that both hydrophobic and basic amino acids contribute to the propensity of the domain to form an α-helix. These same residues are required for membrane bindingin vivo and in vitro and for biological activity. These results support a model in which an amphipathic α-helix within the N-terminal 33-amino acids mediates membrane association of RGS4 and its relatives RGS5 and RGS16. RGS4-GFP, Δ1–33 RGS4-GFP, and GFP were expressed in yeast from a constitutive ADH promoter in pVT102U as described previously (10.Srinivasa S.P. Bernstein L.S. Blumer K.J. Linder M.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5584-5589Crossref PubMed Scopus (131) Google Scholar). Wild type (WT) RGS4-GFP was subcloned as a BamHI-XhoI fragment into pBluescript for construction of the point mutants. K17A/R22A, R14A/K17A/R22A/K29A, and L23Q/L26Q/L27Q were made using the Quikchange mutagenesis kit (Stratagene). Mutant RGS4-GFP constructs were subcloned into pVT102U for expression in yeast for microscopy. For expression from a low copy plasmid in yeast, the ADH promoter, RGS4-GFP coding sequence, and ADH1 3′ region from pVT102U were subcloned as aSphI fragment into the SphI site of YCp50. For expression of RGS4 in Escherichia coli, WT RGS4 was amplified by polymerase chain reaction as aNcoI-SmaI fragment and subcloned into H6TEVpQE60 (19.Lee E. Linder M.E. Gilman A.G. Methods Enzymol. 1994; 237: 146-164Crossref PubMed Scopus (247) Google Scholar). This strategy required the addition of an alanine residue following the initial methionine of RGS4 to complete the NcoI site. For subcloning of the mutant RGS4 clones, a modified H6TEVpQE60 vector was created using His6pQE60 (19.Lee E. Linder M.E. Gilman A.G. Methods Enzymol. 1994; 237: 146-164Crossref PubMed Scopus (247) Google Scholar) as a template. A double stranded oligonucleotide encoding the TEV protease site (ENLYFQG) andBamHI site and SpeI restriction sites was ligated with H6pQE60 digested with NcoI andHinDIII. The sequences of the oligonucleotides were 5′-CATGGCTGAGAATCTTTATTTTCAGGGATCCTAGACTAGTTAATAACCCGGGTAATA-3′ and 5′-AGCTTATTACCCGGGTTATTAACTAGTCTAGGATCCCTGAAAATAAAGATTCTCAGC-3′. The RGS4 mutants were then subcloned from RGS4-GFP-pBS asBamHI-XbaI fragments, removing the GFP tag and allowing ligation into the BamHI and SpeI sites of the new vector to create H6TEV-RGS4-pQE60. As an artifact of this subcloning, restriction sites contributed extra nucleotides at both ends of the RGS4 cloning sequence. After cleavage with TEV, WT RGS4 had an additional two amino acids (GA) at the N terminus. Mutant RGS4 proteins had an additional four amino acids at the N terminus (GSGT) and two amino acids at the C terminus (SS). The integrity of all RGS4 constructs was verified by DNA sequence analysis (Washington University Protein and Nucleic Acid Laboratory). H6TEV-RGS4-pQE60 constructs were transformed into the bacterial strain JM109. 1–2 L of transformed bacteria were grown to A 600 of 0.5–0.8 and induced at 30 °C with 500 μmisopropyl-1-thio-β-d-galactopyranoside. Bacterial pellets were lysed in 50 mm sodium phosphate buffer, pH 8, containing 50 mm KCl, 10 mmβ-mercaptoethanol, and 2 mg/ml aprotinin by freeze-thaw and sonication. His6-TEV-RGS4 proteins were purified by chromatography on Ni2+-nitrilotriacetic acid agarose beads (Qiagen). The proteins were eluted with 100–250 mmimidazole in 50 mm Tris-HCl, pH 6.6, 50 mmNaCl, and 2.5 mm DTT. Peak fractions were dialyzed against 20 mm sodium phosphate buffer, pH 7.2, containing 150 mm NaCl and 10 mm β-mercaptoethanol (NaPi buffer). Purified protein (0.5–2 mg) was digested with His-tagged TEV protease (Life Technologies, Inc.) according to the manufacturer's instructions. His-tagged TEV protease and uncleaved RGS4 were separated from cleaved RGS4 by Ni2+-nitrilotriacetic acid agarose chromatography. Cleaved RGS4 was collected in the flow-through and in sequential washes with NaPi buffer containing 0, 5, and 20 mmimidazole. Protein was dialyzed against HEDG buffer (20 mmHepes, pH 8, 1 mm EDTA, 2 mm DTT, 10% glycerol) and concentrated to 0.3–2.3 mg/ml. GAP activity of WT and mutant RGS4 protein preparations was determined using a solution based single-turnover GTP hydrolysis assay modified from Linderet al. (20.Linder M.E. Ewald D.A. Miller R.J. Gilman A.G. J. Biol. Chem. 1990; 265: 8243-8251Abstract Full Text PDF PubMed Google Scholar). Recombinant myristoylated Goα(200 nm) was incubated with 1 μm[γ-32P]GTP (10,000 cpm/pmol) (NEN Life Science Products) in 50 mm NaHepes, pH 8.0, 5 mm EDTA, 1 mm DTT, and 0.05% Lubrol for 20 min at 25 °C then returned to 4 °C. GTP, MgSO4 and RGS4 were added to final concentrations of 150 μm, 15 mm and 20 nm, respectively, to initiate GTP hydrolysis. Aliquots (50 μl) were taken at 15-s intervals for the first minute and then every minute for 5 min. Samples were processed as described (20.Linder M.E. Ewald D.A. Miller R.J. Gilman A.G. J. Biol. Chem. 1990; 265: 8243-8251Abstract Full Text PDF PubMed Google Scholar). Vesicles were prepared using an adaptation of previously described methods (21.Kim J. Blackshear P.J. Johnson J.D. McLaughlin S. Biophys. J. 1994; 67: 227-237Abstract Full Text PDF PubMed Scopus (146) Google Scholar, 22.Vergeres G. Ramsden J.J. Biochem. J. 1998; 330: 5-11Crossref PubMed Scopus (31) Google Scholar, 23.MacDonald R.C. MacDonald R.I. Menco B.P.M. Takeshita K. Subbarao N.K. Hu L.-r. Biochim. Biophys. Acta. 1991; 1061: 297-303Crossref PubMed Scopus (1389) Google Scholar). Lipids (3 mg) (Avanti Polar Lipids) and 1 μCi of [3H]phosphatidylcholine tracer (NEN Life Science Products) were lyophilized and suspended in 400 μl of 10 mm MOPS, pH 7.4, 0.1 mm EGTA, 170 mm sucrose, 1 mm DTT (buffer B). The suspension was sonicated briefly to break up aggregates and subjected to five cycles of freeze/thaw using liquid nitrogen and a 37 °C bath. The mixture was extruded through two 100-nm pore polycarbonate membranes using the LiposoFast Basic and Stabilizer (Avestin, Inc.) (15–19 passes). The vesicles were diluted 5-fold in buffer C (10 mm MOPS, pH 7.4, 100 mm KCl, 0.1 mmEGTA), incubated for 15 min at room temperature, and collected by centrifugation at 100,000 × g for 1 h at 22 °C. The pellet containing sucrose-loaded vesicles was suspended in 100 μl of buffer C. The lipid concentration of the vesicles was calculated based on the recovery of [3H]phosphatidylcholine in the vesicle pellet. Recombinant protein (0.2 μm) and lipid vesicles (1 mm) were incubated in 100 μl of buffer C for 15 min at room temperature. The reaction was subjected to centrifugation at 100,000 × g for 1 h at 22 °C. The supernatant (90 μl, 90% of the total) was removed, and the pellet was suspended in 90 μl of buffer C. For immunoblots, both fractions were solubilized overnight in SDS sample buffer. Samples were resolved by SDS-polyacrylamide gel electrophoresis (13% gel) and transferred to nitrocellulose (Micron Separations Inc.). After incubation in blocking buffer (50 mm Tris-HCl, pH 8, 2 mm CaCl2, 80 mm NaCl, 5% nonfat dry milk, 0.2% (v/v) Nonidet P-40, and 0.02% sodium azide) for 30 min, the blot was probed with the polyclonal antiserum WU872 (1:1000 in Tris-buffered saline containing 0.1% Tween-20) for 1 h; WU872 was generated against a peptide (NH2-SFKLKSEFSEENIEFWLAC-COOH) derived from the core domain of RGS1 and is cross-reactive with the core domains of RGS4 and other RGS proteins. Immunoreactive proteins were detected by incubation with horseradish peroxidase-conjugated goat anti-rabbit (Cappel) and visualized by Supersignal Chemiluminescent Substrate (Pierce). For dot-blot analysis, vesicles were added to the supernatant fraction equivalent to the amount added to the initial reaction so that both fractions had the same lipid composition. An aliquot (25 μl) of each fraction was spotted in duplicate onto nitrocellulose using a dot-blot vacuum manifold. Each well was washed once with 100 μl of buffer C. The nitrocellulose membrane was incubated in blocking buffer for 30 min, followed by a 1-h incubation with antiserum WU872. After washing with blocking buffer, the nitrocellulose was incubated for 1 h with125I-conjugated goat anti-rabbit antibody (ICN). After washing, the nitrocellulose was exposed to a phosphorimaging screen for 12–24 h. The image was scanned using a Molecular Dynamics PhosphorImager, and the images were processed using ImageQuant software. Bovine brain membranes were prepared as described (24.Sternweis P.C. Robishaw J.D. J. Biol. Chem. 1984; 259: 13806-13813Abstract Full Text PDF PubMed Google Scholar). Recombinant protein (0.4 μm) and bovine brain membranes (200 μg) were incubated in TED (20 mm Tris, pH 8, 1 mm EDTA, 1 mm DTT) for 1 h at 4 °C. The reaction was loaded at the bottom of a three-step sucrose gradient (1.4, 1.2, and 0.5 m) and centrifuged in a TLS-55 rotor (Beckman) at 35,000 rpm for 1 h at 4 °C. The membranes floated up to form a band at the 1.2–0.5 m interface. The membrane band (pellet) and the layers below the band were collected (soluble fraction). The membranes were diluted 5-fold in TED and collected by centrifugation at 200,000 × g in a TL-100 rotor (Beckman). The soluble fraction was precipitated with 4 volumes of methanol at −20 °C for 1 h and then collected by centrifugation in a microcentrifuge. Both membrane and soluble fractions were suspended in SDS sample buffer and subjected to immunoblot analysis as above. Peptides corresponding to the first 31 amino acids of WT and mutant RGS4 were synthesized and purified by reverse phase high pressure liquid chromatography (BioMolecules Midwest). Residues 32 and 33 were excluded from the peptides to simplify the synthesis. The molecular weights of the peptides were determined by electrospray mass spectrometry for WT (3376), K17A/R22A (3234), R14A/K17A/R22A/K29A (3091), and L23Q/L26Q/L27Q (3422) peptides. These values corresponded to the predicted molecular weights of the sequences. The peptides were stored as a solid at −20 °C in a dessicator. A stock solution for each peptide was made by solubilizing ∼15 mg of solid peptide in 1 ml of double distilled water except the K17A/R22A peptide (15 mg/10 ml of water). The concentration of each peptide stock solution was determined by amino acid analysis. Stock solutions were aliquoted, stored at −20 °C, and thawed only once. 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DPPG) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL) and stored in chloroform stocks under argon at −80 °C. Small unilamellar liposomes were prepared by a method modified from Johnson and Cornell (25.Johnson J.E. Cornell R.B. Biochemistry. 1994; 33: 4327-4335Crossref PubMed Scopus (67) Google Scholar). Lipids from chloroform stocks were mixed in small glass vials and dried by nitrogen evaporation followed by high vacuum for a minimum of 1 h. The lipids were hydrated in 20 mm sodium phosphate, pH 7.0, to a lipid concentration of 20 mm. The suspension was sonicated in a bath sonicator (Laboratory Supply Co.) until the solution cleared. The sonicated solution was centrifuged at 1500 × g for 5 min in a Beckman TL-100 Ultracentrifuge to remove debris and multilamellar vesicles. The small unilamellar vesicles in the supernatant were used for CD spectroscopy the same day as prepared. Liposomes were examined by high performance thin layer chromatography (26.Macala L.J., Yu, R.K. Ando S. J. Lipid. Res. 1983; 24: 1243-1250Abstract Full Text PDF PubMed Google Scholar) to monitor the ratio of lipids. Far UV CD spectra were taken on a Jasco J600 Spectropolarimeter and recorded on a Dell 486D/50 personal computer for data processing. Spectra were recorded from 250 to 190 nm in 0.4-nm steps at 50 nm/min, with five spectra being averaged together. A 1-mm-path length quartz cell was used with the instrument at ambient temperature. For measurements, peptide and liposome stocks were diluted in 20 mm sodium phosphate, pH 7.0, to final concentrations of 30 μm and 4 mm, respectively (16.Dunne S.J. Cornell R.B. Johnson J.E. Glover N.R. Tracey A.S. Biochemistry. 1996; 35: 11975-11984Crossref PubMed Scopus (91) Google Scholar, 25.Johnson J.E. Cornell R.B. Biochemistry. 1994; 33: 4327-4335Crossref PubMed Scopus (67) Google Scholar). Background spectra of lipid vesicles alone gave a minimal signal and were subtracted from peptide spectra. Final data are expressed as mean residue molar ellipticity (deg cm2/dmol). The percentage of α-helix was estimated from the molar ellipticity at 222 nm (θ222) using the equation f h = (θ222/θh222∞) + (iκ/N) where f h is the fraction in α-helical form, θ222 is the mean residue molar ellipticity at 222 nm, θh222∞ is the molar ellipticity at 222 nm for an infinitely long α-helix (−39,500 deg cm2/dmol), i is number of helices (assumed to be one), κ is a wavelength specific constant (2.6 at 222 nm), andN is the number of residues in the peptide (31 residues) (16.Dunne S.J. Cornell R.B. Johnson J.E. Glover N.R. Tracey A.S. Biochemistry. 1996; 35: 11975-11984Crossref PubMed Scopus (91) Google Scholar, 27.Chang C.T. Wu C.C. Yang J.T. Anal. Biochem. 1978; 91: 13-31Crossref PubMed Scopus (1026) Google Scholar). The yeast strain used for microscopy was SWY518 (Mataura3-1 his3-11, 15 trp1-1 leu2-3, 112 can1) (28.Bucci M. Wente S.R. J. Cell Biol. 1997; 136: 1185-1199Crossref PubMed Scopus (96) Google Scholar) and for pheromone response assay was BC180 (MATa leu2-3, 112 ura3-52 his3Δ1 ade2-1 sst2-Δ2) (9.Srinivasa S.P. Watson N. Overton M.C. Blumer K.J. J. Biol. Chem. 1998; 273: 1529-1533Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Yeast cells were grown and pheromone response assays were performed as described previously (10.Srinivasa S.P. Bernstein L.S. Blumer K.J. Linder M.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5584-5589Crossref PubMed Scopus (131) Google Scholar). Previously we demonstrated that when expressed in yeast, RGS4 requires its N-terminal domain for targeting to the plasma membrane. Although RGS4 is palmitoylated at cysteine residues in the N-terminal domain, membrane association is independent of the post-translational modification (10.Srinivasa S.P. Bernstein L.S. Blumer K.J. Linder M.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5584-5589Crossref PubMed Scopus (131) Google Scholar). Therefore, to determine whether the association of RGS4 with membranes occurs via direct lipid-protein interactions, we examined the ability of RGS4 to bind to chemically defined sucrose-loaded lipid vesicles. RGS4 and the N-terminal deletion mutant, Δ1–33, were expressed as recombinant proteins with a cleavable hexahistidine tag and purified by nickel chelate chromatography. The tag was removed by cleavage with TEV protease, yielding the purified protein preparations shown in Fig. 5. Proteins were incubated with synthetic sucrose-loaded liposomes containing 33% brain phosphatidylserine (PS) and 67% liver phosphatidylcholine (PC). Vesicle-bound RGS4 was separated from soluble RGS4 by centrifugation. Both WT and Δ1–33 RGS4 were soluble in the absence of vesicles (Fig. 2 A). The small amount of RGS4 found in the pellet fraction was due to incomplete removal of the supernatant. In the presence of the PS:PC (1:2) vesicles, nearly all of the WT RGS4 was found in the vesicle pellet (Fig. 2 A). In contrast, the Δ1–33 mutant remained in the supernatant, indicating that the N-terminal domain is necessary for lipid-protein interaction. Next, we tested the dependence of RGS4 binding on the presence of anionic phospholipids in the vesicles. PS was titrated into PC vesicles from 0 to 60%. RGS4 bound poorly to vesicles containing pure PC but efficiently to vesicles containing 20% or more PS (data not shown). The Δ1–33 mutant required higher concentrations of PS (40% or greater) before appearing in the vesicle pellet (data not shown). We conclude that RGS4 interacts directly with lipids and does not require any protein factor to mediate its binding to a membrane in the presence of physiologically relevant concentrations of anionic phospholipids (29.Devaux P.F. Annu. Rev. Biophys. Biomol. Structure. 1992; 21: 417-439Crossref PubMed Scopus (187) Google Scholar).Figure 2RGS4 binds directly to anionic lipids in the membrane. A, binding of recombinant wild type RGS4 and Δ1–33 RGS4 to PS:PC (1:2) sucrose loaded vesicles. Pellet (P) and soluble (S) fractions of proteins in the presence (+) or absence (−) of vesicles were visualized by immunoblot. A mobility shift was seen for the WT protein because of the presence of vesicles in the gel. B, binding of recombinant wild type and Δ1–33 RGS4 to bovine brain membranes.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine whether our artificial liposomes adequately model cellular membranes, we examined the binding of recombinant RGS4 to bovine brain membranes. Greater than 50% of WT RGS4 bound to membranes, whereas none of the N-terminal deletion mutant bound (Fig. 2 B). Thus, qualitatively it appears that our observations of RGS4 association with liposomes are representative of its interactions with biological membranes. Our hypothesis predicts that the N terminus of RGS4 forms an amphipathic α-helix. Structural information for this region of the protein was not available from the x-ray crystal structure (30.Tesmer J.J.G. Berman D.M. Gilman A.G. Sprang S.R. Cell. 1997; 89: 251-261Abstract Full Text Full Text PDF PubMed Scopus (691) Google Scholar). Therefore, we used CD spectroscopy to examine the structure of a peptide corresponding to the first 31 residues of RGS4. Residues 32 and 33 of the N-terminal domain were omitted from the peptide to simplify the synthesis. Far UV circular dichroism is useful for determining the secondary structure elements of proteins such as β-sheet, α-helix, and random coil. These types of secondary structure can be distinguished by the characteristic shape of the CD spectrum. In aqueous solution, the RGS4 peptide adopted a random coil conformation as seen by the single minimum at 200 nm (Fig.3). To evaluate the conformation of the RGS4 peptide in the presence of liposomes, vesicles were prepared with DPPG and DPPC. This lipid composition was chosen because vesicles containing DPPG and DPPC gave no significant si
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