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• W1990457307 abstract "Guanine nucleotide exchange in heterotrimeric G proteins catalyzed by G protein-coupled receptors (GPCRs) is a key event in many physiological processes. The crystal structures of the GPCR rhodopsin and two G proteins as well as binding sites on both catalytically interacting proteins are known, but the temporal sequence of events leading to nucleotide exchange remains to be elucidated. We employed time-resolved near infrared light scattering to study the order in which the Gα and Gγ C-terminal binding sites on the holo-G protein interact with the active state of the GPCR rhodopsin (R*) in native membranes. We investigated these key binding sites within mass-tagged peptides and G proteins and found that their binding to R* is mutually exclusive. The interaction of the holo-G protein with R* requires at least one of the lipid modifications of the G protein (i.e. myristoylation of the Gα N terminus and/or farnesylation of the Gγ C terminus). A holo-G protein with a high affinity Gα C terminus shows a specific change of the reaction rate in the GDP release and GTP uptake steps of catalysis. We interpret the data by a sequential fit model where (i) the initial encounter between R* and the G protein occurs with the Gβγ subunit, and (ii) the Gα C-terminal tail then interacts with R* to release bound GDP, thereby decreasing the affinity of R* for the Gβγ subunit. The mechanism limits the time in which both C-terminal binding sites of the G protein interact simultaneously with R* to a short lived transitory state. Guanine nucleotide exchange in heterotrimeric G proteins catalyzed by G protein-coupled receptors (GPCRs) is a key event in many physiological processes. The crystal structures of the GPCR rhodopsin and two G proteins as well as binding sites on both catalytically interacting proteins are known, but the temporal sequence of events leading to nucleotide exchange remains to be elucidated. We employed time-resolved near infrared light scattering to study the order in which the Gα and Gγ C-terminal binding sites on the holo-G protein interact with the active state of the GPCR rhodopsin (R*) in native membranes. We investigated these key binding sites within mass-tagged peptides and G proteins and found that their binding to R* is mutually exclusive. The interaction of the holo-G protein with R* requires at least one of the lipid modifications of the G protein (i.e. myristoylation of the Gα N terminus and/or farnesylation of the Gγ C terminus). A holo-G protein with a high affinity Gα C terminus shows a specific change of the reaction rate in the GDP release and GTP uptake steps of catalysis. We interpret the data by a sequential fit model where (i) the initial encounter between R* and the G protein occurs with the Gβγ subunit, and (ii) the Gα C-terminal tail then interacts with R* to release bound GDP, thereby decreasing the affinity of R* for the Gβγ subunit. The mechanism limits the time in which both C-terminal binding sites of the G protein interact simultaneously with R* to a short lived transitory state. IntroductionIn eukaryotes, signal transduction across cell membranes is in many cases based on the interplay between G protein-coupled receptors (GPCRs) 1The abbreviations used are: GPCR, G protein-coupled receptor; R*, signaling active receptor conformation of rhodopsin; Gt, transducin; Gtβγ, (farnesylated) heterodimeric βγ subunit of transducin; CT, C-terminal tail; far, farnesyl; MBP, maltose-binding protein; GTPγS, guanosine 5′-O-(thiotriphosphate); TLCK, 1-chloro-3-tosylamido-7-amino-2-heptanone; HPLC, high performance liquid chromatography. and heterotrimeric guanine nucleotide-binding proteins (G proteins, Gαβγ). Binding of extracellular signaling molecules like hormones, neurotransmitters, or odorants to GPCRs triggers structural rearrangements in the receptor, such that its intracellular domain becomes competent to catalyze nucleotide exchange in the heterotrimeric G protein (1Lu Z.L. Saldanha J.W. Hulme E.C. Trends Pharmacol. Sci. 2002; 23: 140-146Google Scholar).Rhodopsin is the visual pigment in retinal rod photoreceptors, those cells responsible for seeing under dim light conditions, and is the prototypical GPCR of the large family of rhodopsin-like GPCRs. Rhodopsin's ligand, the chromophore 11-cis-retinal, is covalently bound and recognizes a photon as an extracellular signal. Within 200 femtoseconds, the energy of the photon causes cis → trans isomerization of the retinal, thereby triggering the conversion of inactive dark-adapted rhodopsin into the active receptor conformation (R*), which is reached after milliseconds and is capable of interacting with transducin, the G protein of the rod cell (2Okada T. Ernst O.P. Palczewski K. Hofmann K.P. Trends Biochem. Sci. 2001; 26: 318-324Google Scholar).High resolution structures of transducin (Gt (3Lambright D.G. Sondek J. Bohm A. Skiba N.P. Hamm H.E. Sigler P.B. Nature. 1996; 379: 311-319Google Scholar) and the closely related heterotrimeric G protein Giα1β1γ2 (4Wall M.A. Coleman D.E. Lee E. Iniguez-Lluhi J.A. Posner B.A. Gilman A.G. Sprang S.R. Cell. 1995; 83: 1047-1058Google Scholar)) and rhodopsin (in the dark-adapted 11-cis-retinal bound state (5Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A. Le Trong I. Teller D.C. Okada T. Stenkamp R.E. Yamamoto M. Miyano M. Science. 2000; 289: 739-745Google Scholar)) are available (Fig. 1). However, static crystal structures alone cannot elucidate the dynamics of the receptor-G protein interaction. Previous studies have focused on identifying structural domains involved in catalysis. The key binding sites on transducin are the C-terminal tails (CT) of the Gα subunit and the farnesylated Gγ subunit of the Gβγ dimer (CTα and CTγ-far, respectively), which specifically recognize and bind to R* (6Hamm H.E. Deretic D. Arendt A. Hargrave P.A. Koenig B. Hofmann K.P. Science. 1988; 241: 832-835Google Scholar, 7Kisselev O.G. Ermolaeva M.V. Gautam N. J. Biol. Chem. 1994; 269: 21399-21402Google Scholar, 8Kisselev O.G. Meyer C.K. Heck M. Ernst O.P. Hofmann K.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4898-4903Google Scholar). In current models of nucleotide exchange, it is assumed that both of these sites act simultaneously on the distant nucleotide binding domain by a pull or lever mechanism (8Kisselev O.G. Meyer C.K. Heck M. Ernst O.P. Hofmann K.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4898-4903Google Scholar, 9Natochin M. Moussaif M. Artemyev N.O. J. Neurochem. 2001; 77: 202-210Google Scholar, 10Rondard P. Iiri T. Srinivasan S. Meng E. Fujita T. Bourne H.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6150-6155Google Scholar, 11Marin E.P. Krishna A.G. Sakmar T.P. J. Biol. Chem. 2001; 276: 27400-27405Google Scholar, 12Cherfils J. Chabre M. Trends Biochem. Sci. 2003; 28: 13-17Google Scholar, 13Hamm H.E. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4819-4821Google Scholar, 14Gautam N. Structure. 2003; 11: 359-360Google Scholar).In this work, we have investigated the unknown temporal sequence of interaction between the CTα and CTγ-far sites and R*. We used “mass-tagged” peptides (in which the key CTs are fused to functionally neutral maltose-binding protein (MBP)), modified G proteins (wild type and modified in their attached lipids or C-terminal Gα amino acid sequence), and a kinetic near infrared light scattering assay (15Heck M. Pulvermüller A. Hofmann K.P. Methods Enzymol. 2000; 315: 329-347Google Scholar, 16Ernst O.P. Bieri C. Vogel H. Hofmann K.P. Methods Enzymol. 2000; 315: 471-489Google Scholar, 17Heck M. Hofmann K.P. J. Biol. Chem. 2001; 276: 10000-10009Google Scholar) to monitor protein-protein interactions in real time. This approach allowed us to investigate how individual receptor binding sites on the G protein are functionally linked with each other. Based on the experimental data, we propose that nucleotide exchange requires a sequential two-step interaction of the G protein with R*. An encounter of CTγ-far with R* initiates the interaction and thereby makes CTα available for binding to R*. In the second step, R* shifts interaction from CTγ-far toward CTα. Now the nucleotide binding site has low affinity for GDP and is prepared for the uptake of GTP.EXPERIMENTAL PROCEDURESNative and Modified Gα Subunits and Gβγ Dimers—G proteins used in this study were either the G protein of the rod photoreceptor cell (transducin, purified from bovine eyes), purified recombinant Giα1, or a Gtα construct. Because of the known very low expression of the soluble transducin Gα subunit (Gtα) in Escherichia coli and Sf9 cells, single amino acid substitutions were introduced into Gtα to yield a Gtα/Giα1 chimera that contained 16 residues of Giα1 in a Gtα background (18Natochin M. Granovsky A.E. Muradov K.G. Artemyev N.O. J. Biol. Chem. 1999; 274: 7865-7869Google Scholar). Giα1 is not present in photoreceptor cells but belongs to the same Gα subfamily as Gtα and couples to light-activated rhodopsin. Gα with an N-terminal myristoyl modification was obtainable for native Gtα and for recombinant Giα1 (which contains an internal His6 tag) but not for the Gtα/Giα1 chimera (which contains an N-terminal His6 tag). Most experiments were performed in parallel with Gtα purified from bovine retinae, the Gtα/Giα1 chimera, and Giα1 with similar results (see figure legends). All Gβγ dimers consisted of the β1γ1 isoform, the Gβγ dimer of transducin (Gtβγ), and were expressed in Sf9 cells or purified from bovine retinae. Farnesyl-free Gβγ was obtained by expression of the recombinant Gγ1C71S mutant concomitantly with Gβ1 in Sf9 cells or proteolysis of Gβγ obtained from bovine retinae. The results obtained with both preparations were similar.Cloning, Expression, and Purification of Chimeric Gtα/Giα1 and Giα1 Subunits—The expression vector pHis6-Gtα*, which was generously provided by M. Natochin and N. Artemyev, contains a chimeric bovine Gtα/rat Giα1 cDNA sequence, preceded by a nucleotide sequence encoding six histidines as an affinity tag (18Natochin M. Granovsky A.E. Muradov K.G. Artemyev N.O. J. Biol. Chem. 1999; 274: 7865-7869Google Scholar). The chimeric Gα protein contains only 16 residues from Giα1 and was shown to be similar to native Gtα in receptor interaction and basal nucleotide exchange. For construction of the mutants, the SpeI-HindIII fragment of pHis6-Gtα* was introduced into the pLitmus 38 cloning vector (New England Biolabs), yielding the precursor pL-Gtα*. An oligonucleotide duplex with the respective mutation was cloned into the Tth111I and SapI restriction sites of pL-Gtα*, and the modified SpeI-HindIII fragment was subcloned into SpeI/HindIII-digested pHis6-Gtα*. Recombinant Giα1 was expressed and purified (19Lee E. Linder M.E. Gilman A.G. Methods Enzymol. 1994; 237: 146-164Google Scholar) using the plasmid pQE-60 (Qiagen) harboring the rat Giα1 coding sequence (20Kozasa T. Gilman A.G. J. Biol. Chem. 1995; 270: 1734-1741Google Scholar). This protein was designed to contain an internal His6 epitope (amino acid sequence GGHHHHHHGGGMTA) after position 121, where the homologous GPA1 from yeast has a long insert compared with mammalian Gα subunits. For expression of myristoylated Giα1 and mutants thereof, the respective G protein-encoding plasmids were cotransfected with pBB131 (coding for yeast N-myristoyltransferase; a generous gift of Jeffrey Gordon (21Duronio R.J. Jackson-Machelski E. Heuckeroth R.O. Olins P.O. Devine C.S. Yonemoto W. Slice L.W. Taylor S.S. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1506-1510Google Scholar)) into E. coli JM109. Cultures were grown at 30 °C, induced with 100 μm isopropyl-1-thio-β-d-galactopyranoside at an A600 of 0.8 and harvested 12–14 h later. Expression and purification of the Gtα/Giα1 chimera was performed as described (18Natochin M. Granovsky A.E. Muradov K.G. Artemyev N.O. J. Biol. Chem. 1999; 274: 7865-7869Google Scholar, 19Lee E. Linder M.E. Gilman A.G. Methods Enzymol. 1994; 237: 146-164Google Scholar, 22Skiba N.P. Bae H. Hamm H.E. J. Biol. Chem. 1996; 271: 413-424Google Scholar). E. coli BL21(DE3) harboring plasmids encoding Gα subunits were induced with 100 μm isopropyl-1-thio-β-d-galactopyranoside at an A600 of 0.8 and harvested 5–7 h later (20 °C). A TALON metal affinity resin (BD Biosciences Clontech) and an imidazole gradient were used for purification.Expression and Purification of Gβγ Dimers—Nonfarnesylated Gβγ complexes were expressed in and purified from Sf9 cells infected with the respective baculoviruses. Baculoviruses encoding Gβ1 and Gγ1C71S were a generous gift from A. G. Gilman and P. Gierschik, respectively. Gβ1γ1C71S was recovered from the cytosolic fraction of Sf9 lysates as described (23Weitmann S. Schultz G. Kleuss C. Biochemistry. 2001; 40: 10853-10858Google Scholar) and purified by Ni2+-nitrilotriacetic acid co-chromatography with His-tagged Giα1 (20Kozasa T. Gilman A.G. J. Biol. Chem. 1995; 270: 1734-1741Google Scholar).Proteolytic Defarnesylation of Gtβγ—The GGC-farnesyl fragment was removed from purified native Gtβγ by proteolysis as described earlier (24Sondek J. Bohm A. Lambright D.G. Hamm H.E. Sigler P.B. Nature. 1996; 379: 369-374Google Scholar). Digests with Endo-Lys-C (sequencing grade; Roche Applied Science) were carried out in 25 mm Tris-HCl, 1 mm EDTA (pH 8.5) at a protease/Gβγ ratio of 1:100 (w/w) at 4 °C overnight. Reactions were stopped by the addition of 1-chloro-3-tosylamido-7-amino-2-heptanone hydrochloride (TLCK) at a final concentration of 56 μg/ml. In a control sample, TLCK was present during the incubation with protease and prevented proteolysis. The proteolyzed products were analyzed by HPLC (25Matsuda T. Fukada Y. Methods Enzymol. 2000; 316: 465-481Google Scholar) to verify completion of the reaction, using a C18 column (GROM-Sil 300 ODS-5 ST, 4.6 × 150, Grom Analytik + HPLC, Herrenberg, Germany) and a linear gradient of acetonitrile (10–80%, 1%/min) in 0.1% trifluoroacetic acid. Subsequent mass spectrometric analysis was performed with a Voyager-DE Elite matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (Applied Biosystems) using a sinapinic acid matrix (Fluka).Cloning, Expression, and Purification of MBP Fusion Proteins—The expression vector pMal-c2x was purchased from New England Biolabs. Synthetic oligonucleotide duplexes encoding a Gly-Gly linker followed by peptide sequences derived from the C-terminal regions of Gα or Gγ (Fig. 2A) were ligated into EcoRI/BamHI-digested pMal-c2x. Expression and purification of the fusion proteins was performed as described (26Martin E.L. Rens-Domiano S. Schatz P.J. Hamm H.E. J. Biol. Chem. 1996; 271: 361-366Google Scholar). MBP-CTγ-far was prepared by intein-mediated protein ligation (27Xu M.Q. Evans Jr., T.C. Methods. 2001; 24: 257-277Google Scholar) using the protein splicing element Mxe GyrA intein excised from vector pTXB3 (New England Biolabs) and the synthetic peptide CDKNPFKELKGGC-farnesyl. After on-column ligation (chitin beads; New England Biolabs), the protein was purified by chromatography on an amylose resin (New England Biolabs). MBP-CTγ-far was identified by mass spectrometry and had a purity of >85% according to SDS-PAGE analysis.Fig. 2Binding of C-terminal tails of G protein α and γ subunits to light-activated rhodopsin can be monitored by kinetic light scattering. A, the amino acid sequences of the C-terminal tails studied either as synthetic peptides, fused to MBP or contained in the different G protein subunits (see “Experimental Procedures”) are as follows: CTαt (residues 340–350 of bovine Gtα), CTαi (residues 344–354 of rat Giα1), and Gα high affinity (HA) analog sequences, CTαHA1 (26Martin E.L. Rens-Domiano S. Schatz P.J. Hamm H.E. J. Biol. Chem. 1996; 271: 361-366Google Scholar) and CTαHA2. CTγ-far corresponds to amino acids 60–71 of bovine Gγ1 and was farnesylated at Cys-71. B, flash illumination (arrow) of disk membranes containing rhodopsin triggers complex formation between soluble MBP-CT fusion proteins and light-activated rhodopsin (R*) as seen in the increase of the relative intensity of scattered light (ΔI/I, binding signal (15Heck M. Pulvermüller A. Hofmann K.P. Methods Enzymol. 2000; 315: 329-347Google Scholar)). As an example, binding of MBP-CTαHA1 (0.5 μm) to R* (1 μm) is shown in the upper trace. Under the experimental conditions, the assay is not sensitive enough to monitor binding of R*-interacting small synthetic peptides (lower two traces; 1 μm R*, 50 μm peptide). The binding of MBP-CT fusion proteins to R* can be inhibited by corresponding short synthetic peptides (shown for the CTα-HA1 peptide (50 μm), middle trace). The interaction can also be terminated by inactivating R* with hydroxylamine (20 mm), which hydrolyzes the retinal Schiff base in a competing reaction (second trace from top). MBP-CTαHA2 and MBP-CTγ-far show the same basic binding behavior (data not shown).View Large Image Figure ViewerDownload (PPT)Peptide Synthesis—Peptide synthesis, farnesylation, and purification were carried out as described before (28Ernst O.P. Meyer C.K. Marin E.P. Henklein P. Fu W.Y. Sakmar T.P. Hofmann K.P. J. Biol. Chem. 2000; 275: 1937-1943Google Scholar). The amino acid sequences of the peptides are given in Fig. 2A. The amino termini of the peptides were unmodified, and carboxyl termini of the Gγ-derived peptides were amidated. The peptide CDKNPFKELKGGC-farnesyl used for preparation of MBP-CTγ-far was synthesized as C-terminal amide on a Rink resin (loading 0.25 mg/mol; Rapp Polymere, Tübingen, Germany) with a Pioneer Synthesizer (Applied Biosystems) using an Fmoc (N-(9-fluorenyl)methoxycarbonyl) strategy. The C-terminal Cys-sulfur was protected by a trityl group, and the N-terminal Cys-sulfur, which was used for intein-mediated ligation, was protected by an S-(tert-butylsulfenyl) group. All other side chain functions were protected with trifluoroacetic acid labile groups. The peptide was cleaved from the resin with 95% trifluoroacetic acid using triisopropylsilane as a scavenger. The crude peptide was precipitated twice with ether and lyophilized from acetonitrile/water (1:3). For farnesylation, 100 mg of the crude peptide (0.065 mmol), dissolved in 3 ml of N,N-dimethylformamide, was treated with 1.4 eq of farnesyl bromide (0.09 mmol) and an equal amount of diisopropyl ethylamine (0.16 mmol). The formation of oxidation products was suppressed by degassing with N2. After 1 h, the crude farnesylation mixture was purified by preparative HPLC. For deprotection of the S-(tert-butylsulfenyl) group from cysteine, 20 eq of tris(2-carboxyethyl)-phosphine hydrochloride were added to a solution of the peptide in 50% acetonitrile/water. The mixture was adjusted to pH 5 with NH3 and stirred at room temperature for 5 h and then separated by HPLC. The farnesylated peptide obtained was characterized by mass spectrometry and analytical HPLC.Disk Membrane and Transducin Preparation—Preparations were performed as described previously (17Heck M. Hofmann K.P. J. Biol. Chem. 2001; 276: 10000-10009Google Scholar).Measurement of G Protein Conformational Changes by Fluorescence—The basal GDP/GTP exchange of Gα subunits (2 μm) in the absence of Gβγ and rhodopsin was monitored by detection of fluorescence emission at 340 nm (excitation at 300 nm, measured at 20 °C with constant stirring) using a SPEX fluorolog II spectrofluorometer (see Ref. 16Ernst O.P. Bieri C. Vogel H. Hofmann K.P. Methods Enzymol. 2000; 315: 471-489Google Scholar and references therein). Activation of Gα was started by adding GTPγS (10 μm final concentration). Activation of the whole Gα pool was completed by adding NaF and then AlCl3 (yielding 100 μmAlF4− final concentration). Traces were normalized to show the same total increase of intensity of fluorescence emission induced by GTPγS and AlF4−.Kinetic Light Scattering—Changes in intensities of scattered near infrared light were measured as described before (17Heck M. Hofmann K.P. J. Biol. Chem. 2001; 276: 10000-10009Google Scholar). Light-induced binding of proteins from solution to activated rhodopsin in disk membranes leads to an increase of the size of the scattering particle and concomitant increase of the intensity of scattered light. The sensitivity of the light scattering assay is given by the measuring conditions and the experimental setup (15Heck M. Pulvermüller A. Hofmann K.P. Methods Enzymol. 2000; 315: 329-347Google Scholar, 16Ernst O.P. Bieri C. Vogel H. Hofmann K.P. Methods Enzymol. 2000; 315: 471-489Google Scholar, 17Heck M. Hofmann K.P. J. Biol. Chem. 2001; 276: 10000-10009Google Scholar). As employed in the present study, the light scattering change is proportional to the gain of mass and monitors binding of “mass-tagged” peptides (MBP-peptide fusion proteins) to R* but not binding of short synthetic peptides to R* because of the small peptide mass. All measurements were performed in 10-mm path cuvettes at pH 7.4 (20 mm bis-tris propane, 130 mm NaCl, 1 mm MgCl2, pH 7.5) and 23 ± 1 °C. Reactions were triggered by flash photolysis of rhodopsin (3 μm) using flashes of green light (500 ± 20 nm). Binding signals were recorded with 32% flash-activated rhodopsin. For data evaluation, see “Appendix.”RESULTSMonitoring the Interaction between R* and C-terminal Binding Sites of the G Protein by Kinetic Light Scattering—To monitor in real time binding between R* in disk membranes isolated from the rod outer segment and interacting CTs of the heterotrimeric G protein (namely CTα and CTγ-far; Figs. 1 and 2A), the kinetic near infrared light scattering assay was employed (see “Experimental Procedures” and Refs. 15Heck M. Pulvermüller A. Hofmann K.P. Methods Enzymol. 2000; 315: 329-347Google Scholar and 16Ernst O.P. Bieri C. Vogel H. Hofmann K.P. Methods Enzymol. 2000; 315: 471-489Google Scholar). “Mass-tagged” peptides, which can be obtained by expression and purification of MBP-peptide fusion proteins, allow convenient measurement of the binding to R* (Fig. 2B).A typical binding signal triggered by flash activation of rhodopsin is shown in Fig. 2B for MBP-CTαHA1 (top trace). This MBP fusion protein contains the well known Gtα high affinity analog CT sequence (Fig. 2A, CTαHA1), which was identified by Hamm and co-workers (26Martin E.L. Rens-Domiano S. Schatz P.J. Hamm H.E. J. Biol. Chem. 1996; 271: 361-366Google Scholar). Since both the small synthetic and MBP “mass-tagged” CTαHA1 peptides bind to R*, binding of MBP-CTαHA1 to R* can be inhibited by an excess of the competing small synthetic CTαHA1 peptide (Fig. 2B, middle trace). A further control showing that the assay monitors the interaction between R* and MBP-CTαHA1 is provided by the effect of hydroxylamine, which affects this interaction by hydrolyzing the retinal Schiff base in a competing reaction (Fig. 2B, second trace from top). Hence, the degradation of R* results in a transient interaction, which is reflected in a transient increase of the intensity of scattered light.By testing peptides with modified CTαt sequences, we were able to identify a single amino acid exchange (K341L in Gtα) that increases the affinity for R* of the corresponding CTα peptide (termed CTαHA2) to a level similar to CTαHA1 (details to be published). “Mass-tagging” of CT peptides with MBP was extended to CTαt and CTαHA2 and the farnesylated CTγ-far. All of these MBP-CT fusion proteins showed binding to R*, were sensitive to competition by the corresponding synthetic CT peptide, and were sensitive to the effect of hydroxylamine (Fig. 3C and data not shown). MBP lacking a fused G protein CT peptide tail showed no binding to R* (data not shown).Fig. 3The C-terminal tails of G protein α and γ subunits do not bind simultaneously to light-activated rhodopsin. A, the CTγ-far peptide reduces the binding signal of MBP-CTαHA1 (0.45 μm; KD = 0.2 μm) and R* (1 μm) measured by light scattering in a concentration-dependent manner (concentrations as indicated). Affinity and competition results are similar for MBP-CTαHA2. CTγ-far peptide can also effectively inhibit MBP-CTαt (data not shown). B, biochemical competition measurement. Rhodopsin in disk membranes (10 μm) was incubated with MBP-CTαHA1 (0.5 μm) and pelleted in the dark or light in the absence or presence of CTγ-far peptide (concentrations as indicated). Supernatants (s) and pellets (p) were analyzed by SDS-PAGE and proteins were visualized by Coomassie Blue staining. C, competition experiment under reversed conditions. MBP-CTγ-far was produced semisynthetically by intein-mediated protein ligation (27Xu M.Q. Evans Jr., T.C. Methods. 2001; 24: 257-277Google Scholar). The binding signal of 60 μm MBP-CTγ-far (1 μm R*) is inhibited in a concentration-dependent manner by the presence of CTαHA1 peptide (concentrations as indicated). Also CTαHA2 and CTαt peptides can inhibit binding of MBP-CTγ-far to R* (data not shown).View Large Image Figure ViewerDownload (PPT)Only One of the C-terminal Peptides, CTα or CTγ-far, Can Bind to R* at One Time—To investigate whether CTα and CTγ-far interact simultaneously with R* (see Fig. 1), competition of the two CTs for binding to R* was investigated with the kinetic light scattering assay. The interaction between the MBP-CTαHA1 fusion protein and R* could be inhibited in a concentration-dependent manner by the presence of a synthetic peptide corresponding to CTγ-far (Fig. 3A). Analogously, binding of MBP-CTαHA2 and MBP-CTαt to R* could be suppressed by excess CTγ-far peptide (data not shown). The competition between the MBP-CTαHA1 fusion protein and the CTγ-far peptide for R* could be supported by a centrifugation assay (Fig. 3B). In this biochemical binding assay, rhodopsin in disk membranes is pelleted in the presence of MBP-CT fusion proteins in the dark or light, followed by subsequent analysis of the pellet and supernatant by SDS-PAGE and protein staining. The centrifugation assay also showed that the MBP-CTα fusion proteins investigated (Fig. 2A) as well as a control MBP protein lacking a CT sequence did not bind to the disk membranes in the dark (data not shown).Next, we measured binding of the MBP-CTγ-far fusion protein to R* by kinetic light scattering. As observed with the MBP-CTα fusion proteins, a binding signal was obtained with MBP-CTγ-far (Fig. 3C). The farnesyl moiety of the MBP-CTγ-far fusion proteins results in considerable interaction of the fusion protein with the disk membrane already in the dark, which was detected by the centrifugation assay (data not shown). This is reminescent to physiologically completely lipid-modified Giα1/Gtβγ that shows binding to the disk membrane in dark and light (Fig. 4, top). Since only fusion proteins, which bind from solution to the scattering disk vesicle, contribute to the increase of intensity of scattered light, the intensity change is smaller for MBP-CTγ-far.Fig. 4The G protein's lipid modification is mandatory for interaction with activated rhodopsin. Interaction of light-activated rhodopsin (1 μm R*; the arrow indicates time of activation) with 0.6 μm Gαβγ (see “Experimental Procedures”) was measured by kinetic light scattering as described in Fig. 2. The amount of G protein bound to disk membranes (3 μm rhodopsin) in the dark (D) and light (L) was determined by pelleting the disk membranes, subsequent SDS-PAGE analysis of the pellet, and protein staining with Coomassie Blue (G protein bands are boxed; SDS-PAGE data are part of an extensive study on G protein lipid modifications) (M. Heck, O. P. Ernst, R. Herrmann, K. P. Hofmann, and C. Kleuss, manuscript in preparation). Samples contained lipid modifications as indicated (myristoylation (myr) of Giα1 or farnesylation (far) of Gγ). Under the experimental conditions the Gα/Gβγ combinations shown formed holoproteins (data not shown), which were not bound to the disk membrane when at least one lipid modification was lacking. The amplitude of the binding signal of the +myr/+far combination is reduced, because this holoprotein shows like native transducin considerable binding to the disk membrane in the dark, an effect, which is more pronounced for the holoprotein containing Giα1. Consequently, the concentration of soluble G protein, which is available to contribute to changes of the intensity of scattered light (17Heck M. Hofmann K.P. J. Biol. Chem. 2001; 276: 10000-10009Google Scholar), is reduced. Similar results were obtained for transducin when heterogeneously fatty acylated native Gtα, the nonmyristoylated Gtα/Giα1 chimera and Gtβγ with and without farnesyl moiety were used (data not shown). No difference in behavior was observed when enzymatically defarnesylated Gtβγ or the recombinant Gβγ(C71S) mutant was used.View Large Image Figure ViewerDownload (PPT)When we performed the competition experiment with the MBP-CTγ-far fusion protein and CTα peptides, an analogous inhibition by the CTα peptide in a concentration-dependent manner was seen in the light scattering assay (shown for CTαHA1 in Fig. 3C). At higher concentrations of the competing CTα peptide (>1 μm), a transient small binding signal becomes visible, which sits on the normal light scattering change with competitively reduced amplitude. It may reflect a delayed action of the CTα peptide, in agreement with the data presented below. The binding of MBP-CTγ-far to the disk membrane in the dark causes a high background in the centrifugation assay, thereby limiting the use of this assay in competition experiments involving MBP-CTγ-far (data not shown). Taken together, the competition experiments show that R* can interact with only one C-terminal binding domain of the G protein at one time, either CTα or CTγ-far.The First Interaction Step Requires Lipid-modified G Protein—The competition experi" @default.
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