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• W2042132016 abstract "Arrestin blocks the interaction of rhodopsin with the G protein transducin (Gt). To characterize the sites of arrestin that interact with rhodopsin, we have utilized a spectrophotometric peptide competition assay. It is based on the stabilization of the active intermediates metarhodopsin II (MII) and phosphorylated MII by Gt and arrestin, respectively (extra MII monitor). The protocol involves native disc membranes and three sets of peptides 10–30 amino acids in length spanning the arrestin sequence. In the absence of arrestin, not one of the peptides by itself had an effect on the amount of MII formed. However, inhibition of arrestin-dependent extra MII was found for the peptides at residues 11–30 and 51–70 (IC50 < 100 μm) and residues 231–260 (IC50 < 200 μm). A similar pattern of inhibition by arrestin peptides was seen when arrestin was replaced by Gt or the farnesylated Gtγ C-terminal peptide. Only arrestin-(11–30) inhibited MII·Gt less (IC50 = 300 μm) than phosphorylated MII·arrestin. We interpreted the data by competition of the arrestin peptides for interaction sites at rhodopsin, exposed in the MII conformation and specific for both arrestin and Gt. The arrestin sites are located in both the C- and N-terminal domains of the arrestin structure. Arrestin blocks the interaction of rhodopsin with the G protein transducin (Gt). To characterize the sites of arrestin that interact with rhodopsin, we have utilized a spectrophotometric peptide competition assay. It is based on the stabilization of the active intermediates metarhodopsin II (MII) and phosphorylated MII by Gt and arrestin, respectively (extra MII monitor). The protocol involves native disc membranes and three sets of peptides 10–30 amino acids in length spanning the arrestin sequence. In the absence of arrestin, not one of the peptides by itself had an effect on the amount of MII formed. However, inhibition of arrestin-dependent extra MII was found for the peptides at residues 11–30 and 51–70 (IC50 < 100 μm) and residues 231–260 (IC50 < 200 μm). A similar pattern of inhibition by arrestin peptides was seen when arrestin was replaced by Gt or the farnesylated Gtγ C-terminal peptide. Only arrestin-(11–30) inhibited MII·Gt less (IC50 = 300 μm) than phosphorylated MII·arrestin. We interpreted the data by competition of the arrestin peptides for interaction sites at rhodopsin, exposed in the MII conformation and specific for both arrestin and Gt. The arrestin sites are located in both the C- and N-terminal domains of the arrestin structure. metarhodopsin II metarhodopsin I 1,3-bis[tris(hydroxymethyl)methylamino]propane G protein-coupled receptors enable eukaryotic cells to respond to a large variety of extracellular signals, including hormones, odorants, and light (1Baldwin J.M. Curr. Opin. Cell Biol. 1994; 6: 180-190Crossref PubMed Scopus (340) Google Scholar). One of the best studied G protein-coupled receptor-initiated signaling pathways is the visual cascade in retinal rods (2Hargrave P.A. McDowell J.H. FASEB J. 1992; 6: 2323-2331Crossref PubMed Scopus (233) Google Scholar). It is initiated by the absorption of a photon in the visual receptor (rhodopsin) and subsequent isomerization of the 11-cis-retinal, which is covalently attached to Lys296 in the seventh transmembrane helix of the receptor. Via a series of transient intermediates, rhodopsin converts in milliseconds into metarhodopsin II (MII),1 the form of rhodopsin that interacts with the heterotrimeric G protein transducin (Gt). This interaction initiates the intermolecular transduction of the light signal by catalyzing GDP/GTP exchange in the nucleotide-binding site of the G protein and in turn activates the G protein-coupled effector, a cGMP-specific phosphodiesterase. Hydrolysis of cGMP leads to the closure of plasma membrane cation channels and hyperpolarization of the membrane (3Helmreich E.J. Hofmann K.P. Biochim. Biophys. Acta. 1996; 1286: 285-322Crossref PubMed Scopus (127) Google Scholar). The deactivation of rhodopsin starts with the binding of a rhodopsin kinase to photoactivated rhodopsin (4Pulvermüller A. Palczewski K. Hofmann K.P. Biochemistry. 1993; 32: 14082-14088Crossref PubMed Scopus (87) Google Scholar). Rapid phosphorylation of the receptor at C-terminal sites (5Palczewski K. Eur. J. Biochem. 1997; 248: 261-269Crossref PubMed Scopus (100) Google Scholar) increases its affinity for arrestin (6Gibson S.K. Parkes J.H. Liebman P.A. Biochemistry. 2000; 39 (, and references therein): 5738-5749Crossref PubMed Scopus (65) Google Scholar). Biochemical (7Wilden U. Hall S.W. Kühn H. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1174-1178Crossref PubMed Scopus (572) Google Scholar) and electrophysiological (8Xu J. Dodd R.L. Makino C.L. Simon M.I. Baylor D.A. Chen J. Nature. 1997; 389: 505-509Crossref PubMed Scopus (275) Google Scholar) evidence has been accumulated that visual arrestins deactivate the visual cascade by direct competition with the G protein for the active receptor. However, the mechanism of interaction underlying the quench is still not well understood. UV-visible spectroscopy has shown that, to bind arrestin, the light activation of rhodopsin must proceed up to the MII conformation, in which the Schiff base bond of the retinal to the apoprotein is still intact but deprotonated (9Schleicher A. Kühn H. Hofmann K.P. Biochemistry. 1989; 28: 1770-1775Crossref PubMed Scopus (166) Google Scholar). Only after MII formation and phosphorylation of the receptor, arrestin interacts rapidly with the receptor. Although the catalytic activity of MII for the G protein is quenched, arrestin binding (and phosphorylation) stabilizes MII at the expense of its tautomeric forms (6Gibson S.K. Parkes J.H. Liebman P.A. Biochemistry. 2000; 39 (, and references therein): 5738-5749Crossref PubMed Scopus (65) Google Scholar). A conformational difference in arrestin and/or rhodopsin on interaction was earlier suggested by the unusually high apparent activation energy of the rhodopsin-arrestin interaction (9Schleicher A. Kühn H. Hofmann K.P. Biochemistry. 1989; 28: 1770-1775Crossref PubMed Scopus (166) Google Scholar). Later studies have indeed shown that binding of arrestin to the active receptor protects arrestin against limited proteolysis (10Palczewski K. Pulvermüller A. Buczylko J. Hofmann K.P. J. Biol. Chem. 1991; 266: 18649-18654Abstract Full Text PDF PubMed Google Scholar) and lysine acetylation (11Ohguro H. Palczewski K. Walsh K.A. Johnson R.S. Protein Sci. 1994; 3: 2428-2434Crossref PubMed Scopus (83) Google Scholar), suggesting that rhodopsin-bound arrestin adopts a conformation (Ab) that is different from that of free inactive arrestin (Ai). Investigations employing protein engineering (12Gurevich V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar, 13Gurevich V.V. Benovic J.L. Mol. Pharmacol. 1997; 51: 161-169Crossref PubMed Scopus (121) Google Scholar) and phosphorylated peptides (14Puig J. Arendt A. Tomson F.L. Abdulaeva G. Miller R. Hargrave P.A. McDowell J.H. FEBS Lett. 1995; 362: 185-188Crossref PubMed Scopus (70) Google Scholar) have provided evidence for a sequential mechanism, in which the contact of the negatively charged regions of phosphorylated rhodopsin with the cationic region acts as a trigger, switching arrestin into its active conformation and allowing interaction with the rhodopsin-binding sites exposed on photoactivation. A highly cationic region near the center of the arrestin sequence, beginning with residue 163, was proposed to mediate the interaction with the phosphorylated rhodopsin site, thus enabling the contact with other interaction sites exposed in the MII state (10Palczewski K. Pulvermüller A. Buczylko J. Hofmann K.P. J. Biol. Chem. 1991; 266: 18649-18654Abstract Full Text PDF PubMed Google Scholar). Arg175 (within the putative recognition site for the phosphorylated site(s) on rhodopsin) was identified as a key residue for arrestin to distinguish between phosphorylated and unphosphorylated rhodopsin (15Gurevich V.V. Benovic J.L. J. Biol. Chem. 1995; 270: 6010-6016Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 16Gray Keller M.P. Detwiler P.B. Benovic J.L. Gurevich V.V. Biochemistry. 1997; 36: 7058-7063Crossref PubMed Scopus (80) Google Scholar). Recent structural assignments (17Granzin J. Wilden U. Choe H.-W. Labahn J. Krafft B. Buldt G. Nature. 1998; 391: 918-921Crossref PubMed Scopus (208) Google Scholar, 18Hirsch J.A. Schubert C. Gurevich V.V. Sigler P.B. Cell. 1999; 97: 257-269Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar) have now identified this residue as part of a “polar core” (18Hirsch J.A. Schubert C. Gurevich V.V. Sigler P.B. Cell. 1999; 97: 257-269Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar), a central region in arrestin localized between the N- and C-terminal domains of the molecule. It may act as a fulcrum for the conversion of inactive arrestin (Ai) to active bound arrestin (Ab) (19Vishnivetskiy S.A. Paz C.L. Schubert C. Hirsch J.A. Sigler P.B. Gurevich V.V. J. Biol. Chem. 1999; 274: 11451-11454Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). The conformational switch is additionally controlled by the arrestin C terminus (20Palczewski K. Buczylko J. Imami N.R. McDowell J.H. Hargrave P.A. J. Biol. Chem. 1991; 266: 15334-15339Abstract Full Text PDF PubMed Google Scholar, 21Gurevich V.V. Benovic J.L. J. Biol. Chem. 1992; 267: 21919-21923Abstract Full Text PDF PubMed Google Scholar). When it is lacking, as in a splice variant of arrestin (p44), the short arrestin binds both phosphorylated and unphosphorylated forms of MII and even C-terminally truncated rhodopsin (22Palczewski K. Buczylko J. Ohguro H. Annan R.S. Carr S.A. Crabb J.W. Kaplan M.W. Johnson R.S. Walsh K.A. Protein Sci. 1994; 3: 314-324Crossref PubMed Scopus (73) Google Scholar, 23Pulvermüller A. Maretzki D. Rudnicka Nawrot M. Smith W.C. Palczewski K. Hofmann K.P. Biochemistry. 1997; 36: 9253-9260Crossref PubMed Scopus (94) Google Scholar). Protein engineering (24Gurevich V.V. Chen C.Y. Kim C.M. Benovic J.L. J. Biol. Chem. 1994; 269: 8721-8727Abstract Full Text PDF PubMed Google Scholar) and spectroscopic data (25Wilson C.J. Copeland R.A. J. Protein Chem. 1997; 16: 755-763Crossref PubMed Scopus (23) Google Scholar) have suggested that in the inactive arrestin state(s), negatively charged C-terminal residues interact with positively charged residues on the N-terminal region and that this interaction is broken upon binding to phosphorylated MII. In this study, we have attempted to identify specific sites of arrestin that become exposed by the conformational switch, leading to their interaction with the respective receptor sites in the MII conformation. The technique applied is based on the spectrophotometric MII stabilization assay (9Schleicher A. Kühn H. Hofmann K.P. Biochemistry. 1989; 28: 1770-1775Crossref PubMed Scopus (166) Google Scholar) and on competition with peptides derived from arrestin and Gt. Peptide competition has already been applied in previous studies to map regions of interaction in the receptor-arrestin complex. Studies with peptides from the surface-exposed sequences of rhodopsin suggested a role of different loop structures in the interaction (26Krupnick J.G. Gurevich V.V. Schepers T. Hamm H.E. Benovic J.L. J. Biol. Chem. 1994; 269: 3226-3232Abstract Full Text PDF PubMed Google Scholar). Regarding the interactive domains in arrestin, Hargrave and co-workers (27Smith W.C. McDowell J.H. Dugger D.R. Miller R. Arendt A. Popp M.P. Hargrave P.A. Biochemistry. 1999; 38: 2752-2761Crossref PubMed Scopus (21) Google Scholar) have employed a library of peptides covering the entire sequence. Based on phage-display and phosphodiesterase activation data, they localized one of the principal regions of interaction within residues 109–130 and found indications of additional sites in their data (27Smith W.C. McDowell J.H. Dugger D.R. Miller R. Arendt A. Popp M.P. Hargrave P.A. Biochemistry. 1999; 38: 2752-2761Crossref PubMed Scopus (21) Google Scholar). We will indeed provide evidence that more than one region (namely three) in the arrestin molecule is involved in receptor binding. Employing the sensitive MII stabilization assay and peptide competition not only with arrestin, but also with Gt and Gt-derived peptides, we can measure with unprecedented specificity. Based on the data from three sets of overlapping synthetic 10–30-residue peptides spanning the arrestin sequence, we propose a model locating the receptor-binding regions of arrestin in each of the two domains of the molecule. Radioactive [γ-32P]ATP was purchased from PerkinElmer Life Sciences. All chemicals were purchased from Merck (Darmstadt, Germany), Roche Molecular Biochemicals, or Sigma. 11-cis-Retinal was generously provided by Dr. R. K. Crouch (Medical University of South Carolina). Rod outer segments were purified from fresh dark-adapted bovine retinas obtained from a local slaughterhouse using the discontinuous sucrose gradient method described by Papermaster (28Papermaster D.S. Methods Enzymol. 1982; 81: 48-52Crossref PubMed Scopus (254) Google Scholar). Retinas were dissected, and rod outer segments were isolated under dim red illumination. All subsequent procedures were performed at 0–5 °C, and the rod outer segments were stored frozen at −80 °C until used. Rhodopsin was prepared by removing the soluble and membrane-associated proteins from the disc membrane by repetitive washes with a low ionic strength buffer (29Kühn H. Methods Enzymol. 1982; 81: 556-564Crossref PubMed Scopus (62) Google Scholar). All purification steps were performed under dim red illumination, and the membrane suspension was stored at −80 °C until used. Phosphorylated opsin was prepared from washed disc membranes as described previously by Wilden and Kühn (30Wilden U. Kühn H. Biochemistry. 1982; 21: 3014-3022Crossref PubMed Scopus (269) Google Scholar). To remove retinaloxime from the membrane-bound phosphorylated opsin, the membranes were treated with urea and fatty acid-free bovine serum albumin (31Sachs K. Maretzki D. Hofmann K.P. Methods Enzymol. 2000; 315: 238-251Crossref PubMed Google Scholar). An average stoichiometry of ∼1.5 phosphates/opsin was determined using radioactive [γ-32P]ATP as a tracer. Phosphorylated rhodopsin was prepared by regeneration of phosphorylated opsin with 11-cis-retinal (32Hofmann K.P. Pulvermüller A. Buczylko J. Van Hooser P. Palczewski K. J. Biol. Chem. 1992; 267: 15701-15706Abstract Full Text PDF PubMed Google Scholar). Phosphorylated opsin was suspended in 10 mm BisTris propane (pH 7.5) containing 100 mm NaCl. A 3-fold molar excess of 11-cis-retinal was added in the dark to the sample, followed by incubation for 1 h at room temperature and then overnight at 4 °C. After regeneration, phosphorylated membranes were centrifuged (45,000 ×g for 20 min), washed four times with 10 mmBisTris propane (pH 7.5) containing 100 mm NaCl to remove excess 11-cis-retinal, and stored at −80 °C. Arrestin was purified from frozen dark-adapted bovine retinas as described by Heck et al.(33Heck M. Pulvermüller A. Hofmann K.P. Methods Enzymol. 2000; 315: 329-347Crossref PubMed Google Scholar). Transducin was isolated from frozen dark-adapted bovine retinas according to Heck and Hofmann (34Heck M. Hofmann K.P. Biochemistry. 1993; 32: 8220-8227Crossref PubMed Scopus (57) Google Scholar). Peptides were synthesized using the Fmoc (N-(9-fluorenyl)methoxycarbonyl) strategy withN-[1H-benzotriazol(1-yl)(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate-N-oxide activation (Fastmoc, 0.1-mmol small-scale cycles) on an ABI Model 433A peptide synthesizer. The peptides were purified by reverse phase high performance liquid chromatography, lyophilized, and stored at −20 °C. Immediately before the experiments, the peptides were dissolved in deionized water to obtain stock solutions of 2 mm, and pH was adjusted to 7.0 with NaOH. Farnesylation of Gtγ-derived peptides was carried out as described (35Ernst O.P. Meyer C.K. Marin E.P. Henklein P. Fu W.Y. Sakmar T.P. Hofmann K.P. J. Biol. Chem. 2000; 275: 1937-1943Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). The concentrations of rhodopsin and phosphorylated rhodopsin were determined spectrophotometrically at 498 nm as described previously (23Pulvermüller A. Maretzki D. Rudnicka Nawrot M. Smith W.C. Palczewski K. Hofmann K.P. Biochemistry. 1997; 36: 9253-9260Crossref PubMed Scopus (94) Google Scholar). Purified arrestin was determined spectrophotometrically at 278 nm, assuming a molar absorption coefficient of E 0.1% = 0.638 (36Palczewski K. Riazance-Lawrence J.H. Johnson Jr., W.C. Biochemistry. 1992; 31: 3902-3906Crossref PubMed Scopus (25) Google Scholar) and a molecular mass of 45,300 Da. Purified transducin (Gt) concentration was determined using the Bradford method (37Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (214455) Google Scholar). Formation of the photoproduct metarhodopsin II (γmax = 380 nm) was assayed using the two-wavelength technique (9Schleicher A. Kühn H. Hofmann K.P. Biochemistry. 1989; 28: 1770-1775Crossref PubMed Scopus (166) Google Scholar, 38Ernst O.P. Bieri C. Vogel H. Hofmann K.P. Methods Enzymol. 2000; 315: 471-489Crossref PubMed Google Scholar). This technique minimizes scattering artifacts by comparing the flash-induced changes in the absorbance at 380 and 417 nm. The absorbance change at 417 nm (MI isosbestic to MII) serves as a reference for determining the level of MII. The two-wavelength spectrophotometer (2-nm slit width; UV 300, Shimadzu Scientific Instruments, Inc., Kyoto, Japan) was equipped with thermostatted cuvettes (2-mm path length), temperature regulation (circulator G/D8, Haake GmbH, Karlsruhe, Germany), and a green photoflash (filtered to 500 ± 20 nm). When photolyzed rhodopsin in its native disc membrane is cooled to temperatures at which the equilibrium is on the MI side (below 5 °C and pH 8.0) (39Parkes J.H. Gibson S.K. Liebman P.A. Biochemistry. 1999; 38: 6862-6878Crossref PubMed Scopus (30) Google Scholar), any specific binding of protein or peptide to MII causes enhanced formation of MII (extra MII). Extra MII provides a kinetic and stoichiometric measure for the complex between photoactivated rhodopsin and the interactive polypeptide (40Hofmann K.P. Biochim. Biophys. Acta. 1985; 810: 278-281Crossref PubMed Scopus (36) Google Scholar). The enhancement by arrestin of the MII photoproduct formed after a flash of light (extra MII) is specific for prephosphorylated rhodopsin and is not seen with unphosphorylated rhodopsin (9Schleicher A. Kühn H. Hofmann K.P. Biochemistry. 1989; 28: 1770-1775Crossref PubMed Scopus (166) Google Scholar, 23Pulvermüller A. Maretzki D. Rudnicka Nawrot M. Smith W.C. Palczewski K. Hofmann K.P. Biochemistry. 1997; 36: 9253-9260Crossref PubMed Scopus (94) Google Scholar). Fig.1 shows two examples of how synthetic peptides derived from the arrestin sequence influence the formation of extra MII from prephosphorylated samples. The arrestin peptide comprising residues 11–30 (arrestin-(11–30)) reduced the amount of extra MII formed in a concentration-dependent manner (Fig.1 A, compare traces b and c); at 1000 μm, arrestin-(11–30) abolished extra MII nearly to the control level without arrestin (compare traces c ande). This effect was not observed with arrestin-(291–310); even at 1000 μm peptide, extra MII was formed as with arrestin alone (Fig. 1 B, traces a andc). None of the peptides investigated in this study stabilized MII in the absence of arrestin (e.g. Fig. 1,traces d), in contrast to transducin C-terminal peptides (Gtα-(340–350) and farnesylated Gtγ-(50–71)) (41Kisselev O.G. Meyer C.K. Heck M. Ernst O.P. Hofmann K.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4898-4903Crossref PubMed Scopus (105) Google Scholar). The inhibition of extra MII formation by arrestin peptides indicates that they compete with their parent protein for rhodopsin-binding sites. To analyze this more quantitatively, overlapping sets of arrestin peptides with different lengths were synthesized, and their effect in the extra MII assay was measured. Each of the peptides was titrated up to a final concentration of 1000 μm. Fig.2 shows the data for two arrestin peptides from stretches 51–70 and 231–250 (arrestin-(51–70) and arrestin-(231–250), respectively). Peptides with the original sequence, but not with a scrambled sequence, exhibited a dose-dependent effect on MII formation (Fig.2 A). The hyperbolic fits to the data are consistent with a direct competitive effect of both peptides, although with different efficiency. Three sets of arrestin peptides covering the entire arrestin sequence were tested. Their IC50 values, calculated dissociation constants (K D3; see “”), and apparent Hill coefficients (n H) are listed in Table I. High values ofn H may be due to a tendency of the peptide to aggregate and therefore do not enter the calculation under “.”Figure 2Competition between synthetic arrestin peptides and arrestin or transducin for binding to photoactivated rhodopsin. A, inhibition by synthetic arrestin peptides of arrestin binding to phosphorylated metarhodopsin II. The extra MII assay was carried out as described in the legend to Fig. 1. Experiments were performed with arrestin-(51–70) (Arr(51–70);upper panel) and arrestin-(231–250) (Arr(231–250); lower panel). B, inhibition of transducin binding to metarhodopsin II with the same arrestin peptides as described for A. Data points represent the normalized level of extra MII (extra meta II) formed minus the control MII, as a function of added peptide, in the presence (circles) and absence (squares) of arrestin or Gt. Triangles represent the data for scrambled arrestin peptides with the sequences LVCQVYAEKVFRGSLYKRTG and EIQLKKVYTVTNVLVVKKEV replacing arrestin-(51–70) and arrestin-(231–250), respectively. Filled and empty symbols indicate the results obtained from different sets of experiments. Solid lines represent fits according to the hyperbolic equation f = (a −d)/(1 + (x/c) nH ), where a and d are the asymptotic maximum and minimum, respectively; x is the peptide concentration;c is IC50; and nH is the Hill coefficient. The experiments were otherwise performed as described in the legend to Fig. 1.View Large Image Figure ViewerDownload (PPT)Figure 1Flash-induced arrestin-dependent formation of extra MII in the presence of synthetic arrestin peptides. Prephosphorylated disc membranes were reconstituted with purified arrestin and various amounts of synthetic peptide. The signals represent the absorbance change at 380 nm minus the absorbance change at 417 nm; the abrupt decrease after the flash is due to rapid formation of MII precursors. A, effect of arrestin-(11–30) (Arr(11–30)) on extra MII formation;B, effect of arrestin-(291–310) (Arr(291–310)) on extra MII formation. Traces a, sample without peptide;traces b, with 100 μm peptide; traces c, with 1000 μm peptide; traces d, control without arrestin plus 1000 μm peptide;traces e, control without arrestin and without peptide. In all measurements, the final concentrations in the samples were 5 μm phosphorylated rhodopsin and 1 μmarrestin (except for traces d and e). Experimental conditions were 100 mm HEPES (pH 8.0) at 1 °C, sample volume of 200 μl, and cuvette path length of 2 mm; 12% rhodopsin was photolyzed per flash.View Large Image Figure ViewerDownload (PPT)Table IInhibition of arrestin binding to phosphorylated metarhodopsin II by synthetic arrestin peptidesSet 1Set 2Set 3Peptide (amino acids)IC50n HK D3Peptide (amino acids)IC50n HK D3Peptide (amino acids)IC50n HK D3μmμmμmμmμmμm 1–20>500ND>20011–30763.930 1–30393.116 21–40NDNDND31–502700.9108 65–83>500ND>200 41–601982.07951–70258.610109–130>500ND>200 61–80NDNDND71–90NDNDND231–260992.040 81–100>500ND>20091–110>500ND>200301–310>500ND>200101–120>500ND>200111–130>500ND>200311–320>500ND>200121–140>500ND>200131–150>500ND>200321–330>500ND>200141–160>500ND>200151–1703741.8150161–180>500ND>200171–190>500ND>200181–200>500ND>200191–210NDNDND201–220>500ND>200211–230NDNDND221–240>500ND>200231–2501352.254241–2601671.967251–270>500ND>200261–280NDNDND271–290>500ND>200281–300>500ND>200291–310>500ND>200301–3203631145311–330>500ND>200321–341NDNDND331–351>500ND>200342–362>500ND>200352–372>500ND>200363–383NDNDND373–393>500ND>200384–404>500ND>200IC50 values and the Hill coefficients (n H) were determined employing the extra MII assay (Fig. 2).K D3 values were estimated assuming the reaction scheme (Scheme 1) as described under “.” ND, not determined. Open table in a new tab IC50 values and the Hill coefficients (n H) were determined employing the extra MII assay (Fig. 2).K D3 values were estimated assuming the reaction scheme (Scheme 1) as described under “.” ND, not determined. Transducin forms enhanced MII complexes with photoactivated unphosphorylated rhodopsin. It is further known that arrestin inhibits the activity of photoactivated rhodopsin for the G protein (7Wilden U. Hall S.W. Kühn H. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1174-1178Crossref PubMed Scopus (572) Google Scholar). We therefore investigated whether arrestin peptides can inhibit the transducin-dependent formation of extra MII. As shown in Fig. 2 B, the two peptides that competed with arrestin also competed with Gt with similar relative efficiency, providing a control for direct interaction of the peptides with rhodopsin (see “Discussion”). The question was whether arrestin peptides interfere with Gt-derived peptides in their interaction with photoactivated rhodopsin. One may expect that this can be tested by direct competition because C-terminal peptides from Gt stabilize the MII state as the holoprotein (41Kisselev O.G. Meyer C.K. Heck M. Ernst O.P. Hofmann K.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4898-4903Crossref PubMed Scopus (105) Google Scholar, 42Bartl F. Ritter E. Hofmann K.P. FEBS Lett. 2000; 473: 259-264Crossref PubMed Scopus (32) Google Scholar), but arrestin peptides do not (Fig. 2). Fig.3 shows two examples that arrestin peptides can indeed competitively inhibit the formation of extra MII induced by Gt-derived peptides. The effect was specifically seen with the farnesylated Gtγ peptide, indicating an overlap of the respective binding sites at rhodopsin. No such inhibition was measured for the interaction with the Gtα C-terminal peptide (native and high affinity analog). As shown in Fig. 4, almost all the peptides that competed with arrestin did so with Gt and the Gtγ C-terminal peptide as well. An exception is the region near the N terminus, which showed reduced competition with Gt and the farnesylated Gtγ peptide compared with arrestin (see “Discussion”). The data provide new information on the receptor-binding sites of S-antigen, the arrestin responsible for termination of visual transduction in rods. Using overlapping sets of synthetic arrestin-derived peptides and their competition with the parent protein for the flash-activated phosphorylated receptor, we identified peptides from three regions of the arrestin sequence that competed with values <200 μm. The respective parts of the sequence are located in both the N- and C-terminal domains of the protein structure. The regions of peptide competition mark possible candidates for interaction sites with active phosphorylated rhodopsin. However, it is clear that peptide competition by itself is not specific. For example, the peptides could affect sites of intramolecular interaction, involved in conversions necessary to reach the active conformation. Since we know that such a conformational switch in arrestin occurs (see below), this latter possibility is quite realistic. A criterion was found in the comparison of arrestin with Gt and Gtpeptides. The data in Fig. 4 show that most of the peptides that competed with arrestin also competed with Gt and the farnesylated Gtγ C-terminal peptide. The loss of competition between positions 65 and 83 was also reproduced. Because intramolecular switches in arrestin and Gt are unlikely to involve homologous domains, this similarity can be taken as strong evidence for binding of arrestin and Gt or its farnesylated Gtγ peptide to the rhodopsin surface with sufficient overlap (Fig. 5). Our approach is similar to the one taken by Smith et al. (27Smith W.C. McDowell J.H. Dugger D.R. Miller R. Arendt A. Popp M.P. Hargrave P.A. Biochemistry. 1999; 38: 2752-2761Crossref PubMed Scopus (21) Google Scholar), who employed overlapping synthetic peptides to determine regions of arrestin that bind to rhodopsin. Using a phage-display technique of arrestin fragments and Gt binding and activation assays, these authors identified a stretch comprising residues 109–130 as a site involved in the interaction with rhodopsin. Because of the low affinity of this region (IC50 = 1.1 mm), it was suggested that this portion of arrestin may be only one of several binding sites for rhodopsin (27Smith W.C. McDowell J.H. Dugger D.R. Miller R. Arendt A. Popp M.P. Hargrave P.A. Biochemistry. 1999; 38: 2752-2761Crossref PubMed Scopus (21) Google Scholar). In the present study, using the fundamentally different extra MII assay, we indeed found peptide competition with similar IC50 values in this region (Table I), but stronger competition in other regions, namely residues 11–30 and 51–70 (IC50 < 100 μm) and residues 231–250/241–260 (IC50 < 200 μm). These regions and others with lower IC50 values are located in the arrestin C- and N-terminal domains (Table I and Fig. 4). The reason for detection of these sites by our assay is probably the capturing of the interactions as they occur within seconds after the activating light flash. The region near the N terminus (residues 11–30) shows strong competition, but is conserved only in the visual arrestins, or S-antigens (43Craft C.M. Whitmore D.H. FEBS Lett. 1995; 362: 247-255Crossref PubMed Scopus (65) Google Scholar). This is also the region where the only significant difference between arrestin and Gt competition arises. Possible explanations include a role both in rhodopsin-arrestin interaction and in the switch mechanism (see below), in agreement with the conclusions drawn from mutational studies (19Vishnivetskiy S.A. Paz C.L. Schubert C. Hirsch J.A. Sigler P.B. Gurevich V.V. J. Biol. Chem. 1999; 274: 11451-11454Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). In any case, the lack of conservation in this region would argue for a specific role in visual arrestins. S-antigen and presumably all arrestins share with the G proteins a conformational switch, operated by the contact with the active receptor (3Helmreich E.J. Hofmann K.P. Biochim. Biophys. Acta. 1996; 1286: 285-322Crossref PubMed Scopus (127) Google Scholar, 9Schleicher A. Kühn H. Hofmann K.P. Biochemistry. 1989; 28: 1770-1775Crossref PubMed Scopus (166) Google Scholar, 10Palczewski K. Pulvermüller A. Buczylko J. Hofmann K.P. J. Biol. Chem. 1991; 266: 18649-18654Abstract Full Text PDF PubMed Google Scholar, 12Gurevich V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar, 13Gurevich V.V. Benovic J.L. Mol. Pharmacol. 1997; 51: 161-169Crossref PubMed Scopus (121) Google Scholar). Based on structural assignments, Sigler and co-workers (18Hirsch J.A. Schubert C. Gurevich V.V. Sigler P.B. Cell. 1999; 97: 257-269Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar, 19Vishnivetskiy S.A. Paz C.L. Schubert C. Hirsch J.A. Sigler P.B. Gurevich V.V. J. Biol. Chem. 1999; 274: 11451-11454Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar) have specified a trigger mechanism of the conformational switch, in which the phosphorylated C terminus of the receptor interacts with the polar core, embedded between the N- and C-terminal domains in the fulcrum of the molecule. Upon this interaction, intramolecular interactions, including a hydrogen-bonded network of buried ion pairs and salt bridges between charged side chains, are disrupted, leading to structural changes, possibly involving an en blocrearrangement of the N- and C-terminal domains (18Hirsch J.A. Schubert C. Gurevich V.V. Sigler P.B. Cell. 1999; 97: 257-269Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar). Although the two published structures of arrestin are not in full agreement with each other in important details such as the location of the N and C termini, one may attempt to assign the identified binding sites to the available structural elements. The structures shown in Fig. 6 (17Granzin J. Wilden U. Choe H.-W. Labahn J. Krafft B. Buldt G. Nature. 1998; 391: 918-921Crossref PubMed Scopus (208) Google Scholar, 18Hirsch J.A. Schubert C. Gurevich V.V. Sigler P.B. Cell. 1999; 97: 257-269Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar) are both likely to represent the inactive conformation of the molecule. 2H.-W. Choe, personal communication. The sites identified in the present study are distant and do not form a flat surface. A conformational switch may thus be required to allow their simultaneous interaction with the relevant receptor loop structures. The situation is similar for the G protein Gt, where two distant sites (the C termini of the α- and γ-subunits, which are 45 Å apart) are involved in the signal transfer (41Kisselev O.G. Meyer C.K. Heck M. Ernst O.P. Hofmann K.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4898-4903Crossref PubMed Scopus (105) Google Scholar). For arrestin and Gt (see Refs. 23Pulvermüller A. Maretzki D. Rudnicka Nawrot M. Smith W.C. Palczewski K. Hofmann K.P. Biochemistry. 1997; 36: 9253-9260Crossref PubMed Scopus (94) Google Scholar and 39Parkes J.H. Gibson S.K. Liebman P.A. Biochemistry. 1999; 38: 6862-6878Crossref PubMed Scopus (30) Google Scholar, respectively), the simultaneous binding of two receptors at one molecule is unlikely because the titration of the complexes yields 1:1 ratios. Despite the similarity in conformational interlocking, arrestin and Gt appear to use different mechanisms of microscopic (i.e. site to site) recognition. For the G protein, it is known that the Gtα and farnesylated Gtγ C-terminal sequences have the capacity to recognize the MII species and to distinguish it from the other intermediates (41Kisselev O.G. Meyer C.K. Heck M. Ernst O.P. Hofmann K.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4898-4903Crossref PubMed Scopus (105) Google Scholar, 44Hamm H.E. Deretic D. Arendt A. Hargrave P.A. Koenig B. Hofmann K.P. Science. 1988; 241: 832-835Crossref PubMed Scopus (393) Google Scholar). None of the numerous arrestin peptides examined displayed such specificity for the stabilization of MII (see Figs. 1 and 2 for examples). Although we took advantage of this behavior when measuring competition with the stabilizing Gt peptides, we do not yet understand its molecular basis. All we can say is that the parent arrestin protein reserves the capacity to recognize the MII conformation with high selectivity. We have identified some of the sites involved in the stable protein-protein interaction between arrestin and its receptor. However, it should be noted that this interaction may be short-lived in vivo; recent experiments in mice indicate that MII·arrestin complexes dissociate to allow the reduction of all-trans-retinal (45Palczewski K. Van Hooser J.P. Garwin G.G. Chen J. Liou G.I. Saari J.C. Biochemistry. 1999; 38: 12012-12019Crossref PubMed Scopus (136) Google Scholar). It remains to be studied how the necessary release of arrestin is induced and how it depends on the interaction sites identified here. We thank Dr. Peter Henklein for providing the synthetic arrestin peptides, Dr. Martin Heck for helpful discussions, Dr. Oliver Ernst for reading the manuscript, and Jana Engelmann and Ingrid Semjonow for technical assistance. The following reaction scheme (Scheme 1) was used to determine theK D3 values,MI⇌KA1MIIMII+A⇌KA2MII·AR*+P⇌KA3R*·P(R*=MI+MII)SCHEME1where A is arrestin, R* is photolyzed rhodopsin, and P represents different arrestin peptides. The solution for K D3 requires three equilibrium equations (Equations Equation 1, Equation 2, Equation 3),KA1=[MII][MI]Equation 1 KA2=[MII·A][MII]·[A]Equation 2 KA3=[R*·P][R*]·[P]Equation 3 and three conservation equations (Equations Equation 4, Equation 5, Equation 6),[R*] 0=[MI]+[MII]+[MII·A]+[MI·P]+[MII·P]Equation 4 [A] 0=[A]+[MII·A]Equation 5 [P] 0=[P]+[MI·P]+[MII·P]Equation 6 where [MI], [MII], [A], and [P] represent the molar concentrations of MI, MII, arrestin, and arrestin peptides, respectively. The MI-MII equilibrium constantK A1 was calculated according to Parkeset al. (39Parkes J.H. Gibson S.K. Liebman P.A. Biochemistry. 1999; 38: 6862-6878Crossref PubMed Scopus (30) Google Scholar); K A2 was determined using the K D from the rhodopsin-arrestin complex (23Pulvermüller A. Maretzki D. Rudnicka Nawrot M. Smith W.C. Palczewski K. Hofmann K.P. Biochemistry. 1997; 36: 9253-9260Crossref PubMed Scopus (94) Google Scholar). [R*]0, [A]0, and [P]0 are the total concentrations of photolyzed rhodopsin, arrestin, and arrestin peptides, respectively. By use of Equations Equation 1, Equation 2, Equation 3, Equation 4, Equation 5, Equation 6, K D3 can be expressed as follows (Equation 7).KD3=KA1·KA2KA1+1·[A] 0−[R*] 02−1−1·IC50Equation 7 The IC50 value represents the arrestin peptide concentration at which the extra MII signal is inhibited to 50%. Note that peptide binding to rhodopsin or photolyzed rhodopsin led to very similar K D3 values (errors < 1%)." @default.
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