The Selectivity of Visual Arrestin for Light-activated Phosphorhodopsin Is Controlled by Multiple Nonredundant Mechanisms

Arrestin plays an important role in quenching phototransduction via its ability to bind to the phosphorylated light-activated form of the visual receptor rhodopsin (P-Rh*). Remarkable selectivity of visual arrestin toward this functional form is determined by an elegant sequential multisite binding mechanism. Previous structure-function studies have suggested that the COOH-terminal region of arrestin (residues 356–404) is not directly involved in rhodopsin interaction, but instead plays a regulatory role. This region supports basal arrestin conformation and ensures arrestin's transition into a high affinity rhodopsin-binding state upon an encounter with P-Rh*. Overall, our results corroborate this hypothesis and identify three functional subregions (residues 361–368, 369–378, and 379–404) and individual amino acids involved in the control of arrestin stability and binding selectivity. Two of the most potent mutants, arrestin(1–378) and arrestin(F375A,V376A,F377A) belong to a novel class of constitutively active arrestins with high affinity for P-Rh*, dark P-Rh, and Rh* (but not dark Rh), in contrast to earlier constructed mutants arrestin(R175E) and arrestin(Δ2–16) with high affinity for light-activated forms only. The implications of these findings for the mechanism of arrestin-rhodopsin interaction are discussed in light of the recently determined crystal structure of arrestin. Arrestin plays an important role in quenching phototransduction via its ability to bind to the phosphorylated light-activated form of the visual receptor rhodopsin (P-Rh*). Remarkable selectivity of visual arrestin toward this functional form is determined by an elegant sequential multisite binding mechanism. Previous structure-function studies have suggested that the COOH-terminal region of arrestin (residues 356–404) is not directly involved in rhodopsin interaction, but instead plays a regulatory role. This region supports basal arrestin conformation and ensures arrestin's transition into a high affinity rhodopsin-binding state upon an encounter with P-Rh*. Overall, our results corroborate this hypothesis and identify three functional subregions (residues 361–368, 369–378, and 379–404) and individual amino acids involved in the control of arrestin stability and binding selectivity. Two of the most potent mutants, arrestin(1–378) and arrestin(F375A,V376A,F377A) belong to a novel class of constitutively active arrestins with high affinity for P-Rh*, dark P-Rh, and Rh* (but not dark Rh), in contrast to earlier constructed mutants arrestin(R175E) and arrestin(Δ2–16) with high affinity for light-activated forms only. The implications of these findings for the mechanism of arrestin-rhodopsin interaction are discussed in light of the recently determined crystal structure of arrestin. The visual amplification cascade has long served as a model for G protein-coupled receptor signaling. Light-activated rhodopsin (Rh*) 1The abbreviations used are: Rh*, light-activated rhodopsin; Rh, dark rhodopsin; arr, visual arrestin; G protein, guanine nucleotide-binding protein; P-Rh, phosphorylated rhodopsin; P-Rh*, phosphorylated light-activated rhodopsin; bp, base pair(s). activates visual G protein transducin, which in turn stimulates cGMP phosphodiesterase. The reduction in intracellular cGMP leads to the closing of cGMP-gated sodium channels and hyperpolarization of the rod cell. Quenching of the visual signaling is initiated by rapid phosphorylation of Rh* by rhodopsin kinase, followed by highly selective binding of arrestin to P-Rh* (1Wilden U. Hall S.W. Kuhn H. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1174-1178Crossref PubMed Scopus (603) Google Scholar). Arrestin and transducin binding sites on rhodopsin overlap (2Krupnick 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, 3Krupnick J.G. Gurevich V.V. Benovic J.L. J. Biol. Chem. 1997; 272: 18125-18131Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). As the result, arrestin binding precludes rhodopsin/transducin interaction and effectively shuts down signaling (1Wilden U. Hall S.W. Kuhn H. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1174-1178Crossref PubMed Scopus (603) Google Scholar, 3Krupnick J.G. Gurevich V.V. Benovic J.L. J. Biol. Chem. 1997; 272: 18125-18131Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Previously we developed an assay for assessing arrestin binding to various functional forms of rhodopsin, dark P-Rh, P-Rh*, dark Rh, and Rh* (4Gurevich V.V. Benovic J.L. J. Biol. Chem. 1992; 267: 21919-21923Abstract Full Text PDF PubMed Google Scholar). Binding studies with wild type, mutant, and chimeric arrestins identified several structural and functional regions within the arrestin molecule (4Gurevich V.V. Benovic J.L. J. Biol. Chem. 1992; 267: 21919-21923Abstract Full Text PDF PubMed Google Scholar, 5Gurevich V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar, 6Gurevich V.V. Chen C.-Y. Kim C.M. Benovic J.L. J. Biol. Chem. 1994; 269: 8721-8727Abstract Full Text PDF PubMed Google Scholar, 7Gurevich V.V. Dion S.B. Onorato J.J. Ptasienski J. Kim C.M. Sterne-Marr R. Hosey M.M. Benovic J.L. J. Biol. Chem. 1995; 270: 720-731Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar) (Fig. 1) that have been largely confirmed by recent elucidation of the partial crystal structure of arrestin (8Granzin J. Wilden U. Choe H.-W. Labahn J. Krafft B. Buldt G. Nature. 1998; 391: 918-921Crossref PubMed Scopus (215) Google Scholar). The NH2-terminal half of arrestin contains anactivation-recognition site that interacts with the regions of rhodopsin that change conformation upon light activation, and aphosphorylation-recognition site that interacts with the phosphorylated COOH terminus of rhodopsin (Fig. 1) (5Gurevich V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar). These two regions serve as the primary binding sites. When arrestin encounters P-Rh*, it undergoes a conformational rearrangement (9Schleicher A. Kuhn H. Hofmann K.P. Biochemistry. 1989; 28: 1170-1175Crossref Scopus (176) Google Scholar) that results in mobilization of a secondary binding site (5Gurevich V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar) within the COOH-terminal half of the molecule (Fig. 1). According to the model of sequential multisite binding (5Gurevich V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar), this transition is triggered by simultaneous engagement of both primary sites. We hypothesized that the transition is controlled by constraining intramolecular interactions within arrestin (5Gurevich V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar, 6Gurevich V.V. Chen C.-Y. Kim C.M. Benovic J.L. J. Biol. Chem. 1994; 269: 8721-8727Abstract Full Text PDF PubMed Google Scholar, 7Gurevich V.V. Dion S.B. Onorato J.J. Ptasienski J. Kim C.M. Sterne-Marr R. Hosey M.M. Benovic J.L. J. Biol. Chem. 1995; 270: 720-731Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 10Gurevich V.V. Benovic J.L. J. Biol. Chem. 1995; 270: 6010-6016Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 11Gurevich V.V. Benovic J.L. Mol. Pharmacol. 1997; 51: 161-169Crossref PubMed Scopus (124) Google Scholar, 12Gray-Keller M.P. Detwiler P.B. Benovic J.L. Gurevich V.V. Biochemistry. 1997; 36: 7058-7063Crossref PubMed Scopus (82) Google Scholar). Some of these constraints are disrupted as the result of arrestin binding to phosphates on phosphorhodopsin, while others are released upon arrestin binding to that part of rhodopsin, which changes conformation upon activation. When both constraints are simultaneously relieved, arrestin assumes its high affinity rhodopsin-binding conformation. Two constraining interactions have been tentatively identified, one between the basic NH2 terminus and the acidic COOH terminus (5Gurevich V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar, 6Gurevich V.V. Chen C.-Y. Kim C.M. Benovic J.L. J. Biol. Chem. 1994; 269: 8721-8727Abstract Full Text PDF PubMed Google Scholar, 7Gurevich V.V. Dion S.B. Onorato J.J. Ptasienski J. Kim C.M. Sterne-Marr R. Hosey M.M. Benovic J.L. J. Biol. Chem. 1995; 270: 720-731Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar), and another between Arg-175 and its negatively charged partner(s) (10Gurevich V.V. Benovic J.L. J. Biol. Chem. 1995; 270: 6010-6016Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 11Gurevich V.V. Benovic J.L. Mol. Pharmacol. 1997; 51: 161-169Crossref PubMed Scopus (124) Google Scholar, 12Gray-Keller M.P. Detwiler P.B. Benovic J.L. Gurevich V.V. Biochemistry. 1997; 36: 7058-7063Crossref PubMed Scopus (82) Google Scholar). The crystal structure of arrestin identifies Asp-296, Asp-303, and Asp-30 as likely partners of Arg-175 (8Granzin J. Wilden U. Choe H.-W. Labahn J. Krafft B. Buldt G. Nature. 1998; 391: 918-921Crossref PubMed Scopus (215) Google Scholar). Unfortunately, x-ray crystallography did not reveal any structure of the COOH terminus beyond residue 368. In this study we identify specific residues in the COOH terminus that control the selectivity of arrestin binding. γ-32PATP and 3Hleucine were purchased from NEN Life Science Products. All restriction enzymes were purchased from New England Biolabs. Sepharose 2B and all other chemicals were from sources previously described (5Gurevich V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar). Rabbit reticulocyte lysate and SP6 RNA polymerase were prepared as described previously (13Gurevich V.V. Methods Enzymol. 1996; 275: 382-397Crossref PubMed Scopus (60) Google Scholar). 11-cis-Retinal was generously supplied by Dr. R. K. Crouch. Bovine visual arrestin cDNA (14Shinohara T. Dietzschold B. Craft C.M. Wistow G. Early J.J. Donoso L.A. Horwitz J. Tao R. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6975-6979Crossref PubMed Scopus (185) Google Scholar) was generously supplied by Dr. T. Shinohara. Plasmid pARR-VSP was constructed as described (11Gurevich V.V. Benovic J.L. Mol. Pharmacol. 1997; 51: 161-169Crossref PubMed Scopus (124) Google Scholar). This pGEM2-based plasmid encodes bovine wild type arrestin with an “idealized” 5′-untranslated region (13Gurevich V.V. Methods Enzymol. 1996; 275: 382-397Crossref PubMed Scopus (60) Google Scholar) under control of SP6 promoter. Plasmid pARR-VSP was modified by polymerase chain reaction-based site-directed mutagenesis, as follows. Oligonucleotides 5′-gtc atg ggg ata cta gtg tct tac cag-3′ (codons 320–328) (IV) and 5′-t cgc cac ttc act gga tgtcag ctc tcc cag aag tcc tga cac cg-3′ (anticodons 349–334) were used as forward and reverse primers, respectively, to generate an 88 bp fragment. Oligonucleotide 5′-gag gcc tcc tgg cag ttc ttc at-3′ (codons 191–198) (I) and the 88 bp fragment were used as forward and reverse primers, respectively, to generate a 473-bp fragment. Oligonucleotides 5′-aat ttt gtt ttcgaa gag ttc gcggaagg caa aat ctg aaa g-3′ (codons 374–387) and 5′-agcttaagcttgcggccgcg-3′ (antisense modified 3′-UTR) (II) were used as forward and reverse primers, respectively, to generate a 152-bp fragment. Oligonucleotide 5′-ca tcc agt gaa gtg gcg act gag gtg c-3′ (codons 343–352) (III) and the 152-bp fragment were used as forward and reverse primers, respectively, to generate a 244 bp fragment. The 473- and 244-bp fragments (which have an 18-bp overlap) were used without template to generate a 700-bp “primer-dimer,” which was subsequently reamplified using oligonucleotides I and II, purified, digested with XhoI and NotI, and subcloned into XhoI/NotI-digested pARR-VSP. In the resulting construct (pARR-SC), which was used for all further mutagenesis, several silent mutations were introduced (underlined bases in oligonucleotides) into the visual arrestin open reading frame, creating unique restriction sites SpeI (codons 323–325),BstBI (codons 377–378), NruI (codons 380–382), and sites (codons (codons (codons and (codons All mutations were introduced by polymerase chain using and as forward and reverse primers, respectively, and the 244-bp fragment as a Oligonucleotide and resulting fragments of various were used as forward and reverse primers, respectively, with the 700-bp fragment as a template to generate fragments with These fragments were purified, digested in and subcloned into digested mutants were constructed by fragment and it into digested plasmid mutants were constructed polymerase chain fragments into digested The of polymerase chain of all were confirmed by were with in to arrestin In and were as described previously (5Gurevich V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar, V.V. Methods Enzymol. 1996; 275: 382-397Crossref PubMed Scopus (60) Google Scholar). All arrestin were by of 3Hleucine with specific resulting in the specific of The of of the arrestin mutants used in this study a protein with the on Two were used for the of protein in are to with most likely are by in reticulocyte lysate (13Gurevich V.V. Methods Enzymol. 1996; 275: 382-397Crossref PubMed Scopus (60) Google Scholar). to and are by for As an of a stability we used its by the of protein in the for followed by This for a was as a of that for wild type arrestin. rod were phosphorylated with rhodopsin kinase, and with as described M.P. Detwiler P.B. Benovic J.L. Gurevich V.V. Biochemistry. 1997; 36: 7058-7063Crossref PubMed Scopus (82) Google Scholar). The of phosphorylation for the rhodopsin used in these studies was of of In arrestins were in with of the various functional forms of rhodopsin in a of for in the dark in light (5Gurevich V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar). The were on and under light of Sepharose 2B with arrestin with the rod in the and binding determined in the of of of the binding and of the arrestin in the was The of COOH-terminal amino acids and the stability of arrestin and its selectivity toward P-Rh* (4Gurevich V.V. Benovic J.L. J. Biol. Chem. 1992; 267: 21919-21923Abstract Full Text PDF PubMed Google Scholar, 5Gurevich V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar). the residues involved we restriction sites NruI and to and These mutants were by in and with arrestin and (Fig. a in P-Rh* binding and a in binding to dark and The binding of to both dark and Rh* is that of while its binding to P-Rh* is the The of residues in the binding without further change in selectivity (Fig. The binding of any both its affinity for a form of rhodopsin and its stability under assay its binding to functional forms is not by its the of mutations on binding we used an of We that and stability to and of that for arrestin. it that residues within the COOH-terminal are for arrestin stability and selectivity. of functional of negatively charged residues in the COOH terminus of visual arrestin (5Gurevich V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar, 6Gurevich V.V. Chen C.-Y. Kim C.M. Benovic J.L. J. Biol. Chem. 1994; 269: 8721-8727Abstract Full Text PDF PubMed Google Scholar). we introduced and and these mutants with wild type arrestin and a arrestin(R175E) (10Gurevich V.V. Benovic J.L. J. Biol. Chem. 1995; 270: 6010-6016Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 11Gurevich V.V. Benovic J.L. Mol. Pharmacol. 1997; 51: 161-169Crossref PubMed Scopus (124) Google Scholar) (Fig. mutants into three in the I and result in a of arrestin binding. the to involved in arrestin stability in selectivity The most potent of these mutations the stability by in the and arrestin binding to P-Rh* and its binding to both dark forms of rhodopsin (Fig. of all three and results in a in dark as as a in binding to Rh* (Fig. without P-Rh* and dark of these mutations arrestin of in and arrestin binding to all functional forms of rhodopsin, to dark and Rh* (Fig. The most potent mutations in this are and The of on Rh* and dark binding is the in Rh* binding to (Fig. the other of of within this stimulates binding. these residues in with charged of involved in the interaction with Arg-175 of on structure of arrestin COOH terminus (8Granzin J. Wilden U. Choe H.-W. Labahn J. Krafft B. Buldt G. Nature. 1998; 391: 918-921Crossref PubMed Scopus (215) Google Scholar) not to this the is we 1) mutations have a and the of not these residues with mutations result in a change in arrestin selectivity. these we constructed mutants, with another and mutants with and (Fig. The in the of on Rh* but a change to that of The of are the as of negatively charged residues in this not with likely interacts with further this we constructed in which the is and and with and in which are with the of the of the an binding to all forms of rhodopsin, which is most for dark and Rh* (Fig. stability of is that of The of with without the a in dark and Rh* and a in P-Rh* binding. The of an (Fig. and mutations arrestin stability to and respectively, of that of wild type, and the of residues further it to and The between selectivity of and (which has a is (Fig. while the of this in wild type arrestin is (Fig. This that negatively charged residues in and an individual has a it is the of residues in the of has on binding the of the in is (Fig. dark binding of and arrestin(1–378) are In to other COOH-terminal are the of arrestin (Fig. a of three residues the and the charged of a with were to to of the the binding to dark P-Rh, dark Rh, and Rh* was and respectively, and the binding to P-Rh* was (Fig. The of individual mutations in this are that these residues act in The of and mutations of and (Fig. the is in all to the (Fig. that the as a functional with and a role. Two for binding to dark and Rh*, an in affinity of the primary binding site and the mobilization of the secondary site for the interaction with these forms of rhodopsin (5Gurevich V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar). of the binding to high is of the of the secondary site (5Gurevich V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar, 10Gurevich V.V. Benovic J.L. J. Biol. Chem. 1995; 270: 6010-6016Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 11Gurevich V.V. Benovic J.L. Mol. Pharmacol. 1997; 51: 161-169Crossref PubMed Scopus (124) Google Scholar). We of Rh* and dark binding of mutants the most selectivity changes (Fig. The binding of wild type arrestin to Rh* is by with of its binding to P-Rh* the secondary site is All mutants with Rh* are to is wild type arrestin (Fig. that these mutations result in the mobilization of the secondary site for Rh* binding to various arrestin interactions with dark in The COOH-terminal region of visual arrestin plays an important role in basal arrestin conformation and its selectivity toward P-Rh*. a that any conformational in the arrestin molecule to both stability and selectivity. The of conformational with of a protein to a recent study U. R. H. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar) has that a constitutively active of receptor is wild type This constitutively active was to the conformational change upon by an of receptor to in an state under the This the binding of constitutively active arrestins to P-Rh* determined in our (Fig. the of the we that which result in change in protein stability by a in dark Rh* selectivity in stability that the supports structural of the while changes in binding selectivity the result of the a secondary of a conformation. involved in arrestin stability to COOH-terminal These negatively charged residues in I and in and residues The of mutations are mutations of these residues to via interactions with of charged studies (5Gurevich V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar, 6Gurevich V.V. Chen C.-Y. Kim C.M. Benovic J.L. J. Biol. Chem. 1994; 269: 8721-8727Abstract Full Text PDF PubMed Google Scholar) that interaction with the arrestin NH2 terminus is most likely the recently arrestin crystal structure (8Granzin J. Wilden U. Choe H.-W. Labahn J. Krafft B. Buldt G. Nature. 1998; 391: 918-921Crossref PubMed Scopus (215) Google Scholar) not beyond residue the of the of the COOH-terminal region and of the NH2-terminal region and in (8Granzin J. Wilden U. Choe H.-W. Labahn J. Krafft B. Buldt G. Nature. 1998; 391: 918-921Crossref PubMed Scopus (215) Google supports this mutations in the arrestin COOH terminus not selectivity and structural several residues to involved in selectivity The most changes in arrestin selectivity are by of and by the of the Binding of and are not and The of these two that with a which has both a and a of these in two constraining that the of interaction is to high binding to dark and Rh*, via the mobilization of a secondary binding site (Fig. of arrestin crystal structure and of rhodopsin (8Granzin J. Wilden U. Choe H.-W. Labahn J. Krafft B. Buldt G. Nature. 1998; 391: 918-921Crossref PubMed Scopus (215) Google Scholar) a part of the open for interaction with the secondary binding site of arrestin. The arrestin molecule of two by an region (residues with both (8Granzin J. Wilden U. Choe H.-W. Labahn J. Krafft B. Buldt G. Nature. 1998; 391: 918-921Crossref PubMed Scopus (215) Google Scholar). is to that the secondary binding site is on a of COOH-terminal the conformational rearrangement of the arrestin molecule upon P-Rh* binding (9Schleicher A. Kuhn H. Hofmann K.P. Biochemistry. 1989; 28: 1170-1175Crossref Scopus (176) Google Scholar) and the mobilization of the secondary binding site (5Gurevich V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar) both identified with the of COOH-terminal toward of I to the stability of arrestin. is the most important residue in I (Fig. The stability of with that of (5Gurevich V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar) that this likely further and are involved in selectivity control (Fig. in with high binding to P-Rh*, dark P-Rh, and These two subregions as two regulatory both are to arrestin selectivity toward P-Rh*. Two of that is a structural of arrestin with this determined by The arrestin effectively with arrestin(R175E) for both P-Rh* and it as Gurevich and J. is as as wild type protein, the stability of both and is structural of are of the as as with structural are The most of a on stability upon of amino acids to that both mutations an structural studies arrestin mutants with stability and and The mutants into two 1) high binding to P-Rh*, dark P-Rh, and Rh* and high binding to the light-activated forms of rhodopsin and The model of sequential multisite binding that two of constraining intramolecular interactions in arrestin that are disrupted upon arrestin binding to phosphorylated light-activated mutations of binding of the binding P-Rh* and dark recent study (8Granzin J. Wilden U. Choe H.-W. Labahn J. Krafft B. Buldt G. Nature. 1998; 391: 918-921Crossref PubMed Scopus (215) Google Scholar) identified residues that with the Arg-175 Asp-296, and and two and on the rhodopsin-binding of the NH2-terminal that involved in in its form (5Gurevich V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar, 10Gurevich V.V. Benovic J.L. J. Biol. Chem. 1995; 270: 6010-6016Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar) the model not an of the binding of the of In to these we the of the The of constraining interactions residues that are of the primary binding sites These constraints are primary sites with of P-Rh*. 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