Visual Arrestin Binding to Rhodopsin

Arrestin plays a critical role in quenching phototransduction via its ability to specifically interact with the phosphorylated light-activated form of the visual receptor rhodopsin. In an effort to identify the residues involved in interaction with the phosphorylated C terminus of rhodopsin, we introduced point mutations into a basic region in visual arrestin previously implicated in phosphorylation-recognition (residues 163-189). A total of nine point mutations were made, each substituting a neutral hydrophilic residue for a positively charged Lys, Arg, or His. The functional consequences of these mutations were then analyzed by comparing the binding of full-length and truncated wild-type and mutant arrestin to various functional forms of rhodopsin. These studies demonstrate that Arg-171, Arg-175, and Lys-176 in bovine arrestin play a primary role in phosphate interaction, while Lys-166 and Lys-167 likely play a minor role in phosphate binding. In contrast, Lys-163 and His-179 appear to play a regulatory role, while Arg-182 and Arg-189 are not directly involved in arrestin binding to rhodopsin. Arg-175 also appears to function as a phosphorylation-sensitive trigger since charge neutralization by mutagenesis enables arrestin-R175N to bind to light-activated rhodopsin as well as wild-type arrestin binds to phosphorylated light-activated rhodopsin. The implications of these findings for the sequential multisite binding of arrestin to rhodopsin are discussed. Arrestin plays a critical role in quenching phototransduction via its ability to specifically interact with the phosphorylated light-activated form of the visual receptor rhodopsin. In an effort to identify the residues involved in interaction with the phosphorylated C terminus of rhodopsin, we introduced point mutations into a basic region in visual arrestin previously implicated in phosphorylation-recognition (residues 163-189). A total of nine point mutations were made, each substituting a neutral hydrophilic residue for a positively charged Lys, Arg, or His. The functional consequences of these mutations were then analyzed by comparing the binding of full-length and truncated wild-type and mutant arrestin to various functional forms of rhodopsin. These studies demonstrate that Arg-171, Arg-175, and Lys-176 in bovine arrestin play a primary role in phosphate interaction, while Lys-166 and Lys-167 likely play a minor role in phosphate binding. In contrast, Lys-163 and His-179 appear to play a regulatory role, while Arg-182 and Arg-189 are not directly involved in arrestin binding to rhodopsin. Arg-175 also appears to function as a phosphorylation-sensitive trigger since charge neutralization by mutagenesis enables arrestin-R175N to bind to light-activated rhodopsin as well as wild-type arrestin binds to phosphorylated light-activated rhodopsin. The implications of these findings for the sequential multisite binding of arrestin to rhodopsin are discussed. INTRODUCTIONG protein( 1The abbreviations used are: G proteinguanine nucleotide binding proteinRhdark rhodopsinRh∗light-activated rhodopsinP-Rhphosphorylated rhodopsinP-Rh∗phosphorylated light-activated rhodopsinARR(1-365)arrestin truncated after residue 365. )-coupled receptors enable eukaryotic cells to respond to a wide variety of stimuli including hormones, neurotransmitters, odors, and light. The visual amplification cascade is perhaps the best studied G protein-coupled receptor-initiated signaling system(1Hargrave P.A. McDowell J.H. Friedlander M. Mueckler M. Molecular Biology of Receptors and Transporters: Receptors. Academic Press, Inc., New York1992: 49-98Google Scholar). Signal transduction in retinal rod cells is initiated by photoisomerization of 11-cis-retinal covalently attached to a lysine residue within the seventh transmembrane domain of rhodopsin. Rhodopsin, via a series of transient intermediates, is thus converted into metarhodopsin II, the form of rhodopsin capable of binding the visual G protein transducin. The metarhodopsin II-bound transducin then exchanges GTP for GDP, promoting dissociation of the transducin α•GTP and βγ subunits. α•GTP then binds to the inhibitory γ-subunit of cGMP phosphodiesterase, leading to enzyme activation, decreased cGMP levels, the closing of cGMP-gated sodium channels, and hyperpolarization of the rod cell.Quenching of the visual transduction cascade involves a rapid activation-dependent phosphorylation of rhodopsin by the enzyme rhodopsin kinase(2Shichi H. Somers R.L. J. Biol. Chem. 1978; 253: 7040-7046Abstract Full Text PDF PubMed Google Scholar). This is followed by the highly selective binding of arrestin to activated phosphorylated rhodopsin, a process that appears to attenuate the activation of transducin(3Bennett N. Sitaramayya A. Biochemistry. 1988; 27: 1710-1715Crossref PubMed Scopus (127) Google Scholar, 4Wilden U. Hall S.W. Kuhn H. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1174-1178Crossref PubMed Scopus (570) Google Scholar). Recent mutagenesis studies of visual arrestin have enabled the identification of several important functional regions within arrestin (see Fig. 1) (5Gurevich V.V. Benovic J.L. J. Biol. Chem. 1992; 267: 21919-21923Abstract Full Text PDF PubMed Google Scholar, 6Gurevich V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar, 7Gurevich V.V. Chen C.-Y. Kim C.M. Benovic J.L. J. Biol. Chem. 1994; 269: 8721-8727Abstract Full Text PDF PubMed Google Scholar, 8Krupnick 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). In particular, a relatively short positively charged region encompassing residues 163-191 emerged as a likely domain involved in interaction with the phosphorylated C terminus of rhodopsin. The moderate size of this domain as well as the presence of a number of potentially important basic residues makes this region a suitable target for systematic site-directed mutagenesis.EXPERIMENTAL PROCEDURESMaterialsγ-32PATP, 35SdATP, and 3Hleucine were purchased from DuPont NEN. All restriction enzymes were purchased from Boehringer Mannheim, Promega, or New England Biolabs. Sepharose 2B, Sephadex G-25, and all other chemicals were from Sigma. Rabbit reticulocyte lysate and SP6 RNA polymerase were prepared as described previously(5Gurevich V.V. Benovic J.L. J. Biol. Chem. 1992; 267: 21919-21923Abstract Full Text PDF PubMed Google Scholar). 11-cis-Retinal was generously supplied by Dr. R. K. Crouch, National Institutes of Health. Other reagents were from sources described previously(5Gurevich V.V. Benovic J.L. J. Biol. Chem. 1992; 267: 21919-21923Abstract Full Text PDF PubMed Google Scholar, 6Gurevich V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar, 7Gurevich V.V. Chen C.-Y. Kim C.M. Benovic J.L. J. Biol. Chem. 1994; 269: 8721-8727Abstract Full Text PDF PubMed Google Scholar, 8Krupnick 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).Plasmid Construction and Site-directed MutagenesisA bovine visual arrestin cDNA was generously supplied by Dr. T. Shinohara(9Shinohara 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 (178) Google Scholar). An arrestin construct containing the wild-type terminus was with and and into the V.V. Chen C.-Y. Kim C.M. Benovic J.L. J. Biol. Chem. 1994; 269: 8721-8727Abstract Full Text PDF PubMed Google Scholar). was by a of the arrestin cDNA into from the and restriction by sequential with and followed by In to the point and of each to the region of a mutant were and into A of to the region of the and also mutations the restriction and were and into the In to the point mutations and of to the and regions the mutations or mutations the restriction were and The of was then into This the as well as the the wild-type with mutations the of restriction as in In to a of to the region were and into the a of to the region were and into the a of to the region were and into The of all were by the for each were into and and were with in to full-length truncated were with or to or V.V. Benovic J.L. J. Biol. Chem. 1992; 267: 21919-21923Abstract Full Text PDF PubMed Google Scholar, 6Gurevich V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar). In and were as V.V. Benovic J.L. J. Biol. Chem. 1992; 267: 21919-21923Abstract Full Text PDF PubMed Google Scholar, 6Gurevich V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar, 7Gurevich V.V. Chen C.-Y. Kim C.M. Benovic J.L. J. Biol. Chem. 1994; 269: 8721-8727Abstract Full Text PDF PubMed Google Scholar, 8Krupnick 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 rod were phosphorylated with receptor and with 11-cis-retinal as described V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar). The of phosphorylation for the used in these studies were and of of to of the binding involves a in the of arrestin a receptor binding. binding to rhodopsin, the in were in and with of the various functional forms of rhodopsin in a of for in the or in light. The were then and were a Sepharose with arrestin with the rod in the and in the presence of of not of the total binding and was mutagenesis studies have to the identification of several functional regions within the arrestin V.V. Benovic J.L. J. Biol. Chem. 1992; 267: 21919-21923Abstract Full Text PDF PubMed Google Scholar, 6Gurevich V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar, 7Gurevich V.V. Chen C.-Y. Kim C.M. Benovic J.L. J. Biol. Chem. 1994; 269: 8721-8727Abstract Full Text PDF PubMed Google Scholar, 8Krupnick 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, V.V. Kim C.M. Benovic J.L. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar, V.V. J.J. J. Kim C.M. R. Benovic J.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of arrestin (residues was to involved in with the of rhodopsin that as A in Fig. 1) and phosphorylation-recognition with the phosphorylated C terminus of rhodopsin, as in Fig. V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar, V.V. J.J. J. Kim C.M. R. Benovic J.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). these primary binding are by binding to phosphorylated light-activated rhodopsin arrestin a into a binding V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar, 7Gurevich V.V. Chen C.-Y. Kim C.M. Benovic J.L. J. Biol. Chem. 1994; 269: 8721-8727Abstract Full Text PDF PubMed Google Scholar, V.V. J.J. J. Kim C.M. R. Benovic J.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. Kuhn H. Biochemistry. PubMed Scopus Google Scholar). This in the of a binding as in Fig. The for the of this binding appears to by the interaction of the regulatory and regions and in Fig. V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar, 7Gurevich V.V. Chen C.-Y. Kim C.M. Benovic J.L. J. Biol. Chem. 1994; 269: 8721-8727Abstract Full Text PDF PubMed Google Scholar, V.V. J.J. J. Kim C.M. R. Benovic J.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the of the or region a mutant arrestin that a binding binding not to also to phosphorylated rhodopsin and light-activated rhodopsin V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar, 7Gurevich V.V. Chen C.-Y. Kim C.M. Benovic J.L. J. Biol. Chem. 1994; 269: 8721-8727Abstract Full Text PDF PubMed Google Scholar). The of the primary and binding to interaction appear to V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar, V.V. J.J. J. Kim C.M. R. Benovic J.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of the domain an that is capable of of the activation and phosphorylation of rhodopsin via its primary binding a with full-length arrestin since a binding V.V. Benovic J.L. J. Biol. Chem. 1992; 267: 21919-21923Abstract Full Text PDF PubMed Google Scholar, 6Gurevich V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar, 7Gurevich V.V. Chen C.-Y. Kim C.M. Benovic J.L. J. Biol. Chem. 1994; 269: 8721-8727Abstract Full Text PDF PubMed Google of the of of the interaction involved in phosphorylation-recognition that the charged phosphorylated C terminus of rhodopsin with positively charged residues in V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar). mutagenesis studies that residues 163-191 in arrestin play a role in V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar). In visual this region positively charged of are all arrestin 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 (178) Google Scholar, Benovic J.L. J. PubMed Scopus Google Scholar, R. Gurevich V.V. Donoso L.A. Benovic J.L. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar, A. T. H. G. 1993; PubMed Scopus Google Scholar, M. A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, A. M. R. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, K. J. J. H. 1993; PubMed Scopus Google Scholar, Donoso L.A. B. J. PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google In to the functional role of these charged we point mutations by substituting residues that were capable of lysine was with while and were with or In mutant was also These were then by in and ability to bind to various functional forms of rhodopsin and were with that of wild-type arrestin of arrestin mutations full-length arrestin binding to light-activated or and was with the various arrestin from to for in a or were then and and arrestin were by Sepharose as described The from each in are in a The ability of several of the point to bind to phosphorylated rhodopsin containing of of rhodopsin was with wild-type arrestin binding. and arrestin binding to by Arrestin binding to was by these mutations as well as by binding to rhodopsin containing a of phosphorylation was the of the mutant was 2B, arrestin binding to was for The in binding was with the mutant this mutant binding to all forms of rhodopsin, the is in the binding to these that and are likely involved in phosphate of the point also to arrestin binding to various forms of rhodopsin. arrestin binding to a binding to or arrestin binding to by while binding to and were to an and The binding of arrestin-R175N to was while its binding to was The arrestin binding to that the binding of arrestin-R175N to was with the binding of wild-type arrestin to These that Arg-175 play a critical role in the phosphorylation of the of Arrestin binding of full-length arrestin not its ability to interact with the phosphorylated C terminus of rhodopsin also its ability to binding V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar, 7Gurevich V.V. Chen C.-Y. Kim C.M. Benovic J.L. J. Biol. Chem. 1994; 269: 8721-8727Abstract Full Text PDF PubMed Google Scholar, V.V. J.J. J. Kim C.M. R. Benovic J.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). or of these by a in to directly the of each interaction with the phosphorylated C terminus of rhodopsin, we the binding of wild-type and mutant that were truncated residue previously to its phosphorylation-recognition domain while the binding V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google of mutations truncated binding to and was with the various from to for in a were then and and arrestin were by Sepharose as described The from each in are in a In these the point mutations and were to in the in binding to A and This that these residues play a role in phosphate This is by the mutant the binding to is the of these for is by the that the truncated and bind to as well as wild-type and were to binding to a minor role for these residues in phosphate interaction also binding to while mutations and binding to and and of the of the various mutations full-length and truncated arrestin binding a number of Fig. and Fig. and and the binding of full-length to is while the binding of truncated to is not In contrast, the in binding of and to is the truncated are studied with the full-length of are the since the binding of truncated to is while the binding of full-length to all forms of rhodopsin is The of the appears to for binding is also for and and full-length arrestin binding to functional forms of rhodopsin, to a and a of for binding to was previously for and in V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar, 7Gurevich V.V. Chen C.-Y. Kim C.M. Benovic J.L. J. Biol. Chem. 1994; 269: 8721-8727Abstract Full Text PDF PubMed Google Scholar, 8Krupnick 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). The of these was to a in the of the of arrestin into a binding This in the of the binding not in binding to also in the binding to and V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar, 7Gurevich V.V. Chen C.-Y. Kim C.M. Benovic J.L. J. Biol. Chem. 1994; 269: 8721-8727Abstract Full Text PDF PubMed Google Scholar). this also as a in the of arrestin interaction with and V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar). In to the by and mutations arrestin binding to we the of wild-type and mutant arrestin binding to mutations were to the of arrestin interaction with that of the binding are of full-length and truncated binding to mutant or mutant from to were with of in a for were then and and arrestin were by Sepharose as described binding was and for and The from each in are that Arg-175 and to a His-179 are involved in the of the of arrestin into a binding studies have that interaction the basic in Fig. 1) and the C terminus in Fig. 1) of arrestin in this V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar, 7Gurevich V.V. Chen C.-Y. Kim C.M. Benovic J.L. J. Biol. Chem. 1994; 269: 8721-8727Abstract Full Text PDF PubMed Google Scholar). This the of Arg-175 and His-179 also interact with the C terminus of arrestin or with of the this we studied the binding of containing the wild-type or mutant binds with a the full-length mutant In binding to the binding of wild-type In contrast, the the binding of to all functional forms of rhodopsin the ability of to the of to is while the mutant a that of the and mutant these that while and an interaction that of binding the interaction of these residues within regions of the arrestin with Arg-175 involved in an interaction within the domain of of mutations and of full-length and truncated binding. The functional form of rhodopsin was with the arrestin from to for in a were then and and arrestin were by Sepharose as described The from each in are of in we that are to arrestin while phosphorylation not to V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar). the and receptors of of receptor for binding of and arrestin V.V. Kim C.M. Benovic J.L. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar, V.V. J.J. J. Kim C.M. R. Benovic J.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). residues the C terminus of arrestin and were as the for rhodopsin while phosphorylation was to residues and J.H. P.A. Biochemistry. 1993; PubMed Scopus Google Scholar, Biochemistry. 1993; PubMed Scopus Google Scholar). In to of the positively charged residues within the phosphorylation-recognition region of arrestin are specifically to interact with these we the interaction of the with and phosphorylated to of or of of rhodopsin and Fig. and and arrestin binding to highly phosphorylated rhodopsin to various mutations and and a 2B, In the binding is to was with in all the inhibitory binding to are with appears that residue within the phosphorylation-recognition region of arrestin is specifically involved in interaction with the the rhodopsin C arrestin interaction with these appears to for the of charge in the phosphorylation-recognition thus the inhibitory of point binding of the truncated wild-type and mutant are the of the mutations and binding appear with The inhibitory of binding is A and the of the full-length arrestin is also with A and of Arrestin in these to functional to positively charged residues within truncated arrestin interaction with functional form of rhodopsin, full-length arrestin interaction with this the ability of full-length arrestin to bind to and light-activated by This that Lys-163 not directly in the interaction with the phosphorylated C terminus of rhodopsin, its presence appears to important for the of binding full-length arrestin binding to all functional forms of rhodopsin also a inhibitory truncated arrestin binding to while interaction with These a minor role of Lys-166 in phosphate binding. full-length and truncated arrestin binding to and by while a binding to This a role of Lys-167 in phosphate interaction that not appear for the of binding The inhibitory of full-length and truncated arrestin binding to and light-activated are and a role of in phosphate also truncated arrestin binding to this also full-length arrestin binding to all functional forms of rhodopsin a that Arg-175 plays a role in phosphate interaction and of binding Arg-175 interaction with within the domain to arrestin in a binding binding to a phosphate the C terminus of rhodopsin, the charge of Arg-175 the interaction and arrestin to a binding that with light-activated rhodopsin. of the charge Arg-175 by mutagenesis for phosphate interaction, as a arrestin a binding binding to the other the primary interaction the rhodopsin C terminus and the arrestin phosphorylation-recognition is by the thus truncated arrestin binding to function of Lys-176 appears to to that of The a moderate inhibitory full-length arrestin while truncated arrestin binding to not to Lys-176 appears to of the residues involved in phosphate binding. of these residues Arg-175, and truncated arrestin binding to that V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar, 7Gurevich V.V. Chen C.-Y. Kim C.M. Benovic J.L. J. Biol. Chem. 1994; 269: 8721-8727Abstract Full Text PDF PubMed Google Scholar, 8Krupnick 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 and this not the role of residues the 163-191 region as involved in the of the of the that Arg-171, Arg-175, and Lys-176 play the role in phosphorylation of the full-length arrestin binding are to of this truncated arrestin binding. The binding of to appears to binding to arrestin-R175N interaction with while His-179 not play a role in phosphate in the of arrestin into a binding binding to its by mutagenesis this likely His-179 with a residue within the domain of in to Arg-175, phosphate binding this a truncated arrestin binding to all functional forms of rhodopsin. the full-length this a inhibitory arrestin binding to other functional Arg-182 and Arg-189 not appear to play role in phosphate binding or of binding these charged residues are the the and are the of (residues Fig. and in the of positively charged residues appear to directly involved in phosphate binding. Arg-171, Arg-175, and Lys-176 play a role, and Lys-166 and Lys-167 play a minor role in phosphate The of Lys-163 or Lys-166 appears to for full-length for truncated a role of these residues in the of arrestin into a binding In contrast, the of Arg-175 and His-179 full-length arrestin binding that these residues a function of Arg-175 is of for several sequential multisite binding that the of arrestin into a binding V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar, 7Gurevich V.V. Chen C.-Y. Kim C.M. Benovic J.L. J. Biol. Chem. 1994; 269: 8721-8727Abstract Full Text PDF PubMed Google Scholar). of these binding to a phosphorylated form of rhodopsin, while binding to an activated form of rhodopsin. the the of binding arrestin binding to V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google and V.V. Chen C.-Y. Kim C.M. Benovic J.L. J. Biol. Chem. 1994; 269: 8721-8727Abstract Full Text PDF PubMed Google that Arg-175 in of these that appears to the neutralization of its charge by phosphate binding. a arrestin-R175N in binding to with an with that of wild-type arrestin binding to These the sequential multisite binding and that Arg-175 function as a of the phosphorylation-sensitive These thus the into the of the involved in arrestin binding to A and also involved in the of positively charged residue in the to Arg-175 in visual arrestin is in all thus This that the of a neutral residue for this positively charged residue as a to a This also and the of of receptor of in since a mutant not the presence of the G protein-coupled receptor The of also a to G protein-coupled positively charged residues to directly involved in phosphate binding are within an of the phosphorylation-recognition region This the of the rhodopsin of phosphorylation within a (residues the C terminus of J.H. P.A. Biochemistry. 1993; PubMed Scopus Google Scholar, Biochemistry. 1993; PubMed Scopus Google Scholar). The charged residues appear to play a regulatory role while the and phosphorylation-sensitive are within the of the phosphorylation-recognition all have a potentially and and in visual Fig. 1) to the phosphorylation-recognition The role of these residues in of arrestin also by this we have a number of residues that are directly involved in phosphate binding and residues that appear to function as a phosphorylation-sensitive trigger to the of arrestin into a binding mutagenesis studies of other of the arrestin to the of phosphorylation and the of binding are INTRODUCTIONG protein( 1The abbreviations used are: G proteinguanine nucleotide binding proteinRhdark rhodopsinRh∗light-activated rhodopsinP-Rhphosphorylated rhodopsinP-Rh∗phosphorylated light-activated rhodopsinARR(1-365)arrestin truncated after residue 365. )-coupled receptors enable eukaryotic cells to respond to a wide variety of stimuli including hormones, neurotransmitters, odors, and light. The visual amplification cascade is perhaps the best studied G protein-coupled receptor-initiated signaling system(1Hargrave P.A. McDowell J.H. Friedlander M. Mueckler M. Molecular Biology of Receptors and Transporters: Receptors. Academic Press, Inc., New York1992: 49-98Google Scholar). Signal transduction in retinal rod cells is initiated by photoisomerization of 11-cis-retinal covalently attached to a lysine residue within the seventh transmembrane domain of rhodopsin. Rhodopsin, via a series of transient intermediates, is thus converted into metarhodopsin II, the form of rhodopsin capable of binding the visual G protein transducin. The metarhodopsin II-bound transducin then exchanges GTP for GDP, promoting dissociation of the transducin α•GTP and βγ subunits. α•GTP then binds to the inhibitory γ-subunit of cGMP phosphodiesterase, leading to enzyme activation, decreased cGMP levels, the closing of cGMP-gated sodium channels, and hyperpolarization of the rod cell.Quenching of the visual transduction cascade involves a rapid activation-dependent phosphorylation of rhodopsin by the enzyme rhodopsin kinase(2Shichi H. Somers R.L. J. Biol. Chem. 1978; 253: 7040-7046Abstract Full Text PDF PubMed Google Scholar). This is followed by the highly selective binding of arrestin to activated phosphorylated rhodopsin, a process that appears to attenuate the activation of transducin(3Bennett N. Sitaramayya A. Biochemistry. 1988; 27: 1710-1715Crossref PubMed Scopus (127) Google Scholar, 4Wilden U. Hall S.W. Kuhn H. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1174-1178Crossref PubMed Scopus (570) Google Scholar). Recent mutagenesis studies of visual arrestin have enabled the identification of several important functional regions within arrestin (see Fig. 1) (5Gurevich V.V. Benovic J.L. J. Biol. Chem. 1992; 267: 21919-21923Abstract Full Text PDF PubMed Google Scholar, 6Gurevich V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar, 7Gurevich V.V. Chen C.-Y. Kim C.M. Benovic J.L. J. Biol. Chem. 1994; 269: 8721-8727Abstract Full Text PDF PubMed Google Scholar, 8Krupnick 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). In particular, a relatively short positively charged region encompassing residues 163-191 emerged as a likely domain involved in interaction with the phosphorylated C terminus of rhodopsin. The moderate size of this domain as well as the presence of a number of potentially important basic residues makes this region a suitable target for systematic site-directed