Ca2+-dependent Interaction of Recoverin with Rhodopsin Kinase

Recoverin (Rv) is a myristoylated Ca2+-binding protein present primarily in bovine photoreceptors. It represents a newly identified family of neuronal specific Ca2+-binding proteins that includes neurocalcin, hippocalcin, and guanylyl cyclase-activating protein. To investigate the function of Rv in photoreceptors, we identified proteins that bind immobilized Rv in a Ca2+-dependent manner. Rhodopsin kinase (RK), interphotoreceptor retinoid-binding protein, and tubulin interact with Rv in the presence of Ca2+. The importance of the Rv/RK interaction was further characterized. RK, purified using immobilized Rv as an affinity matrix, catalyzed the light-dependent and Ca2+-independent incorporation of phosphates into rhodopsin when reconstituted with urea-stripped rod outer segment membranes. When only a small fraction (0.04%) of rhodopsin was photolyzed, as many as 700 phosphates were incorporated per photolyzed rhodopsin, a phenomenon known as “high gain” phosphorylation. When recoverin was added, the activity of RK became sensitive to free Ca2+, with EC50 = 3 μM. The N-terminal myristoyl residue of Rv enhances the inhibitory effect of Rv and introduces cooperativity to the Ca2+-dependent inhibition of rhodopsin phosphorylation. Rv neither interacts with other members of the G-protein-coupled receptor kinase family such as β-adrenergic receptor kinase 1 nor inhibits β-adrenergic receptor kinase 1 activity. The specific and Ca2+-dependent Rv/RK interaction is necessary for the inhibitory effect of Rv on rhodopsin phosphorylation and may play an important role in photoreceptor light adaptation. Recoverin (Rv) is a myristoylated Ca2+-binding protein present primarily in bovine photoreceptors. It represents a newly identified family of neuronal specific Ca2+-binding proteins that includes neurocalcin, hippocalcin, and guanylyl cyclase-activating protein. To investigate the function of Rv in photoreceptors, we identified proteins that bind immobilized Rv in a Ca2+-dependent manner. Rhodopsin kinase (RK), interphotoreceptor retinoid-binding protein, and tubulin interact with Rv in the presence of Ca2+. The importance of the Rv/RK interaction was further characterized. RK, purified using immobilized Rv as an affinity matrix, catalyzed the light-dependent and Ca2+-independent incorporation of phosphates into rhodopsin when reconstituted with urea-stripped rod outer segment membranes. When only a small fraction (0.04%) of rhodopsin was photolyzed, as many as 700 phosphates were incorporated per photolyzed rhodopsin, a phenomenon known as “high gain” phosphorylation. When recoverin was added, the activity of RK became sensitive to free Ca2+, with EC50 = 3 μM. The N-terminal myristoyl residue of Rv enhances the inhibitory effect of Rv and introduces cooperativity to the Ca2+-dependent inhibition of rhodopsin phosphorylation. Rv neither interacts with other members of the G-protein-coupled receptor kinase family such as β-adrenergic receptor kinase 1 nor inhibits β-adrenergic receptor kinase 1 activity. The specific and Ca2+-dependent Rv/RK interaction is necessary for the inhibitory effect of Rv on rhodopsin phosphorylation and may play an important role in photoreceptor light adaptation. Photoactivation of rhodopsin stimulates cGMP hydrolysis within vertebrate photoreceptors (for review, see 28Lagnado L. Baylor D. Neuron. 1992; 8: 995-1022Google Scholar and 43Yarfitz S. Hurley J.B. J. Biol. Chem. 1994; 269: 14329-14331Google Scholar). The resulting loss of intracellular cGMP slows the influx of Ca2+ through cGMP-dependent cation channels in the photoreceptor plasma membrane. Under these conditions, unabated Ca2+ efflux through Na+/K+,Ca2+ exchangers lowers the bulk concentration of intracellular free Ca2+ from 550 to 50 nM (13Gray-Keller M.P. Detwiler P.B. Neuron. 1994; 13: 1-20Google Scholar). The light-induced lowering of intracellular free Ca2+ is a signal that promotes recovery from photoexcitation. The calcium signal is decoded by calcium-binding proteins that participate in several biochemical reactions. Low Ca2+ concentrations stimulate resynthesis of cGMP by the action of Ca2+-binding proteins that stimulate guanylyl cyclase (26Koch K.W. Stryer L. Nature. 1988; 334: 64-66Google Scholar; 11Gorczyca W.A. Gray-Keller M.P. Detwiler P.B. Palczewski K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4014-4018Google Scholar; 10Dizhoor A.M. Lowe D. Olshevskaya E. Laura R. Hurley J.B. Neuron. 1994; 12: 1345-1352Google Scholar). Low Ca2+ concentrations also stimulate plasma membrane cation channel activity by relieving the inhibitory effect of Ca2+/calmodulin on cGMP-gated cation channels (15Hsu Y.T. Molday R.S. Nature. 1993; 361: 76-79Google Scholar). Finally, low Ca2+ concentrations facilitate phosphorylation and inactivation of photoactivated rhodopsin (19Kawamura S. Nature. 1993; 362: 855-857Google Scholar; 19Kawamura S. Nature. 1993; 362: 855-857Google Scholar). The effect of Ca2+ on rhodopsin phosphorylation is mediated by a protein referred to as sensitivity-modulating protein (S-modulin) 1The abbreviations used are: S-modulinsensitivity-modulating proteinRvrecoverinROSrod outer segment(s)RKrhodopsin kinaseMOPS3-(N-morpholino)propanesulfonic acidPAGEpolyacrylamide gel electrophoresisIRBPinterphotoreceptor retinoid-binding proteinBr2-BAPTA5,5’-dibromo-1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acidGCAPguanylyl cyclase-activating proteinVILIPvisinin-like proteinNA-Rvnonacylated recoverinC1420-Rvmyristoylated recoverin. or recoverin (Rv) (21Kawamura S. Hisatomi O. Kayada S. Tokunaga F. Kuo C.H. J. Biol. Chem. 1993; 268: 14579-14582Google Scholar). sensitivity-modulating protein recoverin rod outer segment(s) rhodopsin kinase 3-(N-morpholino)propanesulfonic acid polyacrylamide gel electrophoresis interphotoreceptor retinoid-binding protein 5,5’-dibromo-1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid guanylyl cyclase-activating protein visinin-like protein nonacylated recoverin myristoylated recoverin. Recoverin is a Ca2+-binding protein present only in vertebrate photoreceptors (39Ray S. Zozulya S. Niemi G.A. Flaherty K.M. Brolley D. Dizhoor A.M. McKay D.B. Hurley J.B. Stryer L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5705-5709Google Scholar), certain retinal cone bipolar cells, and pineal glands (30Milam A.H. Dacey D.M. Dizhoor A.M. Visual Neurosci. 1993; 10: 1-12Google Scholar). It represents a recently identified family of neuronal specific Ca2+-binding proteins that includes hippocalcin (25Kobayashi M. Takamatsu K. Saitoh S. Miura M. Noguchi T. Biochem. Biophys. Res. Commun. 1992; 189: 511-517Google Scholar), neurocalcin (32Okazaki K. Watanabe M. Ando Y. Hagiwara M. Terasawa M. Hidaka H. Biochem. Biophys. Res. Commun. 1992; 185: 147-153Google Scholar), visinin (42Yamagata K. Goto K. Kuo C.H. Kondo H. Miki N. Neuron. 1990; 4: 469-476Google Scholar), S-modulin (20Kawamura S. Murakami M. Nature. 1991; 349: 420-423Google Scholar), visinin-like protein (VILIP) (29Lenz S.E. Henschel Y. Zopf D. Voss B. Gundelfinger E.D. Brain Res. Mol. Brain Res. 1992; 15: 133-140Google Scholar), frequenin (37Pongs O. Lindemeier J. Zhu X.R. Theil T. Engelkamp D. Krah-Jentgens I. Lambrecht H.G. Koch K.W. Schwemer J. Rivosecchi R. Mallart A. Galceran J. Canal I. Barbas J.A. Ferrus A. Neuron. 1993; 11: 15-28Google Scholar), and guanylyl cyclase activating protein (GCAP) (11Gorczyca W.A. Gray-Keller M.P. Detwiler P.B. Palczewski K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4014-4018Google Scholar). Rv was initially purified from bovine retinas, and it was thought to be a Ca2+-sensitive activator of guanylyl cyclase, but this function has not been confirmed (16Hurley J.B. Dizhoor A.M. Ray S. Stryer L. Science. 1993; 260: 740Google Scholar). Rv was also recognized as an antigen associated with cancer-associated retinopathy (33Palczewski K. Buczylko J. Kaplan M.W. Polans A.S. Crabb J.W. J. Biol. Chem. 1991; 266: 12949-12955Google Scholar). When internally dialyzed into rod outer segments (ROS), Rv prolongs the photoresponse (14Gray-Keller M.P. Polans A.S. Palczewski K. Detwiler P.B. Neuron. 1993; 10: 523-531Google Scholar). This in vivo effect of Rv is consistent with the in vitro observation that S-modulin, a frog homologue of Rv, enhances the effect of light on cGMP phosphodiesterase and inhibits rhodopsin phosphorylation (19Kawamura S. Nature. 1993; 362: 855-857Google Scholar). A similar effect of bovine Rv has been reported (19Kawamura S. Nature. 1993; 362: 855-857Google Scholar), and it has been suggested that Rv interacts with rhodopsin kinase (RK) since Rv coeluted with a 67-kDa protein on a sizing column in the presence of Ca2+ (12Gorodovikova E.N. Phillipov P. FEBS Lett. 1993; 335: 277-279Google Scholar). The NH2 terminus of Rv purified from bovine retinas is heterogeneously acylated by one of four fatty acids, C14:0, C14:1, C14:2, or C12:0 (8Dizhoor A.M. Ericsson L.H. Johnson R.S. Kumar S. Olshevskaya E. Zozulya S. Neubert T.A. Stryer L. Hurley J.B. Walsh K.A. J. Biol. Chem. 1992; 267: 16033-16036Google Scholar). N-acylation of Rv plays an essential role in Ca2+-dependent membrane targeting (9Dizhoor A.M. Chen C.-K. Olshevskaya E. Sinelnikova V.V. Phillipov P. Hurley J.B. Science. 1993; 259: 829-832Google Scholar) through a novel calcium-myristoyl protein switch mechanism (44Zozulya S. Stryer L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11569-11573Google Scholar). However, N-terminal myristoylation is not required for Rv to inhibit rhodopsin phosphorylation (6Chen C.-K. Hurley J.B. Invest. Ophthamol. 35: 1485Google Scholar; 22Kawamura S. Cox J.A. Nef P. Biochem. Biophys. Res. Commun. 1994; 203: 121-127Google Scholar). This suggests that binding of Rv to ROS membranes is not necessary for its inhibitory effect. A recent report demonstrated that an N-terminal myristoyl residue lowers the apparent Ca2+ affinity of Rv, but introduces cooperative binding of Ca2+. Nonacylated Rv (NA-Rv) binds two Ca2+ with affinities of 0.11 and 6.9 μM, respectively. Myristoylated Rv (C14:0-Rv) binds two Ca2+ with an affinity of 17 μM and a Hill coefficient of 1.75. The affinity of C14:0-Rv for Ca2+ in the presence of membranes was calculated to be 4 μM according to a concerted allosteric model (1Ames J.B. Porumb T. Tanaka T. Ikura M. Stryer L. J. Biol. Chem. 1995; 270: 4526-4533Google Scholar). There are disparate reports about the Ca2+ dependence of recoverin's effect on rhodopsin phosphorylation. Original reports indicated that the IC50 for both Rv and S-modulin was 100-200 nM free Ca2+, but a recent study indicates that the IC50 may be significantly higher (24Klenchin V.A. Calvert P.D. Bownds M.D. Biophys. J. 1994; 66 (abstr.): A48Google Scholar). We have characterized recoverin's effect on rhodopsin phosphorylation in vitro to gain insight as to how Rv functions in vivo. Our aim was to identify soluble retinal proteins that interact with Rv, and we found that RK interacts with immobilized Rv in a specific and Ca2+-dependent manner. RK, affinity-purified by immobilized Rv, phosphorylates rhodopsin in response to light when reconstituted with urea-stripped ROS membranes. When only a small fraction of rhodopsin is photolyzed in the reconstituted system, as many as 700 phosphates are incorporated per photolyzed rhodopsin. These results suggest that nonphotolyzed rhodopsins are also being phosphorylated in response to light. Similar “high gain” phosphorylation has been reported by 4Binder B.M. Biernbaum M.S. Bownds M.D. J. Biol. Chem. 1990; 265: 15333-15340Google Scholar using electropermeabilized frog ROS. When Rv is added to our reconstituted system, high gain phosphorylation is reduced in a Ca2+-titratable manner. C14:0-Rv and NA-Rv were produced as described (39Ray S. Zozulya S. Niemi G.A. Flaherty K.M. Brolley D. Dizhoor A.M. McKay D.B. Hurley J.B. Stryer L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5705-5709Google Scholar). The extent of myristoylation of the C14:0-Rv preparation was determined to be >99% using liquid chromatography-coupled electrospray mass spectrometry (data not shown). CNBr-activated Sepharose CL-4B (Sigma) was washed with 50 mM HCl for 30 min and incubated with 100 mM sodium borate (pH 8.5) at room temperature for 1 h. The beads were then washed with 100 mM sodium (pH and mM was added to a concentration of 1 Rv of was then added to the The was at for the was by (pH to a concentration of 100 and was for h. The was The beads were at in mM (pH 30 mM mM mM 1 mM 1 mM and μM with sodium 100 bovine retinas were and in ROS 1 mM and then at at for 30 The was with an of ROS and at at for 30 The was and at at for for retinal was in a room The was to Rv and the were washed with ROS protein washed as by the proteins were with ROS mM from 50 bovine retinas were to a 1 NA-Rv The column was washed with ROS and two high The high of the the described in RK Koch J. J. Biol. Chem. 1994; 269: Scholar) were used as RK was further purified by column the from the NA-Rv column was with mM (pH mM 1 mM and 50 μM and to a column RK was with a of mM in D. ROS membranes were washed and in in mM (pH and mM for min in the H. J. Biol. Chem. Scholar). the ROS membranes were washed with mM (pH and 4 mM RK activity was by incorporation into rhodopsin as a function of RK of purified Rv, RK, and urea-stripped ROS membranes. Rhodopsin phosphorylation was as described The effect of RK concentration on the of incorporated per rhodopsin is when Rv μM NA-Rv or μM C14:0-Rv was The concentrations of free Ca2+, and urea-stripped ROS membranes as concentration of in the were and μM, not inhibit rhodopsin phosphorylation catalyzed by β-adrenergic receptor kinase β-adrenergic receptor kinase 1 not bind β-adrenergic receptor kinase 1 were through immobilized and the binding of β-adrenergic receptor kinase 1 was by by and by using an specific to β-adrenergic receptor kinase 1 1 and of β-adrenergic receptor kinase and of 3 and we the to of β-adrenergic receptor kinase 1 to the β-adrenergic receptor kinase 1 in 4 and are Rv not inhibit the phosphorylation of rhodopsin catalyzed by β-adrenergic receptor kinase RK and β-adrenergic receptor kinase 1 are as of kinase activity at 100 μM when Rv was Similar results were in RK, and urea-stripped ROS membranes were in the with or Rv, μM was added to a of 30 was added, a that rhodopsin was and the was to the for a as in in for min in the at room were used as was by an of The phosphorylation of rhodopsin was by The of incorporated into rhodopsin was by the rhodopsin from the the gel with at for and liquid The a not to light of the activity and of the in to high gain was to light-dependent phosphorylation. ROS urea-stripped ROS membranes μM μM Rv, and 50 nM RK or 100 nM β-adrenergic receptor kinase 1 and purified from J. J. Biol. Chem. 1993; 268: Scholar), with or μM purified from bovine required for activity of β-adrenergic receptor kinase J.A. J. J.B. Science. 1992; Scholar), were in the 50 μM was added to a of was added, a that rhodopsin was and the was to the for min in the at room not to light were used as The was by an of The of rhodopsin phosphorylation was as described that were of the activity were to light-dependent phosphorylation. To identify proteins that bind Rv, C14:0-Rv or NA-Rv was to CNBr-activated Sepharose to an immobilized Rv A retinal in 1 mM Ca2+ was then the The column was with 1 mM Ca2+, and then Ca2+-dependent proteins were with mM proteins and in to immobilized C14:0-Rv in the presence of Ca2+ and when Ca2+ was with the and proteins to immobilized NA-Rv The protein was further purified by liquid and its N-terminal was determined by as of this with identified the protein as bovine interphotoreceptor retinoid-binding protein We also purified the by liquid and found the N-terminal identified this as the N-terminal of tubulin and The of these proteins were further confirmed by with tubulin on (data not shown). The protein was recognized by bovine RK K. Buczylko J. L. Crabb J.W. Polans A.S. J. Biol. Chem. 1993; 268: Scholar; J. J. Biol. Chem. 1992; 267: Scholar). The of the protein as RK was confirmed by it on an NA-Rv column and its to rhodopsin (data not shown). The purified protein also A and consistent with its as RK Johnson J. Biol. Chem. 1990; 265: Scholar). The NA-Rv column RK from retinal RK when Ca2+ was by the column with on this Ca2+-dependent we a to RK from retinal as in The NA-Rv column was a C14:0-Rv column it not bind and it to have a higher for It that purified retinal RK is since it has the as RK (data not and has a from a of RK J.A. J. J.B. Science. 1992; Scholar). The of RK purified by this from retinal from to a for RK, we of RK from 1 of J. E. D. R. J. and J. B. in To the effect of Rv on rhodopsin phosphorylation RK and to this effect soluble protein in to RK, we reconstituted urea-stripped ROS purified RK and C14:0-Rv or RK, when reconstituted with urea-stripped ROS catalyzed the light-dependent incorporation of as many as several phosphates into the rhodopsin for rhodopsin that was This indicates that nonphotolyzed rhodopsin is being phosphorylated in response to light. This high gain phosphorylation be in The extent of high gain phosphorylation as RK concentration was gain phosphorylation was when only a small fraction of rhodopsin was the presence of Rv and Ca2+, the high gain phosphorylation to be and When the effect of recoverin was with free Ca2+ by mM both NA-Rv and C14:0-Rv an EC50 for free Ca2+ of 3 μM, but the presence of a myristoyl residue to cooperativity to the inhibitory effect The Hill coefficient of the Ca2+ effect for NA-Rv is and the for C14:0-Rv is The myristoyl residue also enhances the inhibitory effect of The IC50 for is μM, and the IC50 for NA-Rv is μM at Ca2+ concentration of free Ca2+ concentration on the inhibitory effect of Rv on RK activity. Rhodopsin phosphorylation was as described The free Ca2+ concentration was by with mM and was by a Ca2+-sensitive using The effect of Ca2+ on the of incorporated per rhodopsin is when Rv μM NA-Rv or μM C14:0-Rv was The concentrations of RK, and urea-stripped ROS membranes were μM, and μM, respectively. Similar results were from two of rhodopsin phosphorylation by Rhodopsin phosphorylation was as described The effect of NA-Rv and C14:0-Rv concentrations on RK activity when Rv was is The concentrations of free Ca2+, and urea-stripped ROS membranes were and μM, respectively. RK was present at or Similar results were from two Our results using immobilized recoverin a and specific interaction Rv and RK, but not the importance of the Rv/RK interaction for the inhibitory effect of Rv on rhodopsin phosphorylation. It is that inhibition of rhodopsin phosphorylation may of Rv, with rhodopsin A.M. Ray S. Kumar S. Niemi M. Brolley D. Walsh K.A. Hurley J.B. Stryer L. Science. 1991; Scholar) or with (8Dizhoor A.M. Ericsson L.H. Johnson R.S. Kumar S. Olshevskaya E. Zozulya S. Neubert T.A. Stryer L. Hurley J.B. Walsh K.A. J. Biol. Chem. 1992; 267: 16033-16036Google Scholar; A.M. Chen C.-K. Olshevskaya E. Sinelnikova V.V. Phillipov P. Hurley J.B. Science. 1993; 259: 829-832Google Scholar). To investigate the mechanism of recoverin's we to Rv interacts with other members of the G-protein-coupled receptor kinase family such as β-adrenergic receptor kinase 1 J. Koch J. Biol. Chem. 1993; 268: Scholar). receptor kinase RK, phosphorylates rhodopsin in a light-dependent in vitro F. Nature. Scholar). that β-adrenergic receptor kinase 1 not bind to immobilized recoverin. be from the in the phosphorylation of photoactivated rhodopsin by β-adrenergic receptor kinase 1 is not by Rv These results were in the presence of the bovine a required for β-adrenergic receptor kinase 1 but similar results were also in the of (data not shown). These results that the interaction Rv and RK is essential for the Ca2+-dependent inhibition of rhodopsin phosphorylation. Our results for the that is a Ca2+-dependent interaction Rv and RK The Rv/RK interaction is specific Rv not interact with other members of the G-protein-coupled receptor kinase family such as β-adrenergic receptor kinase 1 a reconstituted rhodopsin phosphorylation system, we have that RK, purified using immobilized Rv as an affinity matrix, is and the incorporation of as many as several rhodopsin into the rhodopsin gain Rv inhibits rhodopsin phosphorylation in this reconstituted system, other soluble is required for the inhibitory effect of of the extent of RK binding to immobilized C14:0-Rv NA-Rv suggests that the myristoyl on Rv with RK A of this is in A for this is that may be RK and for binding on immobilized It also be that immobilized NA-Rv has a higher affinity for RK immobilized We found that Rv also binds to and Ca2+-dependent binding of to Rv N-terminal myristoylation of It has been reported that binds a of fatty B. J. Biol. Chem. 260: Scholar). This suggests that binds to the N-terminal myristoyl residue of Rv that when Rv binds Ca2+ (9Dizhoor A.M. Chen C.-K. Olshevskaya E. Sinelnikova V.V. Phillipov P. Hurley J.B. Science. 1993; 259: 829-832Google Scholar), as by the calcium-myristoyl protein switch However, it is that interacts with Rv within the in the interphotoreceptor A.H. J. Biol. Scholar), Rv primarily within photoreceptors. The binding of tubulin the binding of RK and in it be by high We have not to or not the interaction is A interaction Rv and RK was suggested by a report in Rv coeluted with a 67-kDa protein, RK, gel (12Gorodovikova E.N. Phillipov P. FEBS Lett. 1993; 335: 277-279Google Scholar). Our that Rv interacts with RK in a Ca2+-dependent manner. We also found that C14:0-Rv is a This suggests that the interaction of Rv with ROS membranes enhances the inhibitory effect. The observation that β-adrenergic receptor kinase not bind Rv, is not by Rv suggests that the Rv/RK interaction is necessary for the inhibitory effect of recoverin. It has been reported that S-modulin and Rv inhibit rhodopsin with = 100 nM free Ca2+ (9Dizhoor A.M. Chen C.-K. Olshevskaya E. Sinelnikova V.V. Phillipov P. Hurley J.B. Science. 1993; 259: 829-832Google Scholar). the Ca2+ dependence was reported to be significantly higher (24Klenchin V.A. Calvert P.D. Bownds M.D. Biophys. J. 1994; 66 (abstr.): A48Google Scholar). These used or to Ca2+. and not 1 μM of high affinity for Ca2+. We free Ca2+ with = μM for Scholar) in our The EC50 for the Rv effect on rhodopsin phosphorylation is 3 μM for both NA-Rv and the presence of a myristoyl residue not the but introduces an apparent A similar effect of myristoylation on the affinity of Rv for Ca2+ has recently been reported (1Ames J.B. Porumb T. Tanaka T. Ikura M. Stryer L. J. Biol. Chem. 1995; 270: 4526-4533Google Scholar). the Ca2+ affinity for C14:0-Rv and the Ca2+ dependence of the inhibitory effect of Rv Ca2+ concentrations significantly higher the bulk intracellular free Ca2+ by in vertebrate photoreceptors (11Gorczyca W.A. Gray-Keller M.P. Detwiler P.B. Palczewski K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4014-4018Google Scholar). for this apparent is that Rv functions in a to the plasma the free Ca2+ concentration may be from the bulk free Ca2+ may be that membranes the of Rv by binding the myristoyl to a concerted allosteric model that was to the calcium-myristoyl protein switch mechanism (8Dizhoor A.M. Ericsson L.H. Johnson R.S. Kumar S. Olshevskaya E. Zozulya S. Neubert T.A. Stryer L. Hurley J.B. Walsh K.A. J. Biol. Chem. 1992; 267: 16033-16036Google Scholar) to the in Ca2+ binding to NA-Rv and the presence of a membrane the affinity of C14:0-Rv for Ca2+ (1Ames J.B. Porumb T. Tanaka T. Ikura M. Stryer L. J. Biol. Chem. 1995; 270: 4526-4533Google Scholar). It be necessary to an in Ca2+ affinity for Rv in the presence of membranes RK to this Rv may other proteins from our reconstituted to in the of free Ca2+ It was reported that light of one rhodopsin stimulates the phosphorylation of of nonphotolyzed rhodopsins in electropermeabilized frog ROS B.M. Biernbaum M.S. Bownds M.D. J. Biol. Chem. 1990; 265: 15333-15340Google Scholar). This high gain phosphorylation was to the of ROS in the The kinase for high gain phosphorylation was not and it was suggested that protein kinase be for high gain phosphorylation J. Biol. Chem. 1993; 268: Scholar). Our that purified RK high gain phosphorylation when reconstituted with urea-stripped ROS membranes. The extent of high gain phosphorylation is to the of RK present The for the reported by 4Binder B.M. Biernbaum M.S. Bownds M.D. J. Biol. Chem. 1990; 265: 15333-15340Google Scholar may of RK when ROS are It has also been reported that RK a from the of rhodopsin when RK is by a of photolyzed rhodopsin (33Palczewski K. Buczylko J. Kaplan M.W. Polans A.S. Crabb J.W. J. Biol. Chem. 1991; 266: 12949-12955Google Scholar). The mechanism by RK is to high gain phosphorylation is not our reconstituted system, the extent of high gain phosphorylation is not at 1 μM RK the of photolyzed rhodopsin is only This a mechanism by photolyzed rhodopsin stimulates RK a affinity of RK for photolyzed rhodopsin. The of high gain phosphorylation using purified RK reported in this the for a study of the role of high gain phosphorylation in photoreceptor light adaptation. that the the extent of rhodopsin phosphorylation and the of are in to high gain phosphorylation the gain of Finally, the specific and Ca2+-dependent Rv/RK interaction may a model interaction that the members of the newly identified neuronal specific Ca2+-binding protein family by Rv and the G-protein-coupled receptor kinase family by We A. M. T. J. K. A. M. and for We S. Kumar for protein and acid A. for the mass of and for We also K. Palczewski for and RK S. for and for