RECOVERIN AND CA2+ IN VERTEBRATE PHOTOTRANSDUCTION
James B. Hurley
Recoverin, visinin and S-modulin were first identified as retina-specific Ca2+-binding proteins. They are realtively abundant in photoreceptor cells, but their functions there remain controversial. Several highly conserved recoverin homologues have been identified in retina and other tissues suggesting that this family of proteins is important for cellular function. This paper reviews recent studies of recoverin and related proteins with an emphasis on their possible role in photoreceptors.
During the excitation phase of the vertebrate rod photoresponse light stimulates an enzymatic cascade that culminates in the hydrolysis of cyclic GMP (cGMP) (Stryer, 1991; Lagnado and Baylor, 1992). Photoconversion of rhodopsin to metarhodopsin II within the rod disk membranes stimulates GTP-binding to the a subunit of the heterotrimeric G-protein, transducin. Light activated transducin a (Ta-GTP) dissociates from the Tbg complex, then stimulates cGMP phosphodiesterase (PDE) activity by relieving inhibition imposed on the PDE catalytic subunits by a small inhibitory subunit, PDEg. The mechanism of PDE activation appears to involve formation of a complex between Ta-GTP and PDEg. The ensuing hydrolysis of intracellular cGMP reduces cGMP gated cation channel activity in the rod outer segment plasma membrane.
Several processes contribute to the recovery phase of the photoresponse. First, photolyzed rhodopsin is phosphorylated by rhodopsin kinase. This reduces the ability of the photolyzed rhodopsin to stimulate transducin. It also stimulates binding of arrestin to the photolyzed rhodopsin. Arrestin binding further quenches the ability of the photolyzed rhodopsin to activate transducin. Activated transducin a subunits (Ta) already formed by photolyzed rhodopsin deactivate when their bound GTP is hydrolyzed to GDP. Ta-GDP then reassociates with Tbg and releases the PDEg subunit which re-inhibits PDE activity.
Light-stimulated hydrolysis of cGMP within rod photoreceptors reduces the activity of cGMP-gated cation channels in the photoreceptor plasma membrane. Since these channels are the major route by which Ca2+ enters the photoreceptor, the intracellular concentration of Ca2+ falls. Lowered Ca2+ levels stimulate a photoreceptor guanylyl cyclase to resynthesize cGMP (Koch and Stryer, 1988). In addition, lowered Ca2+ speeds the rate of deactivation of cGMP PDE (Kawamura and Murakami, 1991). Recently, it has also been reported that Ca2+ may reduce the affinity of the cGMP-gated plasma membrane cation channel for cGMP (Hsu and Molday, 1993). Each of these effects of Ca2+ may help promote recovery of the photoreceptor following photoexcitation.
The mechanisms by which Ca2+ regulates photoreceptor guanylyl cyclase and cGMP PDE are not completely understood. However, a 23 kDa Ca2+-binding protein specifically found in photoreceptors has been implicated in both of these regulatory processes (Kawamura and Murakami, 1991; Dizhoor et al., 1991; Lambrecht and Koch, 1991). Initial reports suggested that one form of this protein, recoverin, isolated from bovine retinas, is a regulator of photoreceptor guanylyl cyclase (Dizhoor et al., 1991; Lambrecht and Koch, 1991). However, it now appears that those initial reports were incorrect (Hurley et al., 1993). Other observations have upheld the view that this protein imparts Ca2+-sensitivity to the cGMP PDE (Kawamura and Murakami, 1991; Kawamura, 1993). Here I describe some characteristics of this protein and its homologs. The protein has been given several different names by the laboratories that discovered it. Isolates from chicken were referred to as visinin, isolates from bovine retinas were referred to either as recoverin or p26 and isolates from frog retinas were referred to as S-modulin.
Members of the recoverin family: Visinin: Visinin was identified as a 24 kDa cone protein from chicken retinas (Yamagata et al., 1990). Its cDNA was isolated and used to produce visinin-specific antibodies. These antibodies were used to localize visinin to chicken cone photoreceptors. The nucleotide sequence of visinin cDNA was found to encode a protein with three potential Ca2+-binding sites known as "EF-hands". Ca2+-binding to visinin was confirmed using visinin expressed in E. coli. Although the function of visinin has not been specifically investigated, it has been reported that visinin dialyzed into rod photoreceptors affects photoresponse lifetime in the same way as recoverin (Gray-Keller et al., 1993).
Recoverin: Recoverin was originally identified as a protein with an apparent molecular weight of 26 kDa, that bound to a column made from detergent solubilized rhodopsin immobilized onto concanavalin A-Sepharose (Dizhoor et al., 1991). Recoverin immunoreactivity has been detected in rod and cone photoreceptors and in a class of bipolar cells in the retina (Milam et al., 1992). The amino acid sequence of recoverin was determined by Edman degradation of proteolytic fragments (Dizhoor et al., 1991). The sequence included Ca2+-binding sites and strong homology with visinin. A cDNA encoding recoverin was isolated and it has been used to express recoverin in E. coli (Ray et al., 1992). Both recoverin isolated from bovine retinas and recoverin expressed in E. coli bind Ca2+. Recently, the crystal structure of recoverin was determined by X-ray analysis. Recoverin is a compact molecule with a concave hydrophobic cleft between two Ca2+-binding domains (Flaherty et al., 1993).
Initial reports suggested that recoverin is a Ca2+-sensitive stimulator of photoreceptor guanylyl cyclase. However, more recent findings have not confirmed this conclusion (Hurley et al., 1993). The relationship between recoverin and photoreceptor guanylyl cyclase will be described in a later section of this paper.
S-modulin: S-modulin was identified as a protein that binds in a Ca2+-depedent manner to frog photoreceptor membranes (Kawamura and Murakami, 1991). This property was used to purify S-modulin from frog photoreceptors. The activity of cGMP PDE in frog photoreceptors is sensitive to Ca2+. Kawamura and Murakami found that a factor responsible for this Ca2+-sensitivity eluted from truncated photoreceptors when the free Ca2+ concentration was lowered to 20 nM (Kawamura and Murakami, 1991). Reconstituting truncated photoreceptors or photoreceptor homogenates with purified S-modulin restored Ca2+-sensitivity to the PDE.
Recent reports (Kawamura, 1993; Kawamura et al., 1993) have clarified the effect of S-modulin and recoverin on photoreceptor cGMP PDE. An efficient method for purifying S-modulin/recoverin allowed reconstitution experiments using concentrations of S-modulin/recoverin that were significantly higher than were used in earlier experiments. The results demonstrated clearly that S-modulin/recoverin prolongs the lifetime of activated PDE following a light flash. The up to four fold effect occurred at free Ca2+ concentrations in the submicromolar range. Further analyses revealed that Ca2+ and S-modulin/recoverin partially suppressed light-stimulated phosphorylation of rhodopsin.
More recently, it has been reported that recoverin forms a Ca2+-dependent complex with rhodopsin kinase (Chen and Hurley, 1994; Subbaraya et al., 1994). There have been no reports of recoverin interactions with other soluble photoreceptor-specific proteins such as phosphodiesterase subunits. Although the detection of a recoverin/rhodopsin kinase complex suggests that recoverin may regulate rhodopsin kinase directly, these findings do not rule out other interactions of recoverin, perhaps with membranes or membrane proteins, that might also occur and influence rhodopsin phosphoryaltion.
p26 (CAR-antigen): p26 was identified in screens for proteins that reacted with antisera from human patients with retinas degenerating from cancer associated retinopathy (CAR) (Polans et al., 1991; Thirkill et al., 1992). In retinas of CAR patients, photoreceptors are degraded through an autoimmune reaction presumably stimulated by a tumor in a non-retinal tissue. Sera from some CAR patients showed immunoreactivity with a retina specific protein of apparent size 26 kDa. The protein was purified and a partial amino acid sequence was determined. The sequence included three potential Ca2+-binding sites and it was found to be homologous to chicken visinin and identical to bovine recoverin. The mechanisms by which some tumors induce production of antibodies that recognize recoverin have not yet been seriously addressed. However, the existence of homologs of recoverin in other tissues raises the possibility that proteins expressed in some tumors could induce the production of antibodies that cross-react with retinal recoverin.
Recoverin homologs in other tissues: Recoverin immunoreactivity has been detected in both rods and cones (Dizhoor et al., 1991; Milam et al., 1992). However, immunological studies using a variety of antibodies raised against recoverin and against homologs from different animal species have not yet shown whether or not rods and cones express the same or different forms of recoverin (Polans et al., 1993).
A variety of recoverin homologs including neurocalcin, hippocalcin and Villip from non-retinal tissues have also been identified. Neurocalcin has been detected in bovine brain (Okazaki et al., 1992) and in retinal amacrine cells and ganglion cells (Nakano et al., 1992). Vilip was detected in chicken brain and retina (Lenz et al., 1992) and hippocalcin was detected in rat hippocampus (Kobayashi et al., 1992). Functional studies for these recoverin homologs have not been reported.
Post-translational modifications of recoverin. Recoverin expressed in E. coli was found to have properties that are quite different from the properties of recoverin purified from bovine retinas (Ray et al., 1992). For example, the fluorescence emission spectrum of bovine retinal recoverin red shifts when the concentration of Ca2+ is raised from 70 nM to 10 mM while the emission of recombinant recoverin undergoes only a minor change in intensity. Furthermore, the mobilities of retinal and recombinant recoverins are quite different in native gel electrophoresis and in isoelectric focussing (Lambrecht and Koch, 1992; Ray et al., 1992).
To investigate the cause of these differences, Dizhoor et al. (Dizhoor et al., 1992) used electro-spray mass spectrometry to examine recoverin purified from bovine retinas for post-translational modifications. The mass of retinal recoverin was found to be 207.7 atomic mass units larger than the mass of recombinant recoverin. This difference is consistent with the amino-terminal methionine of recoverin being replaced by a short chain fatty acyl residue such as a myristoyl group. This finding is consistent with the consensus sequence for N-terminal myristoylation present at the recoverin amino-terminus. Closer examination of the recoverin amino-terminus by mass spectrometric analyses of proteolytic fragments revealed four different types of amino-termini. The N-terminal glycine of recoverin was found linked to either 12:0; 14:2 cis,cis D5D8; 14: 1cis D5 or 14:0 fatty acyl residues. The identities of the fatty acyl residues and the positions and configurations of the double bonds were confirmed by a variety of methods including gas phase chromatography, ozonolysis and tandem mass spectrometry. Similar heterogeneity was also detected at the amino-terminus of another photoreceptor protein, the transducin a subunit (Neubert et al., 1992). The functional significance of photoreceptor protein heterogeneity is unknown but it is currently being investigated.
The functional significance of the fatty acyl residue on the recoverin amino-terminus was investigated by comparing the biochemical properties of myristoyated and non-acylated recoverin expressed in E. coli (Ray et al., 1992). Myristoylated recoverin was produced in E. coli by coexpressing recoverin with yeast N-myristoyl transferase and myristic acid. In addition to differences in fluoresence emission and electrophoretic mobility, non-acylated recoverin has a significantly higher affinity for Ca2+ than acylated recoverin. Affinities of acylated and non-acylated recoverins for membranes were also investigated. Kawamura and Murakami (Kawamura and Murakami, 1991) had shown previously that S-modulin, a recoverin homologue from frog photoreceptors, binds to photoreceptor membranes only in the presence of >1mM Ca2+. Ca2+ also promotes binding of recoverin to ROS membranes (Dizhoor et al., 1993; Zozulya and Stryer, 1992). Myristoylated recombinant recoverin was found to bind to ROS membranes more efficiently than non-acylated recombinant recoverin. These findings suggested that the fatty acid on the recoverin amino-terminus may serve as a Ca2+-dependent membrane anchor. To test this model, recoverin was prepared with a [3H] myristic acid residue at its amino-terminus. The labelled recoverin, either in its Ca2+-liganded form or its Ca2+-free form, was then exposed to trypsin under non-denaturing conditions. The labelled amino-terminus of Ca2+-free recoverin was not cleaved by trypsin. The labelled amino-terminus of Ca2+-recoverin was rapidly cleaved suggesting that Ca2+ binding to recoverin induces a conformational change that exposes the amino-terminus. Recoverin lacking a tetrapeptide from its amino-terminus failed to bind to ROS membranes. These findings suggest that Ca2+ binding frees the hydrophobic acylated amino-fatty acid residue to make it accessible for membrane interactions. The site on the membranes with which recoverin interacts has not been identified.
Recoverin and guanylyl cyclase. Photoreceptor guanylyl cyclase in rod outer segment homogenates is active only at concentrations of free Ca2+ less than approximately ~200 mM. Koch and Stryer (Koch and Stryer, 1988) found that a soluble factor required for the Ca2+-sensitivity of guanylyl cyclase could be eluted from rod outer segment membranes. When recoverin was first isolated and characterized (Dizhoor et al., 1991) it was apparent that it had properties suggesting that it might regulate guanylyl cyclase. Preparations of purified recoverin were assayed and were found to stimulate guanylyl cyclase in a Ca2+-sensitive manner. These results together with findings from another laboratory (Lambrecht and Koch, 1991) led to the conclusion that recoverin was the soluble factor that imparted Ca2+-sensitivity to the photoreceptor guanylyl cyclase. However, the following recent findings suggest that recoverin is not the soluble factor that stimulated guanylyl cyclase in the original recoverin preparations (Hurley et al., 1993). i. Reconstitution experiments using purified recombinant recoverin showed that neither myristoylated nor non-myristoylated recombinant recoverin stimulate photoreceptor guanylyl cyclase. ii. Highly purified recoverin isolated from bovine retinas by several fractionation methods does not stimulate photoreceptor guanylyl cyclase. iii. Recoverin dissociates from photoreceptor membranes under conditions that stimulate membrane associated guanylyl cyclase activity. The Ca2+-dependent membrane-binding properties of recoverin appear to be inconsisent with a role in guanylyl cyclase regulation. iv. Recoverin separates from other fractions containing guanylyl cyclase stimulatory activity during several fractionation procedures. The most highly purified fractions of guanylyl cyclase stimulatory activity contain nearly undetectable amounts of recoverin.
Recent observations from other another laboratory also suggest that recoverin does not stimulate photoreceptor guanylyl cyclase activity. Gray-Keller et al. (Gray-Keller et al., 1993) have shown that purified recoverin dialyzed into rod cells slows recovery. These findings are inconsistent with stimulation of photoreceptor guanylyl cyclase by recoverin.
Summary: Recoverin is a 23 kDa Ca2+-binding protein specifically found in vertebrate photoreceptor cells. The role of recoverin in phototransduction may be to increase photosensitivy by prolonging the lifetime of photolyzed rhodopsin in darkness when intracellular Ca2+ concentrations are high. The Ca2+-bound form of recoverin appears to inhibit deactivation of photolyzed rhodopsin by phosphorylation. Following illumination, lowered intracellular Ca2+ levels may promote recovery by allowing more rapid phosphorylation and inactivation of rhodopsin. Recoverin homologs in other tissues may impart Ca2+-sensitivity to other signalling mechanisms in a similar way.
I wish to thank all of the members of my laboratory for many stimulating discussions. I wish to particularly thank Alexander M. Dizhoor and Jason Chen who are primarily responsible for many of the findings from my laboratory discussed in this review. Their work was supported by grant RO1 EYO6641 from the National Eye Institute.
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