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Recoverin is a neuronal calcium sensor protein that controls the activity of rhodopsin kinase in a Ca(2+)-dependent manner. Mutations in the EF-hand Ca2+ binding sites are valuable tools for investigating the functional properties of recoverin. In the recoverin mutant E121Q (Rec E121Q ) the high-affinity Ca2+ binding site is disabled. The non-myristoylated form of Rec E121Q binds one Ca2+ via its second Ca(2+)-binding site (EF-hand 2), whereas the myristoylated variant does not bind Ca2+ at all. Binding of Ca2+ to non-myristoylated Rec E121Q apparently triggers exposure of apolar side chains, allowing for association with hydrophobic matrices. Likewise, an interaction surface for the recoverin target rhodopsin kinase is constituted upon Ca2+ binding to the non-acylated mutant. Structural changes resulting from Ca(2+)-occupation of EF-hand 2 in myristoylated and non-myristoylated recoverin variants are discussed in terms of critical conditions required for biological activity.

Cholesterol-rich membranes or detergent-resistant membranes (DRMs) have recently been isolated from bovine rod outer segments and were shown to contain several signaling proteins such as, for example, transducin and its effector, cGMP-phosphodiesterase PDE6. Here we report the presence of rhodopsin kinase and recoverin in DRMs that were isolated in either light or dark conditions at high and low Ca2+ concentrations. Inhibition of rhodopsin kinase activity by recoverin was more effective in DRMs than in the initial rod outer segment membranes. Furthermore, the Ca2+ sensitivity of rhodopsin kinase inhibition in DRMs was shifted to lower free Ca2+ concentration in comparison with the initial rod outer segment membranes (IC50=0.76 microm in DRMs and 1.91 microm in rod outer segments). We relate this effect to the high cholesterol content of DRMs because manipulating the cholesterol content of rod outer segment membranes by methyl-beta-cyclodextrin yielded a similar shift of the Ca2+-dependent dose-response curve of rhodopsin kinase inhibition. Furthermore, a high cholesterol content in the membranes also increased the ratio of the membrane-bound form of recoverin to its cytoplasmic free form. These data suggest that the Ca2+-dependent feedback loop that involves recoverin is spatially heterogeneous in the rod cell.

Recoverin, a retina-specific Ca2+-binding protein, is one of the paraneoplastic antigens (PNAs) which are normally present in neurons, but can also be aberrantly expressed in malignant tumors localized outside the nervous system. In this study, we have analyzed 16 small cell lung carcinoma (SCLC) and 12 non-small cell lung carcinoma cell lines and found that none of them is capable of expressing recoverin in vitro. However, two small cell lung carcinoma lines, NCI-H69 and NCI-H82, became recoverin-positive after cultivation in the presence of butyrate. Recoverin expression in the butyrate-treated cells has been detected by immunoblotting with polyclonal (monospecific) antibodies against recoverin and confirmed by the analysis of recoverin mRNA expression. To our knowledge, this work is the first to demonstrate stimulation of the aberrant expression of recoverin in cancer cell lines in vitro. This result opens the way to investigation of the mechanisms underlying the aberrant expression of recoverin, as well as other paraneoplastic antigens, in tumor cells.

Using immunoblotting with recombinant recoverin as an antigen, we have examined 279 serum samples from individuals with small cell lung carcinoma (SCLC, 99 patients), non-small cell lung carcinoma (NSCLC, 44 patients), and non-malignant pulmonary disorders (86 patients) as well as sera from 50 healthy donors. Autoantibodies against recoverin (anti-Rc) were detected in sera from 15 patients with SCLC (15% of cases) and from 9 patients with NSCLC (about 20% of cases). Only two anti-Rc positive cases were detected in patients with non-malignant pulmonary disorders, while no such cases were found in healthy individuals. Immunohistochemical investigation of paraffin sections of 44 SCLC and 40 NSCLC tumors revealed recoverin-positive reaction in 30 SCLC (68%) and 34 NSCLC (85%) sections. Despite the high specificity (98%), the low sensitivity (less than 20%) does not allow serum anti-Rc to be considered as a valuable marker of lung cancer. However, taking into account the high occurrence of aberrant expression of recoverin in lung tumors, this PNA could be considered as a potential target for immunotherapy of lung cancer.

The goal of the present study was to analyze serum and tumor tissue of a patient with non-small cell lung cancer (NSCLC) for the presence of autoantibodies against recoverin (anti-Rc) and recoverin expression, correspondingly. Using immunoblotting with recombinant recoverin as an antigen, we have detected anti-Rc in serum of the patient. At the same time, the patient did not manifest any signs of cancer-associated retinopathy (CAR). Polyclonal (monospecific) antibodies against recoverin used for immunohistochemical analysis of the patient's tumor revealed recoverin expression in the tumor sections. To our knowledge, this is the first case of the presence of serum anti-Rc in NSCLC patients in the absence of paraneoplastic retina degeneration.

Recoverin is a neuronal calcium sensor protein that plays a crucial role in vertebrate phototransduction. It undergoes a Ca(2+)-myristoyl switch when Ca(2+) binds to its two functional EF-hand motifs (EF-hands 2 and 3), each present in one of recoverin's two domains. Impairment of Ca(2+)-binding in recoverin leads to a disturbance of the Ca(2+)-myristoyl switch and loss of its regulatory properties, i.e. inhibiton of rhodopsin kinase. We have engineered recoverin mutants with either of the two functional EF-hands disabled, but with a functional Ca(2+)-binding site in EF-hand 4. While a defect in EF-hand 2 could not be rescued by the additional EF-hand 4, the impairment of EF-hand 3 was powerfully compensated by Ca(2+)-binding to EF-hand 4. For example, the myristoylated form of the latter mutant bound to membranes in a Ca(2+)-dependent way and was able to inhibit rhodopsin kinase in a way similar to that of the wild-type protein. Thus, for recoverin to undergo a Ca(2+)-myristoyl switch, it is necessary and sufficient to have either of the two EF-hands in the second domain in a functional state. On the basis of these results and inspection of published three-dimensional structures of recoverin, we propose a model highlighting the mutual interdependence of sterical configurations in EF-hands 3 and 4 of recoverin.

Recoverin is a Ca2+-regulated signal transduction modulator found in vertebrate retina that has been shown to undergo dramatic conformational changes upon Ca2+ binding to its two functional EF-hand motifs. To elucidate the differential impact of the N-terminal myristoylation as well as occupation of the two Ca2+ binding sites on recoverin structure and function, we have investigated a non-myristoylated E85Q mutant exhibiting virtually no Ca2+ binding to EF-2. Crystal structures of the mutant protein as well as the non-myristoylated wild-type have been determined. Although the non-myristoylated E85Q mutant does not display any functional activity, its three-dimensional structure in the presence of Ca2+ resembles the myristoylated wild-type with two Ca2+ but is quite dissimilar from the myristoylated E85Q mutant. We conclude that the N-terminal myristoyl modification significantly stabilizes the conformation of the Ca2+-free protein (i.e. the T conformation) during the stepwise transition toward the fully Ca2+-occupied state. On the basis of these observations, a refined model for the role of the myristoyl group as an intrinsic allosteric modulator is proposed.

Recoverin is an N-myristoylated 23 kDa calcium-binding protein from retina, which modulates the Ca2+-sensitive deactivation of rhodopsin via Ca2+-dependent inhibition of rhodopsin kinase. It was shown by intrinsic and bis-ANS probe fluorescence, circular dichroism, and differential scanning calorimetry that myristoylated recombinant recoverin interacts specifically with zinc ions. Similar to the calcium binding, the binding of zinc to Ca2+-loaded recoverin additionally increases its alpha-helical content, hydrophobic surface area, and environmental mobility/polarity of its tryptophan residues. In contrast to the calcium binding, the binding of zinc decreases thermal stability of the Ca2+-loaded protein. Zn2+-titration of recoverin, traced by bis-ANS fluorescence, reveals binding of a single Zn2+ ion per protein molecule. It was shown that the double-mutant E85Q/E121Q with inactivated Ca2+-binding EF-hands 2 and 3 (Alekseev, A. M.; Shulga-Morskoy, S. V.; Zinchenko, D. V.; Shulga-Morskaya, S. A.; Suchkov, D. V.; Vaganova, S. A.; Senin, I. I.; Zargarov, A. A.; Lipkin, V. M.; Akhtar, M.; Philippov, P. P. FEBS Lett. 1998, 440, 116-118), which can be considered as an analogue of the apo-protein, binds Zn2+ ion as well. Apparent zinc equilibrium binding constants evaluated from spectrofluorimetric Zn2+-titrations of the protein are 1.4 x 10(5) M(-1) (dissociation constant 7.1 microM) for Ca2+-loaded wild-type recoverin and 3.3 x 10(4) M(-1) (dissociation constant 30 microM) for the E85Q/E121Q mutant (analogue of apo-recoverin). Study of the binding of wild-type recoverin to ROS membranes showed a zinc-dependent increase of its affinity for the membranes, without regard to calcium content, suggesting further solvation of a protein myristoyl group upon Zn2+ binding. Possible implications of these findings to the functioning of recoverin are discussed.

Recoverin is an EF-hand Ca(2+)-binding protein that is suggested to control the activity of the G-protein-coupled receptor kinase GRK-1 or rhodopsin kinase in a Ca(2+)-dependent manner. It undergoes a Ca(2+)-myristoyl switch when Ca(2+) binds to EF-hand 2 and 3. We investigated the mechanism of this switch by the use of point mutations in EF-hand 2 (E85Q) and 3 (E121Q) that impair their Ca(2+) binding. EF-hand 2 and 3 display different properties and serve different functions. Binding of Ca(2+) to recoverin is a sequential process, wherein EF-hand 3 is occupied first followed by the filling of EF-hand 2. After EF-hand 3 bound Ca(2+), the subsequent filling of EF-hand 2 triggers the exposition of the myristoyl group and in turn binding of recoverin to membranes. In addition, EF-hand 2 controls the mean residence time of recoverin at membranes by decreasing the dissociation rate of recoverin from membranes by 10-fold. We discuss this mechanism as one critical step for inhibition of rhodopsin kinase by recoverin.

Over many years until the middle of the 1980s, the main problem in vision research had been the mechanism of transducing the visual signal from photobleached rhodopsin to the cationic channels in the plasma membrane of a photoreceptor to trigger the electrophysiological response of the cell. After cGMP was proven to be the secondary messenger, the main intriguing question has become the mechanisms of negative feedback in photoreceptors to modulate their response to varying conditions of illumination. Although the mechanisms of light-adaptation are not completely understood, it is obvious that Ca2+ plays a crucial role in these mechanisms and that the effects of Ca2+ can be mediated by several Ca2+-binding proteins. One of them is recoverin. The leading candidate for the role of an intracellular target for recoverin is believed to be rhodopsin kinase, a member of a family of G-protein-coupled receptor kinases. The present review considers recoverin, rhodopsin kinase and their interrelationships in the in vitro as well as in vivo contexts.

To date, many authors have described the presence of autoantibodies against various neuronal proteins, paraneoplastic antigens (PNA), in a serum of patients with different kinds of malignant tumors located outside the nervous system. These autoantibodies may cross-react with the corresponding PNA or their epitopes present in neurons and thus initiate the development of a variety of neurological disorders, paraneoplastic syndromes (PNS), even though the primary tumor and its metastases have not invaded the nervous system. Cancer-associated retinopathy (CAR) is a rare ocular PNS induced by autoantibodies against several retinal antigens, one of which is a photoreceptor calcium-binding protein, recoverin. Only several CAR patients with a few kinds of cancer (endothelial carcinoma, breast cancer, epithelial ovarian carcinoma) have so far been found to contain autoantibodies against recoverin in their sera. As for lung cancer, the majority of CAR cases mediated by anti-recoverin autoantibodies have been revealed in patients with the most malignant lung cancer, small cell lung carcinoma (SCLC), and only one similar case has been described for a patient with non-small lung carcinoma. The common feature of all these anti-recoverin-positive patients, irrespective of the type of cancer, is the presence of both the CAR syndrome and high titres (as a rule, more than 1:1000) of the underlying autoantibodies in their serum. In this study, we have used recombinant myristoylated recoverin to screen serum samples of 50 patients with SCLC by Western blot and revealed 5 individuals with low titres of anti-recoverin antibodies, who have no manifestation of a loss of vision. To our knowledge, this is the first report on the presence of low titre autoantibodies against recoverin in a serum of patients with cancer, but without visual dysfunction.

A molecule of the photoreceptor Ca(2+)-binding protein recoverin contains four potential EF-hand Ca(2+)-binding sites, of which only two, the second and the third, are capable of binding calcium ions. We have studied the effects of substitutions in the second, third and fourth EF-hand sites of recoverin on its Ca(2+)-binding properties and some other characteristics, using intrinsic fluorescence, circular dichroism spectroscopy and differential scanning microcalorimetry. The interaction of the two operating binding sites of wild-type recoverin with calcium increases the protein's thermal stability, but makes the environment around the tryptophan residues more flexible. The amino acid substitution in the EF-hand 3 (E121Q) totally abolishes the high calcium affinity of recoverin, while the mutation in the EF-hand 2 (E85Q) causes only a moderate decrease in calcium binding. Based on this evidence, we suggest that the binding of calcium ions to recoverin is a sequential process with the EF-hand 3 being filled first. Estimation of Ca(2+)-binding constants according to the sequential binding scheme gave the values 3.7 x 10(6) and 3.1 x 10(5) M(-1) for third and second EF-hands, respectively. The substitutions in the EF-hand 2 or 3 (or in both the sites simultaneously) do not disturb significantly either tertiary or secondary structure of the apo-protein. Amino acid substitutions, which have been designed to restore the calcium affinity of the EF-hand 4 (G160D, K161E, K162N, D165G and K166Q), increase the calcium capacity and affinity of recoverin but also perturb the protein structure and decrease the thermostability of its apo-form.

In the early 1970s it became clear that an unknown intracellular messenger participated in signal transduction in retinal rod cells. Ca2+ and cGMP had been considered as candidates for such a messenger until 1985 when the cGMP-gated conductance of the plasma membrane was discovered. However, for the last several years, Ca2+ has taken revenge as a signal molecule which contributed to the processes underlying the photoreceptor cell recovery. This review describes the overall picture of phototransduction molecular mechanisms with special emphasis on participation of Ca2+ in visual cell signalling.

Several EF-hand recoverin mutants were obtained and their abilities to bind to photoreceptor membranes and to inhibit rhodopsin kinase were determined. The mutants with the 'spoiled' 2nd, 3rd or (2nd+3rd) EF-hand structures did not act upon the kinase activity in the microM range of Ca2+ concentrations. Mutations of the 4th EF hand, which 'repaired' its Ca2+-binding activity, resulted in recoverin with three 'working' Ca2+-binding sites. The latter mutant inhibited rhodopsin kinase even more effectively than the wild-type recoverin, containing two working Ca2+-binding structures.

Recoverin, a calcium-binding protein, is supposed to have rhodopsin kinase as a target in the retinal rod cell. In the present work, we show that efficiency of recoverin as an inhibitor of rhodopsin phosphorylation in bovine rod outer segments is inversely proportional to the level of rhodopsin bleaching. These results, together with the data obtained previously in a reconstituted system (Senin et al. (1997) Biochem. J. 321, 551-555), allow us to hypothesize that recoverin might be responsible for a Ca2(+)-dependent regulation of the kinase in vivo, preventing it from participating in the phosphorylation of unbleached rhodopsin.

In its resting state rhodopsin kinase is present in an inactive from and is activated after interaction with light-activated rhodopsin (Rho*). The activated rhodopsin kinase then phosphorylates Rho* but is also able to catalyse the phosphorylation of dark-adapted rhodopsin. A consequence of the latter behaviour of the activated kinase is that at low levels of bleach a large number of phosphoryl groups are incorporated per mol of Rho*. Recoverin- and Ca2+-dependent inhibition of rhodopsin kinase was found to be inversely related to the extent of bleaching; the lower the fraction of rhodopsin bleached, the greater the inhibition. The IC50 of recoverin is approx. 1 microM at a 0.2% level of bleach and about 5 microM in a fully bleached sample. The inhibitory effect of recoverin was studied separately on the phosphorylation of rhodopsin and Rho*. The formation of phosphorylated rhodopsin was inhibited 4.5-fold more strongly than that of phosphorylated Rho*. These results are interpreted to suggest that one of the roles of the recoverin-dependent regulation of the activity of rhodopsin kinase is to prevent the enzyme from participating in the unwanted phosphorylation of dark-adapted rhodopsin, directing it to fulfil its 'correct' function of quenching the transduction activity of Rho*.

Recoverin, a recently identified member of the EF-hand superfamily of Ca(2+)-binding proteins, is capable to inhibit rhodopsin phosphorylation by rhodopsin kinase at high but not at low free [Ca2+]. The N-terminal glycine residue of retinal recoverin is heterogeneously acylated with myristoyl or related N-acyl group. To clarify the role of the N-terminal acylation of recoverin in its inhibitory action upon rhodopsin phosphorylation, we compared the efficiency of myristoylated and non-myristoylated forms of recombinant recoverin as inhibitors of rhodopsin kinase activity. We have found that rhodopsin phosphorylation by purified rhodopsin kinase, which does not depend on free [Ca2+] in the absence of recoverin, is regulated by Ca2+ in the presence of both forms of the recombinant protein. EC50 values for Ca2+ are the same (2 microM) for the myristoylated and non-myristoylated forms; the Hill coefficients of 1.7 and 0.9, respectively, indicate that the effect is cooperative with respect to Ca2+ only for myristoylated recoverin. In the presence of Ca2+, both forms of recoverin taken at saturated concentrations cause an almost equal inhibition of rhodopsin phosphorylation. However, the inhibitory action of the myristoylated form occurs at much lower its concentrations than that of the non-myristoylated form (EC50 are 0.9 and 6.5 microM, respectively).

Rhodopsin phosphorylation in the reconstituted system consisting of urea-washed photoreceptor membranes, rhodopsin kinase and recoverin is regulated by Ca2+: the process takes place at low [Ca2+] but is suppressed at high [Ca2+]. In the absence of recoverin, rhodopsin kinase is active irrespective of the cation concentration used. Hence, recoverin is an inhibitor (at high [Ca2+]) but not an activator of rhodopsin kinase. Based jointly on these data obtained on the reconstituted system and on our preceding experiments on rod outer segments suspension, one may conclude that (i) the function of recoverin in retina rod cells is the Ca(2+)-sensitive control of rhodopsin phosphorylation and (ii) the presence of recoverin is essential and sufficient to provide rhodopsin kinase with the Ca2+ sensitivity.

Rhodopsin phosphorylation and in consequence cGMP hydrolysis in bovine rod outer segments are Ca2+ dependent in the presence of ATP. The level of rhodopsin phosphorylation decreases and the lifetime of active phosphodiesterase increases when the free [Ca2+] is raised from < 1 nM to about 1 microM; in both cases the half-maximal effect was observed at 140-170 nM of free Ca2+. Antibodies to recoverin reverse both effects at high [Ca2+] but have no influence at low [Ca2+]. We conclude that the Ca2+ effects observed are mediated by recoverin which inhibits rhodopsin kinase at a high Ca2+ level.