Table

Recoverin Family of Neural Calcium Binding Proteins

Protein Designation

Recoverin (S-modulin)

Rem-1

Visinin s26 VILIP NCS-1

Occurrence

Photoreceptor cells; mammalian bipolar cells Retinal cells; haematopoietic cells;

gut cells Photoreceptor cells Photoreceptor cells (frog retina) Inner retina

Photoreceptor inner segments; inner plexiform layer; ganglion cells

Ref.

Kawamura et al. (1996) De Raad et al. (1995) De Raad et al. (1995)

these receptors promote guanine nucleotide exchange of guanosine diphosphate (GDP) to GTP on the Ga. This in turn leads to the dissociation of GPy complex from Ga. These then regulate the activity of the target protein, leading eventually to the mobilisation of second messengers. A family of proteins called RGS (regulators of G-protein signalling) can preferentially bind to activated Ga. RGS proteins appear to function as guanosine triphosphatase (GTPase) activating proteins (GAP) (Berman et al. 1996; Watson et al. 1996; Hunt et al. 1996) and attenuate or block the signalling pathway (Popov et al. 1997; Tesmer et al. 1997; Hepler et al. 1997).

The G-protein-dependent and calcium-signalling pathways are often closely allied. For example, neurotransmitters inhibit calcium channel current, which seems to be regulated by the photoreceptor G-protein called transducin (Jeong et al. 1999; also, see below). Similarly, dopamine-coupled receptors inhibit voltage-activated calcium channels (Wolfe and Morris, 1999). Dolphin et al. (1999) have identified the calcium channel-protein domains that might be involved in the modulation of calcium channel currents by G-proteins. In the signalling events associated with the acrosome reaction in spermatozoa, G-proteins require calcium together with PLA2 to induce acrosomal reaction (Dominguez et al. 1999).

Recoverin and Its Function

The transduction of the extracellular sensory signal involves the interaction of activated rhodopsin with the photoreceptor G-protein, transducin. The rhodopsin intermediate meta II activates transducin by catalysing the exchange of GDP to GTP. The RGS protein down-regulates this signalling pathway by promoting Ga GTPase activity and additionally by down-regulating cGMP phosphodiesterase, which effectively enhances GTPase activity. RCN (a homologue of this from the frog is known as S-modulin) is a 23-kDa protein that plays a specific role of phototransduction in mammalian retinal photoreceptors. The transduction of visual signals requires rhodopsin to be activated by phosphorylation. Upon illumination rhodopsin is phos-phorylated by rhodopsin kinase at several serine and threonine residues. The activated rhodopsin is dephosphorylated by phosphatases to return it to the basal state.

RCN is involved in the process of light and dark adaptation by rod cells by regulating rhodopsin phosphorylation, thereby controlling photoreceptor light sensitivity, which is Ca2+ dependent. Several mutant forms of RCN, with mutations in EF-hands 2 and 3, and others with mutation of EF-hand 4, have been isolated recently. Of these, EF-hand 4 RCN mutants were found to be able to inhibit rhodopsin kinase more effectively than could wild-type RCN (Alekseev et al. 1998). Apparently, a decrease in cytoplasmic calcium levels is necessary for light adaptation (Figure 11). Besides RCN, other calcium-binding proteins are involved in this process. Among them are the photoreceptor-specific GCAP and calmodulin, together with their targets, namely rhodopsin kinase, guanylate cyclase, cGMP-gated channel, and nitric oxide synthase (Koch, 1995; Gorczyca et al. 1995).

FIGURE 11 The pathway of sensory signal transduction involving recoverin and RGS (regulator of G-protein signalling) protein.

The RCN gene has been mapped to human chromosome 17p13.1 (J.F. McGinnis et al. 1995). Interestingly, the autosomal-dominant progressive cone dystrophy (CORD5) gene maps to chromosome 17p12-p13. The genes, coding for retinal guanylyl cyclase and pigment epithelium-derived factor, and the retinitis pigmentosa (RP) genes also occur in the RCN region (Balciuniene et al. 1995).

Recoverin occurs predominantly in mammalian photoreceptor cells. It may be found in other retinal cell types and may therefore subserve other functions besides phototransduction (McGinnis et al. 1997). RCN appears to be a highly conserved protein, as suggested by its occurrence in the photoreceptor cells of the lamprey (Dalil-Thiney et al. 1998). Its expression may be developmentally regulated (Yan and Wiechmann, 1997). It is found to be transiently expressed in developing folli-cular and parafollicular pinealocytes in the developing chick embryo (Bastianelli and Pochet, 1994). Four isoforms of RCN occur and each of these shows N-terminal myristoylation. RCN not only participates in phototransduction, a role suggested by its localisation in photoreceptor cells, but it is also associated with the pathogenesis of the autoimmune state of cancer-associated retinopathy and uveoretinitis. However, it is uncertain whether RCN is involved with autosomal recessive RP.

Mode of Action of Recoverin

Recoverin regulates photoreceptor response by inhibiting rhodopsin phosphorylation. Rhodopsin kinase is active in the absence of RCN (Gorodovikova et al. 1994a). Rhodopsin phosphorylation and consequent cGMP hydrolysis are Ca2+- and ATP-dependent processes. When the free Ca2+ level is raised, phosphorylation of rhodop-sin is reduced and there is an increase in the lifetime of phosphodiesterase. This Ca2+ effect is negated by anti-RCN antibodies, which has been interpreted as suggesting that the calcium effects observed are a result of the inhibition of rhodopsin kinase (Gorodovikova et al. 1994b). Upon addition of RCN, rhodopsin kinase becomes sensitive to free Ca2+. Calcium-dependent interaction between RCN and rhodopsin kinase is indeed necessary for the inhibition of rhodopsin phosphorylation by RCN (C.K. Chen et al. 1995). All four isoforms of RCN inhibit rhodopsin phosphorylation in the same free calcium range (0.3 to 0.8 ^M), but they differ with respect to the magnitude of inhibition achieved, which appears to be related to their hydrophobicity (Sanada et al. 1995).

Post-Translational Modification of Recoverin

Four isoforms of RCN have been identified; all appear to be posttranslationally modified at the N-terminal glycine residue with myristic acid or related lipids. Myristoylation is believed to be essential for the function of RCN. Senin et al. (1995) compared the inhibitory effect on rhodopsin phosphorylation of myristoylated and nonmyristoylated forms of recombinant RCN. They found that both forms of RCN inhibit rhodopsin kinase in the presence of Ca2+, but myristoylated RCN was more efficient in inhibiting the kinase. However, others believe that myristoylation is not necessary for the kinase inhibitory effect of RCN and that it only induces a cooperative Ca2+ dependence of the process (Kawamura et al. 1994; Calvert et al. 1995). Ames et al. (1994) have suggested, on the basis of the flexibility of the N-terminal helix in myristoylated calcium-free and nonmyristoylated calcium-bound form, that calcium binding to the EF-hand 3 domain induces EF-hand 2 to adopt a conformation that promotes calcium binding to RCN.

Covalent attachment of a myristoyl or related N-acyl group to the N-terminal glycine appears to promote the binding of RCN to the optic disc membrane when free Ca2+ is raised. The so-called calcium-myristoylation switch is believed to play an essential role in the targeting of RCN to the cell membrane. The addition of the myristoyl group has been found to reduce the calcium affinity of RCN and induce cooperative calcium binding. Two conformational states have been recognised viz. the T and R states. In the T state the myristoyl group is sequestered inside the protein, whereas in the R state it is exposed, and furthermore, calcium binds to the R state several thousand-fold more strongly than to the T state (Ames et al. 1995). Calcium binding to the myristoylated RCN induces its translocation to the membrane. The nonmyristoylated RCN is not translocated in this way (Tanaka et al. 1995). It would appear, therefore, that posttranslational modification of RCN is required for targeting to and interaction of RCN with the membrane. The RCN family protein VILIP also shows calcium-dependent targeting to the cell membrane. It has been found to interact with the actin component of the cytoskeleton in its recruitment to the cell membrane (Lenz et al. 1996). Braunewell et al. (1997) have confirmed that myris-toylated VILIP can be shown to be associated with membranes, whereas nonmyris-toylated VILIP is not. Membrane association may stabilise the RCN-rhodopsin kinase complex, as suggested by experimental demonstration of a preferential association of RCN with cell membrane with concomitant increase in rhodopsin kinase inhibition (Sanada et al. 1996). However, Johnson et al. (1997) found that conformational changes and distribution of RCN in the cell were not influenced greatly by calcium concentration and suggest, therefore, that the calcium-myristoylation switch may not be the only mechanism involved in the targeting of RCN to the cell membrane.

Recoverin and Cancer-Associated Retinopathy

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