Disease relevance of RCV1

• Myristoylated recombinant recoverin formed in this way in E. coli is like retinal recoverin in exhibiting a large calcium-induced shift in its tryptophan fluorescence emission spectrum [1].
• The recoverin coding sequence was inserted into a pET-11a expression vector under control of the T7 phage promoter [1].
• A second expression system, in which the coding sequence was placed under control of the lambda phage PR promoter, gave 10-fold higher yields (10 mg of purified recoverin per liter of Escherichia coli culture) [1].
• Staining proteins with Ponceau S did not interfere with either the radioiodination or trypsin digestion, as indicated by essentially identical peptide patterns being obtained for the internal protein p26 from equine infectious anemia virus, regardless of whether the digests were prepared from polyacrylamide gel slices or nitrocellulose sections [2].
• Bovine recoverin regulates rhodopsin phosphorylation and controls photoreceptor light sensitivity in a Ca(2+)-dependent manner [3].

High impact information on RCV1

• Recoverin, a recently discovered member of the EF hand superfamily, serves as a calcium sensor in vision [4].
• Ca2+ binding to myristoylated, but not unmyristoylated, recoverin induces its translocation to bilayer membranes, indicating that the myristoyl group is essential to the read-out of calcium signals (calcium-myristoyl switch) [5].
• On the basis of these studies, Ca2+ is suggested to play a central role in photorecovery and light adaptation, not only by regulating guanylate cyclase, possibly through recoverin, but also by modulating the cGMP-gated channel through calmodulin interaction with the 240K protein [6].
• Recoverin, a calcium ion (Ca2+)-binding protein of vertebrate photoreceptors, binds to photoreceptor membranes when the Ca2+ concentration is greater than 1 micromolar [7].
• These results suggest that the hydrophobic NH2-terminus is constrained in Ca(2+)-free recoverin and liberated by Ca2+ binding [7].

Biological context of RCV1

• 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 [8].
• The N-terminal myristoyl residue of Rv enhances the inhibitory effect of Rv and introduces cooperativity to the Ca(2+)-dependent inhibition of rhodopsin phosphorylation [9].
• 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 [10].
• 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 [10].
• Heterogeneous acylation was detected at the NH2 terminus of bovine retinal recoverin [11].

Anatomical context of RCV1

• 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 [8].
• To investigate the function of Rv in photoreceptors, we identified proteins that bind immobilized Rv in a Ca(2+)-dependent manner [9].
• The protein is present in both rod and cone photoreceptors and was named recoverin because it promotes recovery of the dark state [12].
• Recoverin and rhodopsin kinase activity in detergent-resistant membrane rafts from rod outer segments [13].
• The pores formed through the plasma membranes of rod cells permit the diffusion of small molecules <2 kDa but prevent the loss of proteins, including recoverin (25 kDa) [14].

Associations of RCV1 with chemical compounds

• RK, purified using immobilized Rv as an affinity matrix, catalyzed the light-dependent and Ca(2+)-independent incorporation of phosphates into rhodopsin when reconstituted with urea-stripped rod outer segment membranes [9].
• Rhodopsin kinase (RK), interphotoreceptor retinoid-binding protein, and tubulin interact with Rv in the presence of Ca2+ [9].
• Recoverin: a calcium sensitive activator of retinal rod guanylate cyclase [12].
• The NH2-terminal glycine of each retinal recoverin molecule is linked to one of four different types of acyl groups [11].
• Furthermore, a high cholesterol content in the membranes also increased the ratio of the membrane-bound form of recoverin to its cytoplasmic free form [13].

Physical interactions of RCV1

• 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 [8].
• Recoverin in every fraction bound Ca2+ as assessed by fluorescence spectroscopy and inhibited the light-dependent rhodopsin phosphorylation in the same range of free Ca2+ concentration (0.3-0.8 microM) [15].

Enzymatic interactions of RCV1

• Finally, we observe that a kinase mutant lacking the N-terminal recoverin binding site is unable to phosphorylate light-activated rhodopsin [16].

Regulatory relationships of RCV1

• Recoverin is a calcium sensor that regulates rhodopsin phosphorylation in a calcium-dependent manner [17].
• The available data suggest that the functions of recoverin myristoylation in the living rod are to induce a sharp Ca2+ dependence of rhodopsin kinase inhibition and to bring this dependence into the rod's physiological Ca2+ concentration range [18].
• The finding that retinal recoverin is myristoylated at its amino terminus led us to coexpress the recombinant protein and N-myristoyltransferase (EC 2.3.1.97) [1].

Other interactions of RCV1

• The importance of the Rv/RK interaction was further characterized [9].
• GCAP-2 differs from recoverin in that the calcium ion binds to EF-4 in addition to EF-2 and EF-3 [19].
• The results suggest that GCIP is a Ca2+-binding protein of the GCAP/recoverin subfamily [20].

Analytical, diagnostic and therapeutic context of RCV1

• Cloning, expression, and crystallization of recoverin, a calcium sensor in vision [1].
• 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 [21].
• How can Ca2+ selectively activate recoverin in the presence of Mg2+? Surface plasmon resonance and FT-IR spectroscopic studies [22].
• Purification of rhodopsin kinase by recoverin affinity chromatography [23].
• To evaluate this, exogenous bovine recoverin and two other homologous Ca(2+)-binding proteins from chicken and Gecko retina were dialyzed into functionally intact Gecko rods using whole-cell recording [24].

References