The reaction catalyzed by all-trans-retinol dehydrogenase of rod outer segments completes the quenching of photoactivated rhodopsin and initiates the cycle of reactions leading to regeneration of visual pigment. The goal of this study was to determine the kinetic parameters of the dehydrogenase at physiological levels of bleaching, to investigate its specificity, and to determine its possible role in modulating phototransduction. Reduction of all-trans-retinal could be measured after bleaching < 0.15% rhodopsin. Kinetic parameters for the forward reaction determined with endogenous all-trans-retinal were Km = 1.1 microM; Vmax = 7 nmol/min/mg rhodopsin. The low enzymatic activity suggests that at high bleach rates, all-trans-retinal could accumulate, increasing the steady state level of bleaching intermediates or promoting formation of pseudophotoproducts. Active pseudophotoproducts, which stimulate Gt activation and opsin phosphorylation by rhodopsin kinase, are formed with opsin and all-trans-retinal as well as retinal analogues lacking the 13 methyl or the terminal two carbons of the polyene chain. Addition of all-trans-retinol, NADP, and [32P]ATP to rod outer segments increased rhodopsin phosphorylation. Kinetic parameters for the reverse reaction determined with exogenous all-trans-retinol were Km = 10 microM; Vmax = 11 nmol/min/mg rhodopsin. Our results support the hypothesis that all-trans-retinol dehydrogenase could influence the phototransduction cascade, including activities of Gt, rhodopsin kinase, and binding of arrestin, by impeding the recycling of rhodopsin at high bleach levels.

Abstract. Cancer-associated retinopathy (CAR), a paraneoplastic syndrome, is characterized by the degeneration of retinal photoreceptors under conditions where the tumor and its metastases have not invaded the eye. The retinopathy often is apparent before the diagnosis of cancer and may be associated with autoantibodies that react with specific sites in the retina. We have examined the sera from patients with CAR to further characterize the retinal antigen. Western blot analysis of human retinal proteins reveals a prominent band at 26 kD that is labeled by the CAR antisera. Antibodies to the 26-kD protein were affinity-purified from complex CAR antisera and used for EMimmunocytochemical localization of the protein to the nuclei, inner and outer segments of both rod and cone cells. Other antibodies obtained from the CAR sera did not label photoreceptors. Using the affinitypurified antibodies for detection, the 26-kD protein, designated p26, was purified to homogeneity from the outer segments of bovine rod photoreceptor cells by Phenyl-Sepharose and ion exchange chromatography. Partial amino acid sequence of p26 was determined by gas phase Edman degradation and revealed extensive homology with a cone-specific protein, visinin. Based upon structural relatedness, both the p26 rod protein and visinin are members of the calmodulin family and contain calcium binding domains of the E-F hand structure.variety of neurodegenerative diseases are known to be associated with different types of cancer, even though the tumor and its metastases have not invaded the nervous system (Brain and Norris, 1965;Brain and Wilkinson, 1965;

In rod and cone photoreceptor cells, activation of particulate guanylate cyclase (retGC1) is mediated by a Ca2+-binding protein termed GCAP1, that detects changes in [Ca2+]free. In this study, we show that N-acylated GCAP1 restored Ca2+ sensitivity of native and recombinant photoreceptor retGC1. ATP increased the affinity of retGC1 for GCAP1 and accelerated catalysis. Using peptides derived from the GCAP1 sequence, we found that at least three regions, encompassing the N-terminus, the EF-1 motif, and the EF-3 motif, were likely involved in the interaction with retGC1. Mutation of 2Gly to Ala (GCAP1-G2A), which abolished myristoylation and a 25 amino acid truncation at the N-terminus (delta25-GCAP1) reduced retGC1-stimulating activity dramatically, while deletion of 10 amino acids (delta10-GCAP1) reduced the specific activity by only approximately 60% and modified the Ca2+ sensitivity. At 10(-6) M [Ca2+]free, in conditions that inactivated native GCAP1, retGC1 showed significant activity in the presence of delta10-GCAP1. Native and all three mutant forms of GCAP1 had similar affinities for Ca2+ as demonstrated by gel filtration and the changes in tryptophan fluorescence. All mutants bound to ROS membranes in a Ca2+-independent manner, except delta25-GCAP1, which was mostly soluble. These findings suggest that the N-terminal region is important in tethering of GCAP1 to the ROS membranes.

Rhodopsin is constrained in an inactive conformation by interactions with 11-cis-retinal including formation of a protonated Schiff base with Lys 296. Upon photoisomerization, major structural rearrangements that involve protonation of the active site Glu 113 and cytoplasmic acidic residues, including Glu 134 , lead to the formation of the active form of the receptor, metarhodopsin II b, which decays to opsin. However, an activated receptor may be generated without illumination by addition of all-trans-retinal or its analogues to opsin, as measured in this study by the increased phosphorylation of opsin by rhodopsin kinase. The potency of stimulation depended on the chemical and isomeric nature of the analogues and the length of the polyene chain with all-trans-C17 aldehyde and all-trans-retinal being the most active and trans-C12 aldehyde being the least active. Certain cis-isomers, 11-cis-13-demethyl-retinal and 9-cis-C17 aldehyde, were also active. Most of the retinal analogues tested did not regenerate a spectrally identifiable pigment, and many were incapable of Schiff base formation (ketone, stable oximes, and Schiff basederivatives of retinal). Thus, receptor activation resulted from formation of non-covalent complexes with opsin. pH titrations suggested that an equilibrium exists between partially active (protonated) and inactive (deprotonated) forms of opsin. These findings are consistent with a model in which protonation of one or more cytoplasmic carboxyl groups of opsin is essential for activity. Upon addition of retinoids, the partially active conformation of opsin is converted to a more active intermediate similar to metarhodopsin II b. The model provides an understanding of the structural requirements for opsin activation and an interpretation of the observed activities of natural and experimental opsin mutants.Highly specific protein-protein recognition allows specific signal transduction pathways to be selected from an immense network of inter-and intracellular communications. Structural and chemical complementaries and hydrophobic and electrostatic properties of interacting domains provide precise docking of two or more proteins. Recognition domains may be permanently present in the interacting proteins, assembled because of posttranslational modifications, induced in one or both proteins by a ligand, or formed temporarily as a result of a photochemical reaction. An examination of the principles of proteinprotein recognition is pivotal for understanding the relationship between structure and function of proteins, their participation in physiologically relevant processes, and their regulation.Rhodopsin (Rho), 1 the transducing molecule of vision and a G protein-coupled receptor, undergoes conformational changes upon illumination that ultimately lead to interaction with and activation of the retinal specific G protein (G t ) (reviewed in Ref. 1). The transiently photoactivated Rho is subsequently phosphorylated by rhodopsin kinase (RK) and binds a regulatory protein, arrestin, before it decays to...

Arrcstin binds to phosphorylavzd rhodopsin in its light-activated form (melarhodopsin II), blocking thcrcby its interaction with the G-protein, mmsducin. In this study, WC show thal highly phosphorylatcd forms of inositol compete against the arrcslin-rhodopsin inlcraction. Competition curves and direct binding assays with free arrcslin consislcntly yield affinities in the micromolar range; for example, inositol 1.3.4.5-telrakisphospharc (InP,) and inosilol hcxakisphosphalc (InP, bind to arrcstin with dissociation constants of 12pM and 5 PM. respectively. Only a small control amount or inosilol phosphates is bound. when arrcstin interacts wilh phosphorylatcd rhodopsin.This argues for a release of bound inositol phosphates by imernclion with rhodopsin. Transducin, rhodopsin kinasc, or cyclic GMP phosphodicsterasc arc noi affected by inositol phosphates. These observations open a new way 10 purify arrcsiin and to inhibh its imcraction with rhodopsin. Their physiological significance dcscrvcs further inscsligalion.

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A BSTR ACTGuanylate cyclase-activating proteins (GCAP1 and GCAP2) are thought to mediate the intracellular stimulation of guanylate cyclase (GC) by Ca 2؉ , a key event in recovery of the dark state of rod photoreceptors after exposure to light. GCAP1 has been localized to rod and cone outer segments, the sites of phototransduction, and to photoreceptor synaptic terminals and some cone somata. We used in situ hybridization and immunocytochemistry to localize GCAP2 in human, monkey, and bovine retinas. In human and monkey retinas, the most intense immunolabeling with anti-GCAP2 antibodies was in the cone inner segments, somata, and synaptic terminals and, to a lesser degree, in rod inner segments and inner retinal neurons. In bovine retina, the most intense immunolabeling was in the rod inner segments, with weaker labeling of cone myoids, somata, and synapses. By using a GCAP2-specific antibody in enzymatic assays, we confirmed that GCAP1 but not GCAP2 is the major component that stimulates GC in bovine rod outer segment homogenates. These results suggest that although GCAP1 is involved in the Ca 2؉ -sensitive regulation of GC in rod and cone outer segments, GCAP2 may have non-phototransduction functions in photoreceptors and inner retinal neurons.In photoreceptor cells, photoactivation of rhodopsin or cone visual pigment results in a transient decrease in the concentrations of Ca 2ϩ and cGMP. These receptors and second messengers are linked through a cascade of specific activation͞ inactivation reactions in phototransduction (1, 2). The levels of Ca 2ϩ and cGMP are strictly controlled and interconnected. cGMP is a gating ligand of the plasma membrane cation channels that are permeable to Ca 2ϩ ions. After cGMP is hydrolyzed, the efflux of Ca 2ϩ exceeds the influx, resulting in decreased [Ca 2ϩ ] within the cell. The lowering of [Ca 2ϩ ] triggers production of cGMP through activation of a photoreceptor-specific particulate guanylate cyclase (GC) (3). The Ca 2ϩ sensitivity of GC (e.g., the higher activity at low levels of [Ca 2ϩ ]) is mediated by one or more Ca 2ϩ -binding proteins, termed guanylate cyclase-activating proteins (GCAPs) (4, 5).Two photoreceptor-specific GCs, GC1 and GC2, have been cloned (6-9). Although the localization of GC1 to rod and cone outer segments and synaptic terminals was established by both biochemical and immunocytochemical methods (5,(10)(11)(12)(13)(14), the localization of GC2 within photoreceptors is not known. Two GCAPs that stimulate GC1 and GC2 have also been cloned (15-17). There is abundant evidence that GCAP1 activates photoreceptor GC: (i) GCAP1 was isolated from rod outer segments (ROS) (4, 18); (ii) GCAP1 mRNA was found in the myoid region of rod and cone photoreceptor cells (15, 19); (iii) GCAP1 was localized to rod and cone outer segments and to some cone somata and synaptic terminals by immunocytochemistry (17, 18); (iv) cross-linking techniques revealed that GCAP1 was complexed with GC1 (20). In addition, the finding that GC1 is not expressed in the retina of the rd...

The inactivation of photolyzed rhodopsin requires phosphorylation of the receptor and binding of a 48-kDa regulatory protein, arrestin. By binding to phosphorylated photolyzed rhodopsin, arrestin inhibits G protein (G,) activation and blocks premature dephosphorylation, thereby preventing the reentry of photolyzed rhodopsin into the phototransduction pathway. In this study, we isolated a 44-kDa form of arrestin, called p4, from fresh bovine rod outer segments and characterized its structure and function. A partial primary structure of p44 was established by a combination of mass spectrometry and automated Edman degradation of proteolytic peptides. The amino acid sequence was found to be identical with arrestin, except that the C-terminal35 residues (positions 370-404) are replaced by a single alanine. p44 appeared to be generated by alternative mRNA splicing, because intron 15 interrupts within the nucleotide codon for 369Ser in the arrestin gene. Functionally, p44 binds avidly to photolyzed or phosphorylated and photolyzed rhodopsin. As a consequence of its relatively high affinity for bleached rhodopsin, pa blocks G, activation. The binding characteristics of p4 set it apart from tryptic forms of arrestin (truncated at the N-and C-termini), which require phosphorylation of rhodopsin for tight binding. We propose that p44 is a novel splice variant of arrestin that could be involved in the regulation of G, activation.