Arrestin quenches signal transduction in rod photoreceptors by blocking the catalytic activity of photoactivated phosphorylated rhodopsin toward the G protein, transducin (Gt). Rod cells also express a splice variant of arrestin, termed p44, in which the last 35 amino acids are replaced by a single Ala. In contrast to arrestin, this protein has been reported to bind to both the phosphorylated and nonphosphorylated forms of the activated receptor. In this study, we analyzed formation of the rhodopsin-p44 complex in vitro. Like arrestin, p44 stabilized the meta II (MII) photoproduct relative to forms MI and MIII and did not interact measurably with the apoprotein opsin. However, several differences between p44 and its parent protein were found: (i) p44 binds to nonphosphorylated MII with a much lower affinity (KD = 0.24 microM) than to phosphorylated MII (P-MII) (KD = 12 nM); arrestin binds only to P-MII (KD = 20 nM); (ii) p44 interacted also with truncated MII (329G-Rho MII), which lacked the sites of phosphorylation; (iii) with both MII and P-MII, the activation energy of complex formation with p44 was lower than that found for arrestin (70 kJ/mol instead of 140 kJ/mol); and (iv) InsP6 inhibited poorly the interaction between p44 and P-MII, but it strongly inhibited the interaction between arrestin and P-MII. Extrapolation of the measured on-rates to physiological conditions yielded reaction times for the binding of p44 to activated rhodopsin. The data suggest that the splice variant, p44, and its parent protein, arrestin, play different roles in phototransduction. The physiological significance of these differences remains to be determined.

Vertebrate rhodopsin consists of the apoprotein opsin and the chromophore 11-cis-retinal covalently linked via a protonated Schiff base. Upon photoisomerization of the chromophore to all-trans-retinal, the retinylidene linkage hydrolyzes, and all-trans-retinal dissociates from opsin. The pigment is eventually restored by recombining with enzymatically produced 11-cis-retinal. All-trans-retinal release occurs in parallel with decay of the active form, metarhodopsin (Meta) II, in which the original Schiff base is intact but deprotonated. The intermediates formed during Meta II decay include Meta III, with the original Schiff base reprotonated, and Meta III-like pseudo-photoproducts. Using an intrinsic fluorescence assay, Fourier transform infrared spectroscopy, and UV-visible spectroscopy, we investigated Meta II decay in native rod disk membranes. Up to 40% of Meta III is formed without changes in the intrinsic Trp fluorescence and thus without all-trans-retinal release. NADPH, a cofactor for the reduction of all-trans-retinal to all-trans-retinol, does not accelerate Meta II decay nor does it change the amount of Meta III formed. However, Meta III can be photoconverted back to the Meta II signaling state. The data are described by two quasiirreversible pathways, leading in parallel into Meta III or into release of all-trans-retinal. Therefore, Meta III could be a form of rhodopsin that is storaged away, thus regulating photoreceptor regeneration.Phototransduction in vertebrate rods starts with the isomerization of the 11-cis-retinal bound to opsin and the formation of the active photoproduct, metarhodopsin (Meta) 1 II. In Meta II, the Schiff base linkage between the all-trans-retinal and Lys 296 is still intact but deprotonated. Catalytic activation of the Gprotein, G t or transducin, leads to a biochemical cascade of reactions, termed phototransduction. These reactions culminate in the hyperpolarization of the photoreceptor cells and ultimately in changes in the rate of neurotransmitter release at the synaptic terminus. The signaling state of Meta II is quenched rapidly by the action of rhodopsin kinase and arrestin. Equally important for vision is the metabolic cycle, which enables the visual system to take away the photolyzed chromophore, all-trans-retinal, and replace it with 11-cis-retinal, thus regenerating the pigment. The decay of Meta II thus provides an interlink among transduction, the quenching by phosphorylation and capping with arrestin, and regeneration (reviewed in Ref. 1).During the decay of Meta II, the Schiff base linkage between the all-trans-retinal and the opsin apoprotein (Lys 296 ) is hydrolyzed. A side product is the bright orange ( max ϳ 470 nm) Meta III, which slowly replaces the pale yellow color of the Meta II product ( max ϭ 380 nm). Although it is not clear whether Meta III represents one homogeneous species, one may define it as the late product in which the chromophore is still bound to its original binding site. In the isolated retina and in intact rod outer segment preparations...

In rhodopsin's function as a photoreceptor, 11-cis-retinal is covalently bound to Lys 296 via a protonated Schiff base. 11-cis/all-trans photoisomerization and relaxation through intermediates lead to the metarhodopsin II photoproduct, which couples to transducin (G t ). Here we have analyzed a different signaling state that arises from noncovalent binding of all-trans-retinal (atr) to the aporeceptor opsin and enhances the very low opsin activity by several orders of magnitude. Like with metarhodopsin II, coupling of G t to opsin-atr is sensitive to competition by synthetic peptides from the COOH termini of both G t ␣ and G t ␥. However, atr does not compete with 11-cis-retinal incorporation into the Lys 296 binding site and formation of the light-sensitive pigment. Blue light illumination fails to photorevert opsin-atr to the ground state. Thus noncovalently bound atr has no access to the light-dependent binding site and reaction pathway. Moreover, in contrast to light-dependent signaling, removal of the palmitoyl anchors at Cys 322 and Cys 323 in the rhodopsin COOH terminus impairs the atrstimulated activity. Repalmitoylation by autoacylation with palmitoyl-coenzyme A restores most of the original activity. We hypothesize that the palmitoyl moieties are part of a second binding pocket for the chromophore, mediating hydrophobic interactions that can activate a large part of the catalytic receptor/G-protein interface.The retinal photoreceptor rhodopsin absorbs photons via a chromophoric ligand, 11-cis-retinal, to trigger the amplifying cascade of vision. The light-sensitive ground state conformation, with a max ϭ 500 nm, is stabilized by a salt bridge between the protonated Schiff base bond of the 11-cis-retinal to Lys 296 and its counterion at Glu 113 (1). Light-induced 11-cis/ all-trans isomerization of the retinal and subsequent relaxation processes result in protonation-dependent conformations of the receptor identified as "metarhodopsin" photointermediates (for details, see Refs. 2 and 3). Metarhodopsin II (Meta II, 1 max ϭ 380 nm) is distinguished by its deprotonated Schiff base linkage and broken Lys 296 /Glu 113 salt bridge. The signaling state for G t arises from Meta II through proton uptake from solution (4), with the conserved Glu 134 as the likely proton acceptor. The apparent pK for this protonation is shifted to pH values higher than the intrinsic pK of a Glu residue, reflected in pH/rate profiles for activation of both G t (1) and rhodopsin kinase ("forced protonation" (5)). Protonated Meta II catalyzes nucleotide exchange in G t at a high rate (for review, see Refs. 2, 6, and 7). Each catalytic coupling of G t requires interaction via the COOH termini of both the ␣ and ␥ subunits (8).This study deals with a fundamentally different class of signaling states. They are generated without light, by mere addition of the diffusible agonist, all-trans-retinal (atr), to the empty aporeceptor, opsin. Free opsin arises from the spontaneous decay of the Meta intermediates by hydrolysis of the deprotonated ...

Deactivation of light-activated rhodopsin (metarhodopsin II) involves, after rhodopsin kinase and arrestin interactions, the hydrolysis of the covalent bond of alltrans-retinal to the apoprotein. Although the long-lived storage form metarhodopsin III is transiently formed, all-trans-retinal is eventually released from the active site. Here we address the question of whether the release results in a retinal that is freely diffusible in the lipid phase of the photoreceptor membrane. The release reaction is accompanied by an increase in intrinsic protein fluorescence (release signal), which arises from the relief of the fluorescence quenching imposed by the retinal in the active site. An analogous fluorescence decrease (uptake signal) was evoked by exogenous retinoids when they non-covalently bound to native opsin membranes. Uptake of 11-cis-retinal was faster than formation of the retinylidene linkage to the apoprotein. Endogenous all-trans-retinal released from the active site during metarhodopsin II decay did not generate the uptake signal. The data show that in addition to the retinylidene pocket (site I) there are two other retinoidbinding sites within opsin. Site II involved in the uptake signal is an entrance site, while the exit site (site III) is occupied when retinal remains bound after its release from site I. Support for a retinal channeling mechanism comes from the rhodopsin crystal structure, which unveiled two putative hydrophobic binding sites. This mechanism enables a unidirectional process for the release of photoisomerized chromophore and the uptake of newly synthesized 11-cis-retinal for the regeneration of rhodopsin.During its function as a photoreceptor, the visual pigment rhodopsin undergoes changes through a cycle of uptake of 11-cis-retinal and release of photoisomerized chromophore, alltrans-retinal (reviewed in Ref. 1). In rhodopsin, 11-cis-retinal is bound to the opsin apoprotein by a Schiff base linkage to Lys 296 . In the dark ground state, the rhodopsin conformation with a max of 500 nm is stabilized by a salt bridge between the protonated Schiff base and the Glu 113 counterion. The activated state metarhodopsin II (Meta II, max ϭ 380 nm) arises from light-induced 11-cis/all-trans-retinal isomerization and conformational changes that break the salt bridge but retain the all-trans-retinylidene linkage in the original binding site (reviewed in Refs. 2-5). Thereby, photoisomerization is coupled to the protein conformational change that leads to a G-proteincoupled signal cascade, eventually setting off neuronal signaling (1).Initial deactivation of Meta II begins with the interaction of active rhodopsin with its receptor kinase, phosphorylation of the receptor, and a tight binding of arrestin to the still activated phosphorylated form of the receptor (6, 7). Full deactivation occurs when rhodopsin is regenerated. This requires the hydrolysis of the all-trans-retinylidene linkage and release of all-trans-retinal from the active site (1). Critical steps include the nucleophilic attack of wa...