Mechanisms of Opsin Activation

Abstract

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...

Signaling States of Rhodopsin

“…There is general agreement that this conversion is irreversible (39,40). In apparent contradiction to this notion, recombination of purified opsin with exogenous all-trans-retinal leads to substantial activity toward the G-protein (16 -20), rhodopsin kinase (11,41), and arrestin (11). However, the activity toward G t was also seen with a permethylated active site (16), and regeneration with 11-cis-retinal was not inhibited in the presence of all-trans-retinal added in excess (20).…”

Light-Dependent Translocation of Arrestin in the Absence of Rhodopsin Phosphorylation and Transducin Signaling

“…Rhodopsin is primarily absent in RPE65 Ϫ/Ϫ mice because of a defect in the visual cycle to regenerate 11-cis-retinal, but the presence of the opsin apoprotein preserves rod outer segment structure (Redmond et al, 1998). Opsin in RPE65 Ϫ/Ϫ mice is constitutively phosphorylated (Ablonczy et al, 2002;Van Hooser et al, 2002), probably as a consequence of the weak constitutive activity of opsin described in vitro (Robinson et al, 1992;Surya et al, 1995;Buczylko et al, 1996). In RPE65 Ϫ/Ϫ mice, opsin appeared to signal constitutively for arrestin translocation to rod outer segments, regardless of the lighting conditions.…”

“…This result indicates that opsin alone, without the retinal chromophore, has some signaling capacity to trigger arrestin translocation. Opsin has been reported to have a small but measurable catalytic activity in vitro (Robinson et al, 1992;Surya et al, 1995;Buczylko et al, 1996). This weak constitutive activity, however, is apparently not sufficient to trigger Tr ␣ translocation to proximal compartments in RPE65 Ϫ/Ϫ mice, because Tr ␣ immunoreactivity is restricted to the outer segments in both darkand light-adapted retinas (Fig.…”

Crystal structure of the ligand-free G-protein-coupled receptor opsin

“…However, the TM6 movement is not enough for keeping the active conformation because the protonation of Glu-134 appears to be a prerequisite for coupling to the G protein [53,76,86,94,131,135,171,172,173,174]. …”