Ligand Channeling within a G-protein-coupled Receptor

Abstract

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...

A Naturally Occurring Mutation of the Opsin Gene (T4R) in Dogs Affects Glycosylation and Stability of the G Protein-coupled Receptor

“…The sample was bleached for 15 s using a Fiber-Lite covered with a longpass wavelength filter (Ͼ490 nm), followed by a 10-min incubation (if not indicated otherwise) with continuous low speed stirring. The intrinsic fluorescence increase from G␣ t was measured with the PerkinElmer Life Sciences LS 50B luminescence spectrophotometer employing excitation and emission wavelengths at 300 and 345 nm, respectively, as described (32)(33)(34)(35)(36). No signals from Rho in the absence of G t were detected in the control experiment.…”

“…A PerkinElmer Life Sciences LS 50B luminescence spectrophotometer was used to measure the increase in intrinsic Trp fluorescence due to hydrolysis of the protonated Schiff base and release of all-trans-retinal from Rho (32)(33)(34). Immunoaffinity-purified Rho was bleached with a Fiber-Lite illuminator for 15 s from a distance of 15 cm, immediately followed by fluorescence measurements.…”

Chromophore Channeling in the G-Protein Coupled Receptor Rhodopsin

“…Furthermore, if retinal is released by this way, it can be enzymatically converted to all-trans retinol in situ, as suggested by Schadel and co-workers. 4 However, this may not be the preferred drug entrance route into GPCR proteins because it requires the passage of the membrane and long distance channeling to the putative binding site near the extracellular side. As our observations from the simulations indicate, this route is notably less preferred than egress pathways A-E from the extracellular side.…”

“…This question is of great interest because understanding ligand entrance routes may help to improve drug efficacy since many of the drugs bind to the same pocket. Although the uptake and release of retinal can be detected by monitoring the intrinsic protein fluorescence, 4 direct evidence of the channeling routes is unavailable. The recent crystal structures showed no obvious channel through which the ligands can pass into the binding sites.…”

Deactivation and Proton Transfer in Light-induced Metarhodopsin II/Metarhodopsin III Conversion

“…The end product of the new light-induced pathway, Meta III, can form up to 80%, depending on the conditions, and has remarkable properties. Because of its long lifetime (up to hours (34)), it excludes the chromophore very efficiently from the regeneration pathway, which has led to the concept of a retinal storage form (34,37,38). Remarkably, the lifetime of Meta III depends on the presence of G-protein (34) and arrestin (39,40), which was interpreted as an "inverse catalysis" of receptor conversion by G-protein.…”