Ligand channeling within a G-protein-coupled receptor. The entry and exit of retinals in native opsin.
Deactivation of light-activated rhodopsin (metarhodopsin II) involves, after rhodopsin kinase and arrestin interactions, the hydrolysis of the covalent bond of all-trans-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.
Schädel
Sandra A
SA
Institut für Medizinische Physik und Biophysik, Universitätsklinikum Charité, Humboldt Universität zu Berlin, Schumannstrasse 20-21, 10098 Berlin, Germany.
Heck
Martin
M
Institut für Medizinische Physik und Biophysik, Universitätsklinikum Charité, Humboldt Universität zu Berlin, Schumannstrasse 20-21, 10098 Berlin, Germany.
Maretzki
Dieter
D
Institut für Medizinische Physik und Biophysik, Universitätsklinikum Charité, Humboldt Universität zu Berlin, Schumannstrasse 20-21, 10098 Berlin, Germany.
Filipek
Slawomir
S
International Institute of Molecular and Cell Biology and the Department of Chemistry, University of Warsaw, 1 Pasteur St, PL-02109 Warsaw, Poland.
Teller
David C
DC
Departments of Biological Structure, Biochemistry, and Biomolecular Structure Center, University of Washington, Seattle, Washington 98195.
Palczewski
Krzysztof
K
Departments of Ophthalmology, Pharmacology, and Chemistry, University of Washington, Seattle, Washington 98195.
Hofmann
Klaus Peter
KP
Institut für Medizinische Physik und Biophysik, Universitätsklinikum Charité, Humboldt Universität zu Berlin, Schumannstrasse 20-21, 10098 Berlin, Germany.
D000818:Animals; D002417:Cattle; D018360:Crystallography, X-Ray; D008024:Ligands; D011487:Protein Conformation; D012172:Retinaldehyde / Q000737:chemistry / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000378:metabolism*; D017299:Rod Opsins / Q000737:chemistry / Q000378:metabolism*
D008024:Ligands; D017299:Rod Opsins; D012172:Retinaldehyde