Hydrophobic amino acids at the cytoplasmic ends of helices 3 and 6 of rhodopsin conjointly modulate transducin activation |
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Authors: | Bosch-Presegué Laia Iarriccio Laura Aguilà Mònica Toledo Darwin Ramon Eva Cordomí Arnau Garriga Pere |
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Institution: | aCentre de Biotecnologia Molecular, Departament d’Enginyeria Química, Universitat Politècnica de Catalunya, 08222 Terrassa, Spain;bLaboratori de Medicina Computacional, Unitat de Bioestadística, Facultat de Medicina, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain |
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Abstract: | Rhodopsin is the visual photoreceptor responsible for dim light vision. This receptor is located in the rod cell of the retina and is a prototypical member of the G-protein-coupled receptor superfamily. The structural details underlying the molecular recognition event in transducin activation by photoactivated rhodopsin are of key interest to unravel the molecular mechanism of signal transduction in the retina. We constructed and expressed rhodopsin mutants in the second and third cytoplasmic domains of rhodopsin – where the natural amino acids were substituted by the human M3 acetylcholine muscarinic receptor homologous residues – in order to determine their potential involvement in G-protein recognition. These mutants showed normal chromophore formation and a similar photobleaching behavior than WT rhodopsin, but decreased thermal stability in the dark state. The single mutant V1383.53 and the multiple mutant containing V2275.62 and a combination of mutations at the cytoplasmic end of transmembrane helix 6 caused a reduction in transducin activation upon rhodopsin photoactivation. Furthermore, combination of mutants at the second and third cytoplasmic domains revealed a cooperative role, and partially restored transducin activation. The results indicate that hydrophobic interactions by V1383.53, V2275.62, V2506.33, V2546.37 and I2556.38 are critical for receptor activation and/or efficient rhodopsin–transducin interaction. |
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Keywords: | G-protein-coupled receptor Rhodopsin Muscarinic receptor G-protein activation Visual phototransduction |
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