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KMID : 0613820070170060783
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Journal of Life Science
2007 Volume.17 No. 6 p.783 ~ p.790
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Chromophore formation and phosphorylation analysis of constitutively active rhodopsin mutants
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Kim Jong-Myung
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Abstract
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G protein coupled receptors (GPCRs) transmit various extracellular signals into the cells. Upon binding of the ligands, conformational changes in the extracellular and/or transmembrane (TM) domains of GPCRs were propagated into the cytoplasmic (CP) domain of the molecule leading to the activation of their cognate heterotrimeric G proteins and kinases. Constitutively active GPCR mutants causing the activation of G Protein signaling even in the absence of ligand binding are of interest for the study of activation mechanism of GPCRs. Two classes of constitutively active mutations, categorized by their effects on the salt bridge between E113 and K296, were found in the TM domain of rhodopsin. Opsin mutants containing combinations of the mutations were constructed to study the conformational changes required for the activation of rhodopsin. Rhodopsin chromophore regenerated with 11-cis-retinal showed a thermal stability inversely correlated with its constitutive activity. In contrast, rhodopsin mutants exhibited a binding affinity to an agonist, all-trans-retinal, in a constitutive activity-dependent manner. In order to test whether the conformational changes responsible for the activation of transducin (Gt) are the same as the conformation required for the recognition of rhodopsin kinase, analysis of the mutants were carried out with phosphorylation by rhodopsin kinase. Rhodopsin mutants containing combinations of different classes of the mutations showed a strong synergistic effect on the phosphorylation of the mutants in the dark as similar to that of Gt activation. The results suggest that at least two or three kinds of segmental and independent conformational changes are required for the activation of rhodopsin and the conformational changes responsible for activating rhodopsin kinase and Gt are similar to each other.
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KEYWORD
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G protein-coupled receptor, signal transduction, constitutive activation, rhodopsin kinase
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