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B. Nickle, J.A. Cowing, D.M. Hunt, S. Wilkie, P. Robinson; Changes in Constitutive Activation Among Vertebrate Opsins . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1695.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Vertebrate opsins are classified into one of five classes based on amino acid similarity and spectral tuning properties. These classes are short wavelength sensitive 1 and 2 (SWS1, SWS2), medium/long wavelength sensitive (M/LWS), and rhodopsin like 1 and 2 (RH1, RH2). Of these classes, the RH1 class has been studied most intensively. This is largely due to the ease and yield of bovine rhodopsin isolation. Recently, a large number of other vertebrate opsins have begun to be studied, many of which use bovine rhodopsin as a model for their results. In rhodopsin, two critical amino acids are Lys–296 and Glu–113. Lys–296 acts as the chromophore binding site, while Glu–113 acts as the counterion to the protonated Schiff’s base. In addition, these two sites form a salt–bridge in the apoprotein which maintains the opsin in an inactive state. Corresponding residues in each of the other vertebrate opsin classes play similar roles. Previous reports have demonstrated that mutations in these critical residues result in constitutive transducin activation for RH1 class opsins in the absence of chromophore. Here we ask, do other classes of vertebrate opsins maintain similar activation characteristics as bovine rhodopsin. We approach this question by making Glu–113–Gln and Lys–296–Ala mutations in opsins belonging to the SWS1, SWS2, M/LWS, and RH2 classes. The mutant opsins are tested for their ability to constitutively activate bovine transducin. Methods: Site directed mutagenesis was performed using QuickchangeTM. Mutant opsins are expressed in COS–1 cells, followed by isolation of the COS–1 membranes. GTPγS binding assays are carried out to assess activation of bovine transducin. Results: In this study we demonstrate that mutations in site 113 and 296, produce constitutive activation in all classes. However, the mutant opsins differ in their ability to be quenched in the dark state by the addition of chromophore as well as in their degree of constitutive activation. Conclusions: The differences in constitutive activation profiles suggest a structural difference exists among the different opsin classes that may translate into a difference in activation properties.
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