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I.V. Peshenko, E.V. Olshevskaya, A.M. Dizhoor; Interactions of Recombinant retGC1 with Ca2+-bound and Ca2+-free GCAPs and the Effect of the R838S Mutation . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1526.
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Purpose: GCAP-1 and -2 activate photoreceptor guanylyl cyclase, retGC-1, in a Ca2+-sensitive manner. Previous studies by other groups demonstrated that several different substitutions of R838 in retGC-1 associated with human cone-rod dystrophy increase retGC-1 activity at higher Ca2+ concentrations, presumably by decreasing affinity of the bound GCAP-1 for Ca2+ [1-3]. The purpose of this study was to further elucidate possible mechanisms that result in abnormal activity of the mutant retGC-1. Methods: Wild type RetGC-1 and its R838S mutant were produced in transfected HEK 293-F cells and reconsituted with myristoylated GCAP-1 and GCAP-2 or their constitutively active Ca2+-insensitive mutants at different free Ca2+ concentrations ([Ca]f). Results: In a good agreement with the results reported in [2,3] the mutation, R838S, decreases the maximal level of retGC-1 activation by GCAP-1 at low [Ca]f and increases its activity at higher [Ca]f. However, we have also found strong indications that fully Ca2+-loaded form of GCAP-1 itself acts as a weak activator for the recombinant retGC-1, to much lesser extent in case of the wild type retGC-1 (~ 1.4 % compared to the Ca-free GCAP-1), but substantially (~15%) in case of the R838S mutant at free Ca concentrations as high as 300 µM. While the apparent affinity of the R838S mutant to the Ca-free GCAP-1 increases at least 6-fold, its affinity to the Ca2+ loaded form of GCAP-2 increases less than 1.5-fold. GCAP-2 activates the R838S mutant with slightly lower efficiency but without significant change in its Ca2+-sensitivity and equally inhibits both mutant and wild type cyclases at [Ca]f > 1 µM . Conclusions: Our data indicate that in case of recombinant retGC the fully Ca2+ loaded GCAP-1 is a "low activator" form of GCAP-1 rather than just inactive or inhibitory form. We consider a model according to which the apparent shift in Ca2+ sensitivity in case of the R838 retGC mutants described in [1-3] can be a combination of a transition from "high activity" into the "lower activity" complex at saturation by Ca2+ (rather than "inhibited" complex), and the change in the affinity to the Ca2+-free GCAP-1 relative to the Ca2+-loaded GCAP-1 rather than decreased affinity of the GCAP-1/retGC-1 complex for Ca2+. References.  Tucker C.L., et al. (1999) Proc. Natl. Acad. Sci. USA 96, 9039-44  Wilkie S.E., et al. (2000) Hum. Mol. Genet. 9, 3065-73  Ramamurthy V., et al. (2001) J. Biol. Chem. 276, 26218-29.
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