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A. Mendez, A. Almuete, K.A. Emelianoff, N. Calero, D.A. Baylor, M.E. Burns, J. Chen; FUNCTIONAL DIFFERENCES BETWEEN P48 AND M44 FORMS OF ARRESTIN IN VIVO . Invest. Ophthalmol. Vis. Sci. 2004;45(13):2209.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: to dissect the functional roles of arrestin splice variants p48 and p44 in the deactivation of photoactivated rhodopsin in vivo. Methods: selective expression of p48 or m44 (murine Arr1–370) splice variants of arrestin in murine rods was achieved by transgenic expression of either form in the arrestin knockout (Arr–/–) background. The content of transgenic arrestin in both lines (relative to endogenous arrestin in wildtype mice) were estimated to be about 140% (p48) and 8% (mArr1–370) in rod outer segment preparations from dark–adapted mice. The effect of each form on the light response kinetics was determined by single cell recordings; and the effect on the susceptibility of Arr–/– mice to light–damage was assayed by morphological analysis. Both p48 and m44 were also expressed in the rhodopsin kinase/arrestin (RK/Arr) double knockout background to determine their ability to quench the unphosphorylated receptor. Results: Rods expressing p48 and m44 yielded dim flash responses whose amplitude and recovery kinetics were indistinguishable from those of wildtype rods. At brighter flashes, the recovery of m44–expressing rods greatly slowed, presumably because the number of photoactivated rhodopsin molecules exceeded that of m44. In agreement with electrophysiological results, both p48 and m44 rescued the light–dependent degeneration observed in arrestin knockout mice reared under normal cyclic light conditions. However, only p48 was able to rescue the light–damage induced by a short exposure to a high intensity background light (2,000 lx). Experiments on RK/Arr double knockout rods suggest that in the absence of rhodopsin phosphorylation, the stochastic termination of the abnormally prolonged responses is likely due to a weak interaction of photoactivated rhodopsin with p48 arrestin in its basal state. In agreement with the described low affinity of p48 for photoactivated unphosphorylated rhodopsin, this binding appears to be reversible in nature at the single photon level. We do not observe this phosphorylation–independent effect on the p44–expressing line. Conclusions: We show that both p48 and p44 splice variants of arrestin can be functional in the intact cell, with p44 being able to completely quench rhodopsin activity under dim light conditions. However, p48 is the likely candidate to normally quench the rhodopsin molecules during the physiological response to light.
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