Interaction between photoactivated rhodopsin and its kinase: Stability and kinetics of complex formation

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Abstract

Rhodopsin phosphorylation is a key event in the deactivation of this G-protein-coupled receptor. Rhodopsin kinase mediates the reaction and requires specific cytoplasmic loop domains on rhodopsin, distinct from the sites of phosphorylation, for binding and activation. In this study, we characterize the formation of a stable complex between photolyzed rhodopsin in native washed disk membranes and its kinase. Centrifugation of the membranes after illumination and subsequent polyacrylamide gel electrophoresis demonstrates light-dependent binding of rhodopsin kinase to the membranes. A real-time monitor for the transition of the solubilized kinase into the bound state is provided by flash-induced light-scattering binding signals. The complex has the following characteristics: (i) the on-rate of the reaction rises in linear proportion to the concentrations of both the kinase and photoactivated rhodopsin; kinetic analysis yields a bimolecular rate constant of kon = 0.5-1 microM-1s-1. (ii) The dissociation constant of the complex is 0.3 < KD < 0.5 microM in the absence of ATP, but with ATP, it decreases by at least a factor of 10; however, phosphorylation of rhodopsin or (auto)phosphorylation of rhodopsin kinase leads to destabilization of the complex. (iii) In contrast to the binding of arrestin and transducin, the binding of rhodopsin kinase to photoactivated rhodopsin does not stabilize the metarhodopsin II photoproduct; however, rhodopsin kinase competes with the G-protein transducin for binding to photoactivated rhodopsin. Extrapolation of the kinetic parameters to cellular concentrations at room temperature suggests that free competitive binding of the kinase would strongly inhibit the G-protein activation process after a few hundred catalytic cycles.

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