Assays of G protein/cGMP-phosphodiesterase interactions

Abstract

Activation of cGMP phosphodiesterase (PDE) by the photoreceptor G protein transducin (G 1 ), is a key event in the vertebrate visual transduction cascade. The assays of cGMP hydrolysis by activated PDE have been a major tool for monitoring the interaction between transducin and PDE, and initial studies mainly relied on such assays. The presence of rod outer segment (ROS) membranes or lipid vesicles significantly enhances the effectiveness of PDE stimulation because of the formation of an active membrane-bound complex between G 1 α and PDE and allows an increase in assay sensitivity. However, membrane-supported activation of PDE by transducin is a complex process that depends on a number of factors such as type and concentration of membranes {vesicles), binding of PDE, binding of G 1 α to membranes, and the intact stale of lipid modifications on Pαβ and G 1 α. Furthermore, PDE activation is not a direct monitor of transducin binding to the enzyme. This is evident from studies of G 1 α mutants that bind but fail to activate PDE, or interact with the effector weakly without causing enzyme activation.