Enhancement of Phototransduction G Protein-Effector Interactions by Phosphoinositides

Abstract

Light responses in photoreceptor cells are mediated by the action of the G protein transducin (Gt) on the effector enzyme cGMP phosphodiesterase (PDE6) at the surface of disk membranes. The enzymatic components needed for phosphoinositide-based signaling are known to be present in rod cells, but it has remained uncertain what role phosphoinositides play in vertebrate phototransduction. Reconstitution of PDE6 and activated G αt, on the surface of large unilamellar vesicles containing D-myo-phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) stimulated PDE activity nearly 4-fold above the level observed with membranes containing no phosphoinositides, whereas G protein-independent activation by trypsin was unaffected by the presence of phosphoinositides. PDE activity was similarly stimulated by D-MYO-phosphatidylinositol-3,4.bisphosphate and D-MYO-phosphatidylinositol-4-phosphate (PI(4)P), but much less by D-myo-phosphatidylinositol-5-phosphate (PI(5)P) Or D-myo-phosphatidylinositol-3,5-bisphosphate. Incubation of rod outer segment membranes with phosphoinositide-specific phospholipase C decreased G protein-stimulated activation of endogenous PDE6, but not trypsin-stimulated PDE activity. Binding experiments using phosphoinositide-containing vesicles revealed patterns of PDE6 binding and PDE6-enhanced Gα t-GTPγS binding, consistent with the activation profile PI(4,5)P2 > PI(4)P > PI(5)P ∼ control vesicles. These results suggest that enhancement of effector-G protein interactions represents a possible mechanism for modulation of phototransduction gain by changes in phosphoinositide levels, perhaps occurring in response to long-term changes in illumination or other environmental cues.