The role of acidic phospholipids in glucagon action on rat liver adenylate cyclase.

We have examined the effects of phospholipase C from Bacillus cereus (Bc) and from Clostridium perfringens (Cp) on various parameters involved in the activity and response of adenylate cyclase to glucagon in rat liver plasma membranes. A crude preparation of Cp-phospholipase C hydrolyzes “neutral” phospholipids (phosphatidylcholine, phosphatidylethanolamine, sphingomyelin) in these membranes. In contrast, highly purified Bc-phospholipase C hydrolyzes acidic phospholipids (phosphatidylserine, phosphatidylinositol) but not sphingomyelin. Treatment of the membranes with either type of phospholipase does not alter basal adenylate cyclase activity or the stimulatory effects of fluoride ion on the enzyme system. Bc-phospholipase C abolishes the effects of glucagon on adenylate cyclase whereas Cp-phospholipase C causes only partial loss of glucagon response even after hydrolysis of 60% of the membrane phospholipids. These findings provide evidence that acidic phospholipids are more specifically involved in glucagon activation of adenylate cyclase. Acidic phospholipids are not directly involved in the binding of glucagon to its receptor. Treatment with Bc-phospholipase C results in a lo-fold reduction in the affinity but not the quantity of specific binding sites for glucagon. Binding of Des-His-glucagon, a competitive, inactive analogue of glucagon is unaffected by Bc-phospholipase C treatment and displays the same apparent affinity as does glucagon for the binding sites in phospholipase-treated membranes. These findings suggest that acidic phospholipids are involved in the liganding of the histidine residue of glucagon to a regulatory site responsible for glucagon action. GTP, which is required for glucagon action on adenylate cyclase and which increases the rate of dissociation of glucagon from its receptor, does not exert these effects in Bc-phospholipase C-treated membranes. GTP also does not alter the rate of dissociation of Des-His-glucagon from the binding sites in either untreated or treated membranes, indicating that the histidine residue of glucagon is required for the effects of GTP to be expressed. It appears, therefore, that GTP and the histidine residue of glucagon bind to a common site involved both in the activation of adenylate cyclase and in the dissociation of glucagon