Effect of G protein-coupled receptor kinase 2 on the sensitivity of M4 muscarinic acetylcholine receptors to agonist-induced internalization and desensitization in NG108-15 cells

NG108-15 cells express predominantly the M4 subtype of the muscarinic recep tor for acetylcholine. Stimulation of these receptors by the agonist carbac hol causes an inhibition of cellular adenylyl cyclase and a consequent fall in the intracellular cyclic AMP concentration. Pretreatment of the cells w ith carbachol caused both internalization and desensitization of the M4 rec eptor. Overexpression of G protein-coupled receptor kinase (GRK) 2 caused a n increase in the rate constant for receptor endocytosis (from 0.06 to 0.18 min(-1)) and a decrease in the EC50 for carbachol stimulation of internali zation (from 15 to 3 mu M). Overexpression of a dominant negative form of G RK2 had more modest effects, reducing the rate constant for endocytosis (fr om 0.11 to 0.07 min(-1)) and increasing the EC50 for carbachol stimulation of internalization (from 8 to 17 mu M) Neither GRK2 nor dominant negative G RK2 overexpression had any effect on the rate constant for receptor recycli ng following agonist removal. The time course and extent of receptor desens itization in control cells were identical to the corresponding values for r eceptor internalization, and the rate and extent of desensitization were ag ain increased by GRK2 overexpression. Exposure of the cells to hyperosmolar sucrose (0.6 M) almost completely blocked agonist-induced receptor interna lization in both control and GRK2-overexpressing cells. Sucrose treatment a lso blocked agonist-induced desensitization. We conclude that the internali zation and desensitization of the M4 muscarinic receptor in NG108-15 cells can be modulated in response to changes in GRK2 activity and also that inte rnalization plays a key role in desensitization.