RECEPTOR INTERNALIZATION DELAYS M4 MUSCARINIC ACETYLCHOLINE-RECEPTOR RESENSITIZATION AT THE PLASMA-MEMBRANE

We analyzed the role of receptor internalization and recycling in musc arinic acetylcholine receptor (mAChR) desensitization and resensitizat ion. Incubation of Chinese hamster ovary cells stably expressing the m 4 mAChR with 1 mM carbachol for 1 hr reduced cell surface receptor num ber by 50-60% with no change in total receptor number. Pretreatment of the cells with 450 mM sucrose, which did not affect the ability of m4 receptors to inhibit forskolin-stimulated cAMP accumulation, complete ly blocked receptor internalization. On the other hand, the carbachol treatment reduced the ability of m4 receptors to inhibit cAMP accumula tion in both sucrose-treated and untreated cells, with a similar onset and to a similar extent. The EC(50) value for carbachol was increased similar to 10-fold, and maximal inhibition determined at 100 mu M car bachol was reduced similar to 50%. In contrast, thrombin-induced inhib ition of cAMP accumulation was not affected. Recycled receptors in cel ls not treated with sucrose remained refractory to carbachol stimulati on for greater than or equal to 2 hr after agonist removal, even thoug h cell surface receptor number had recovered completely within 1 hr. I n contrast, resensitization of receptor function was very rapid in cel ls treated with sucrose. Ten minutes on removal of agonist, mAChRs in the plasma membrane of sucrose-treated cells were fully resensitized. Also, an internalization-defective m4 mAChR mutant, T399A, that was fo und to desensitize similar to the wild-type receptor, resensitized mor e rapidly than the wild-type receptor. We conclude that desensitizatio n and resensitization of m4 mAChRs in Chinese hamster ovary cells can occur at the plasma membrane and that receptor internalization strongl y delays the process of resensitization of desensitized receptors.