Mutating the dileucine motif of the human beta(2)-adrenoceptor reduces the high initial rate of receptor phosphorylation by GRK without affecting postendocytic sorting.

The internalization of beta(2)-adrenoceptors after agonist activation results in a desensitized and phosphorylated receptor that either resensitizes by recycling to the cell surface or becomes degraded by postendocytic sorting to lysosomes. The duration and physiological effects of agonists therefore depend on beta(2)-adrenoceptor sorting, highlighting the importance of sorting signals. Dileucine motifs within other membrane proteins act as signals for endocytosis and/or postendocytic sorting, and the beta(2)-adrenoceptor has a dileucine motif within helix 8 that might play a role in efficient receptor recycling and/or downregulation. beta(2)-adrenoceptor internalization and sorting were studied in HEK293 cells stably expressing wild type or mutant dialanine L339A,L340A beta(2)-adrenoceptors. The mutant beta(2)-adrenoceptors showed a significantly lower initial rate of phosphorylation at the prominent G-protein coupled receptor kinase (GRK) sites Ser355 and 356 compared to wild type beta(2)-adrenoceptors. Furthermore, the agonist-induced endocytic rate constant for L339A,L340A beta(2)-adrenoceptors was reduced to approximately 25% that of wild type beta(2)-adrenoceptors, which resulted in a similar reduction in agonist-induced downregulation. Internalized L339A,L340A beta(2)-adrenoceptors recycled to the surface with a rate and extent similar to that of wild type beta(2)-adrenoceptors. Therefore, although the role of L339,L340 in beta(2)-adrenoceptor recycling or postendocytic sorting seems minimal, we conclude that L339,L340 is required for the initial high rate of phosphorylation by G-protein coupled receptor kinases at Ser355,356, which in turn is required for efficient beta(2)-adrenoceptors endocytosis.