Regulation of CB1cannabinoid receptor internalization by a promiscuous phosphorylation-dependent mechanism

Agonists stimulate cannabinoid 1 receptor (CB1R) internalization. Previous work suggests that the extreme carboxy-terminus of the receptor regulates this internalization - likely through the phosphorylation of serines and threonines clustered within this region. While truncation of the carboxy-terminus (V460Z CB1) and consequent removal of these putative phosphorylation sites prevents endocytosis in AtT20 cells, the residues necessary for CB1R internalization remain elusive. To determine the structural requirements for internalization, we evaluated endocytosis of carboxy-terminal mutant CB1Rs stably expressed in HEK293 cells. In contrast to AtT20 cells, V460Z CB1R expressed in HEK293 cells internalized to the same extent and with similar kinetics as the wild-type receptor. However, mutation of serine and-or threonine residues within the extreme carboxy-terminal attenuated internalization when these receptors were expressed in HEK293 cells. These results establish that the extreme carboxy-terminal phosphorylation sites are not required for internalization of truncated receptors, but are required for internalization of full-length receptors in HEK293 cells. Analysis of beta -arrestin-2 recruitment to mutant CB1R suggests that putative carboxy-terminal phosphorylation sites mediate beta -arrestin-2 translocation. This study indicates that the local cellular environment affects the structural determinants of CB1R internalization. Additionally, phosphorylation likely regulates the internalization of (full-length) CB1Rs.