Regulation of the endocytic adaptor proteins [beta] arrestin and AP-2 during clathrin-mediated internalization of Angiotensin II type 1 receptor

Abstract

G protein-coupled receptors (GPCRs) are the largest family of cell-surface receptors. They transduce the signals mediated by a diverse range of signalling molecules, including ions, amines, and peptides, as well as photons, to mediate intracellular functions. These receptors play a fundamental role in many physiological responses such as cardiovascular functions. To remain responsive to their environment, cells must find a way to rapidly desensitize and resensitize their activated GPCRs. Desensitization of receptors, for instance, involves the phosphorylation of receptors by G protein-coupled receptor kinase (GRKs) followed by the recruitment of betaarrestin. This interferes with the binding of the G protein (the signalling effector). betaarrestin then targets the receptors to the clathrin endocytosis pathway, and serves as an adaptor linking receptors to other signalling pathways. Internalization of receptors serves not only to remove desensitized receptors from the plasma membrane, but also to engage receptors in the resensitization pathway.The internalization of Angiotensin II (Ang II) type 1 receptor (AT1R) is controversial and poorly described. Therefore, our laboratory studies the mechanisms behind AT1R internalization. The agonist-induced internalization of AT1R begins with the formation of a complex including betaarrestin, the clathrin adaptor AP-2, and the tyrosine protein kinase, c-Src. In turn, this c-Src recruitment regulates the clathrin-mediated internalization of AT1R by controlling the formation of endocytic complexes during endocytosis. Indeed, the recruitment of c-Src is involved in the dissociation of AP-2 during receptor internalization. Based on our evidence that AP-2 and c-Src can be found in the same complex, we suggested that AP-2 could be phosphorylated by c-Src. Indeed, we found that Ang II induced the c-Src-mediated tyrosine phosphorylation of the beta-subunit of AP-2 (beta2-adaptin). We were able to map one of the tyrosines in beta2-adaptin and assess its role in regulating the binding of its principal partner: betaarrestin. The phosphorylation state of beta2-adaptin dictates its association profile with betaarrestin: when phosphorylated it reduces its binding to betaarrestin. Finally, we proposed a model for AT1R internalization. Overall, these studies are significant because they allow a better understanding of the underlying mechanism that regulates the initial steps of clathrin-coated vesicle endocytosis of AT1R