β-Arrestin acts as a clathrin adaptor in endocytosis of the β2-adrenergic receptor

Abstract

The ability of a system to regulate its responsiveness in the presence of a continuous stimulus, often termed desensitization, has been extensively characterized for the beta2-adrenergic receptor (beta2AR). beta2AR signalling is rapidly attenuated through receptor phosphorylation and subsequent binding of the protein beta-arrestin. Ultimately the receptor undergoes internalization, and although the molecular mechanism is unclear, receptor phosphorylation and beta-arrestin binding have been implicated in this processs. Here we report that beta-arrestin and arrestin-3, but not visual arrestin, promote beta2AR internalization and bind with high affinity directly and stoichiometrically to clathrin, the major structural protein of coated pits. Moreover, beta-arrestin/arrestin chimaeras that are defective in either beta2AR or clathrin binding show a reduced ability to promote beta2AR endocytosis. Immunofluorescence microscopy of intact cells indicates an agonist-dependent colocalization of the beta2AR and beta-arrestin with clathrin. These results show that beta-arrestin functions as an adaptor in the receptor-mediated endocytosis pathway, and suggest a general mechanism for regulating the trafficking of G-protein-coupled receptors.

Structure and Function of the Third Intracellular Loop of the 5‐Hydroxytryptamine2A Receptor: The Third Intracellular Loop Is α‐Helical and Binds Purified Arrestins

“…These findings are important, because they represent the first demonstration that GPCRs and arrestins are colocalized in neurons. Additionally, these studies support notions derived from previous in vitro studies that suggest that arrestins are involved in regulating the trafficking of GPCRs (Ferguson et al, 1996;Goodman et al, 1996). Whether arrestins regulate the internalization of 5-HT,, receptors or other 5-HT,, re-ceptor functions is currently under investigation.…”

Paradoxical trafficking and regulation of 5-HT2A receptors by agonists and antagonists

“…Additionally, a dominant-negative mutant of arrestin-2 attenuated agonist-induced internalization in transiently transfected HEK-293 cells [26]. Subsequently, it was found that purified arrestin-2 bound directly to clathrin and clathrin-coated vesicles [39] and the domains of arrestin-2 that are essential for clathrin binding have been identified [38,39,57]. Recently, arrestin-2 has been shown to also interact with adaptor protein 2 (AP-2) [60], a protein that plays a critical role in the recruitment of clathrin and assembly of clathrincoated pits (Fig.…”

Inhibitors of phosphoinositide 3-kinase cause defects in the postendocytic sorting of β2-adrenergic receptors

“…Similar to other GPCRs, phosphorylated β 2 ARs then bind with high affinity to β-arrestin, which fully uncouples receptors from G proteins [4] and also interacts with clathrin and adaptor proteins, thereby facilitating rapid receptor endocytosis [5,6]. Internalized receptors traffic through early endosomes, localizing with transferrin receptors [7] and rab5a [8] before returning to the cell surface (recycling).…”