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First published online 24 April 2007
doi: 10.1242/jcs.03444

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Src-dependent phosphorylation of β2-adaptin dissociates the β-arrestin–AP-2 complex

Delphine Fessart^1,*, May Simaan^1,*, Brandon Zimmerman^1,2, Jonathan Comeau³, Fadi F. Hamdan⁴, Paul W. Wiseman^3,5, Michel Bouvier⁴ and Stéphane A. Laporte^1,2,

¹ Hormones and Cancer Research Unit, Department of Medicine, Royal Victoria Hospital, 687, Pine Avenue West, Montréal, QC, H3A 1A1, Canada
² Department of Pharmacology and Therapeutics, Royal Victoria Hospital, 687, Pine Avenue West, Montréal, QC, H3A 1A1, Canada
³ Department of Chemistry, McGill University, 801 Sherbrooke West, Montréal, QC, H3A 2K6, Canada
⁴ Department of Biochemistry and Institute for Research in Immunology and Cancer, Université de Montréal, Montréal, QC, H3C 3J7, Canada
⁵ Department of Physics, McGill University, 3600 University Street, Montréal, QC, H3A 2T8, Canada

Author for correspondence (e-mail: stephane.laporte{at}mcgill.ca)

Accepted 14 March 2007

β-arrestins are known to act as endocytic adaptors by recruiting the clathrin adaptor protein 2 (AP-2) complex to G-protein-coupled receptors (GPCRs), linking them to clathrin-coated pits (CCPs) for internalization. They also act as signaling molecules connecting GPCRs to different downstream effectors. We have previously shown that stimulation of the angiotensin II (Ang II) type 1 receptor (AGTR1, hereafter referred to as AT1R), a member of the GPCR family, promotes the formation of a complex between β-arrestin, the kinase Src and AP-2. Here, we report that formation of such a complex is involved in the AT1R-mediated tyrosine phosphorylation of β2-adaptin, the subunit of AP-2 involved in binding β-arrestin. We identify a crucial tyrosine residue in the ear domain of β2-adaptin and show in vitro that the phosphorylation of this site regulates the interaction between β-arrestin and β2-adaptin. Using fluorescently tagged proteins combined with resonance energy transfer and image cross-correlation spectroscopy approaches, we show in live cells that β2-adaptin phosphorylation is an important regulatory process for the dissociation of β-arrestin–AP-2 complexes in CCPs. Finally, we show that β2-adaptin phosphorylation is involved in the early steps of receptor internalization. Our findings not only unveil β2-adaptin as a new Src target during AT1R internalization, but also support the role of receptor-mediated signaling in the control of clathrin-dependent endocytosis of receptors.

Key words: Src, β-arrestin, AP-2, GPCR, BRET, Image cross-correlation spectroscopy

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