Src-dependent phosphorylation of beta2-adaptin dissociates the beta-arrestin-AP-2 complex.
Beta-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 beta-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 beta2-adaptin, the subunit of AP-2 involved in binding beta-arrestin. We identify a crucial tyrosine residue in the ear domain of beta2-adaptin and show in vitro that the phosphorylation of this site regulates the interaction between beta-arrestin and beta2-adaptin. Using fluorescently tagged proteins combined with resonance energy transfer and image cross-correlation spectroscopy approaches, we show in live cells that beta2-adaptin phosphorylation is an important regulatory process for the dissociation of beta-arrestin-AP-2 complexes in CCPs. Finally, we show that beta2-adaptin phosphorylation is involved in the early steps of receptor internalization. Our findings not only unveil beta2-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.
Fessart
Delphine
D
Hormones and Cancer Research Unit, Department of Medicine, Royal Victoria Hospital, Montréal, QC, H3A 1A1, Canada.
Simaan
May
M
Zimmerman
Brandon
B
Comeau
Jonathan
J
Hamdan
Fadi F
FF
Wiseman
Paul W
PW
Bouvier
Michel
M
Laporte
Stéphane A
SA
D033962:Adaptor Protein Complex 2 / Q000378:metabolism*; D033966:Adaptor Protein Complex beta Subunits / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D019390:Arrestins / Q000378:metabolism*; D001665:Binding Sites / Q000502:physiology; D019556:COS Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D022163:Clathrin-Coated Vesicles / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology*; D006801:Humans; D010766:Phosphorylation; D017434:Protein Structure, Tertiary / Q000502:physiology; D044140:Receptor, Angiotensin, Type 1 / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism* / Q000648:ultrastructure; D015398:Signal Transduction / Q000502:physiology; D014443:Tyrosine / Q000378:metabolism; D000071557:beta-Arrestins; D019061:src-Family Kinases / Q000378:metabolism*
D033962:Adaptor Protein Complex 2; D033966:Adaptor Protein Complex beta Subunits; D019390:Arrestins; D044140:Receptor, Angiotensin, Type 1; D011956:Receptors, Cell Surface; D000071557:beta-Arrestins; D014443:Tyrosine; D019061:src-Family Kinases