c-Src-mediated phosphorylation of AP-2 reveals a general mechanism for receptors internalizing through the clathrin pathway

Cell Signal. 2009 Jan;21(1):103-10. doi: 10.1016/j.cellsig.2008.09.013. Epub 2008 Oct 1.

Abstract

Clathrin-mediated endocytosis is a complex process regulated at many different levels. We showed previously that activation of the angiotensin type 1 receptor (AT1R), which belongs to the G protein-coupled receptor (GPCR) family, leads to c-Src-dependent tyrosine phosphorylation of beta2-adaptin, a subunit of the clathrin adaptor AP-2. The phosphorylation of beta2-adaptin on tyrosine residue 737 (Y737) negatively regulates its interaction with betaarrestin, another important clathrin adaptor for GPCR internalization. Here we sought to determine whether AP-2 phosphorylation represents a general mechanism for different receptors internalizing through the clathrin pathway. Using a specifically designed antibody against the phosphorylated form of Y737 on beta2-adaptin, we demonstrate that this residue is phosphorylated by AT1R in different cell types like HEK293, COS-7 and vascular smooth muscle cells. Using RNA interference approaches, we reveal that this agonist-mediated event is both betaarrestin- and c-Src-dependent, and that it occurs at the plasma membrane in clathrin-coated vesicles (CCVs). We further show that this is not only a common event employed by other GPCRs like the beta2-adrenergic, vasopressin V2, bradykinin type 2, platelet-activating factor and endothelin A receptors but that the epidermal growth factor receptor is capable of eliciting the phosphorylation of AP-2 in CCVs. Our results imply that tyrosine phosphorylation of Y737 on beta2-adaptin is a common regulatory mechanism employed by different receptors undergoing clathrin-dependent endocytosis, and suggest a wider function for this event than originally anticipated.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Protein Complex 2 / metabolism*
  • Adaptor Protein Complex beta Subunits / agonists
  • Adaptor Protein Complex beta Subunits / metabolism
  • Amino Acid Sequence
  • Angiotensin II / pharmacology
  • Animals
  • Arrestins / metabolism
  • COS Cells
  • CSK Tyrosine-Protein Kinase
  • Cell Line
  • Chlorocebus aethiops
  • Clathrin / metabolism*
  • Clathrin-Coated Vesicles / metabolism
  • Endocytosis
  • ErbB Receptors / metabolism
  • Fluorescent Antibody Technique, Direct
  • Gene Knockdown Techniques
  • Humans
  • Phosphorylation
  • Protein-Tyrosine Kinases / metabolism*
  • Receptor, Angiotensin, Type 1 / metabolism
  • Receptors, G-Protein-Coupled / metabolism
  • src-Family Kinases

Substances

  • Adaptor Protein Complex 2
  • Adaptor Protein Complex beta Subunits
  • Arrestins
  • Clathrin
  • Receptor, Angiotensin, Type 1
  • Receptors, G-Protein-Coupled
  • Angiotensin II
  • ErbB Receptors
  • Protein-Tyrosine Kinases
  • CSK Tyrosine-Protein Kinase
  • src-Family Kinases
  • CSK protein, human