c-Src regulates clathrin adapter protein 2 interaction with beta-arrestin and the angiotensin II type 1 receptor during clathrin- mediated internalization

Mol Endocrinol. 2005 Feb;19(2):491-503. doi: 10.1210/me.2004-0246. Epub 2004 Oct 21.


Beta-arrestins are multifunctional adapters involved in the internalization and signaling of G protein-coupled receptors (GPCRs). They target receptors to clathrin-coated pits (CCPs) through binding with clathrin and clathrin adapter 2 (AP-2) complex. They also act as transducers of signaling by recruiting c-Src kinase to certain GPCRs. Here we sought to determine whether c-Src regulates the recruitment of AP-2 to beta-arrestin and the angiotensin II (Ang II) type 1 receptor (AT1R) during internalization. We show that the agonist stimulation of native AT1R in vascular smooth muscle cells (VSMCs) induces the formation of an endogenous complex containing c-Src, beta-arrestins and AP-2. In vitro studies using coimmunoprecipitation experiments and a yeast three-hybrid assay reveal that c-Src stabilizes the agonist-independent association between beta-arrestin2 and the beta-subunit of AP-2 independently of the kinase activity of c-Src. However, although c-Src expression promoted the rapid dissociation of AP-2 from both beta-arrestin and AT1R after receptor stimulation, a kinase-inactive mutant of c-Src failed to induce the dissociation of AP-2 from the agonist-occupied receptor. Thus, the consequence of c-Src in regulating the dissociation of AP-2 from the receptor was also examined on the internalization of AT1R by depleting c-Src in human embryonic kidney (HEK) 293 cells using a small interfering RNA strategy. Experiments in c-Src depleted cells reveal that AT1R remained mostly colocalized with AP-2 at the plasma membrane after Ang II stimulation, consistent with the observed delay in receptor internalization. Moreover, coimmunoprecipitation experiments in c-Src depleted HEK 293 cells and VSMCs showed an increased association of AP-2 to the agonist-occupied AT1R and beta-arrestin, respectively. Together, our results support a role for c-Src in regulating the dissociation of AP-2 from agonist-occupied AT1R and beta-arrestin during the clathrin-mediated internalization of receptors and suggest a novel function for c-Src kinase in the internalization of AT1R.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Protein Complex 2 / metabolism*
  • Animals
  • Arrestins / metabolism*
  • COS Cells
  • Cell Line
  • Cell Membrane / metabolism
  • Cells, Cultured
  • Endocytosis
  • Green Fluorescent Proteins / metabolism
  • Humans
  • Immunoprecipitation
  • Microscopy, Fluorescence
  • Models, Biological
  • Myocytes, Smooth Muscle / metabolism
  • Oligonucleotides, Antisense / pharmacology
  • Plasmids / metabolism
  • Protein Binding
  • Protein Structure, Tertiary
  • Proto-Oncogene Proteins pp60(c-src) / metabolism*
  • RNA, Small Interfering / metabolism
  • Receptor, Angiotensin, Type 1 / metabolism*
  • Time Factors
  • Transfection
  • Two-Hybrid System Techniques
  • beta-Arrestins


  • Adaptor Protein Complex 2
  • Arrestins
  • Oligonucleotides, Antisense
  • RNA, Small Interfering
  • Receptor, Angiotensin, Type 1
  • beta-Arrestins
  • Green Fluorescent Proteins
  • Proto-Oncogene Proteins pp60(c-src)