c-Src regulates clathrin adapter protein 2 interaction with beta-arrestin and the angiotensin II type 1 receptor during clathrin- mediated internalization.
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.
Fessart
Delphine
D
Hormones and Cancer Research Unit, Department of Medicine, McGill University, Royal Victoria Hospital, 687 Pine Avenue West, Montréal, Québec, Canada H3A 1A1.
Simaan
May
M
Laporte
Stéphane A
SA
D033962:Adaptor Protein Complex 2 / Q000378:metabolism*; D000818:Animals; D019390:Arrestins / Q000378:metabolism*; D019556:COS Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D004705:Endocytosis; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D032389:Myocytes, Smooth Muscle / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism; D044140:Receptor, Angiotensin, Type 1 / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection; D020798:Two-Hybrid System Techniques; D000071557:beta-Arrestins
D033962:Adaptor Protein Complex 2; D019390:Arrestins; D016376:Oligonucleotides, Antisense; D034741:RNA, Small Interfering; D044140:Receptor, Angiotensin, Type 1; D000071557:beta-Arrestins; D049452:Green Fluorescent Proteins; D016392:Proto-Oncogene Proteins pp60(c-src)