Nedd4 mediates agonist-dependent ubiquitination, lysosomal targeting, and degradation of the beta2-adrenergic receptor.
Agonist-stimulated beta(2)-adrenergic receptor (beta(2)AR) ubiquitination is a major factor that governs both lysosomal trafficking and degradation of internalized receptors, but the identity of the E3 ubiquitin ligase regulating this process was unknown. Among the various catalytically inactive E3 ubiquitin ligase mutants that we tested, a dominant negative Nedd4 specifically inhibited isoproterenol-induced ubiquitination and degradation of the beta(2)AR in HEK-293 cells. Moreover, siRNA that down-regulates Nedd4 expression inhibited beta(2)AR ubiquitination and lysosomal degradation, whereas siRNA targeting the closely related E3 ligases Nedd4-2 or AIP4 did not. Interestingly, beta(2)AR as well as beta-arrestin2, the endocytic and signaling adaptor for the beta(2)AR, interact robustly with Nedd4 upon agonist stimulation. However, beta(2)AR-Nedd4 interaction is ablated when beta-arrestin2 expression is knocked down by siRNA transfection, implicating an essential E3 ubiquitin ligase adaptor role for beta-arrestin2 in mediating beta(2)AR ubiquitination. Notably, beta-arrestin2 interacts with two different E3 ubiquitin ligases, namely, Mdm2 and Nedd4 to regulate distinct steps in beta(2)AR trafficking. Collectively, our findings indicate that the degradative fate of the beta(2)AR in the lysosomal compartments is dependent upon beta-arrestin2-mediated recruitment of Nedd4 to the activated receptor and Nedd4-catalyzed ubiquitination.
Shenoy
Sudha K
SK
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA. sudha@receptor-biol.duke.edu
Xiao
Kunhong
K
Venkataramanan
Vidya
V
Snyder
Peter M
PM
Freedman
Neil J
NJ
Weissman
Allan M
AM
D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D019390:Arrestins / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D045643:DNA Repair Enzymes; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D008247:Lysosomes / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D051736:Proto-Oncogene Proteins c-mdm2 / Q000378:metabolism; D034622:RNA Interference; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D054875:Ubiquitination / Q000187:drug effects; D000071557:beta-Arrestins
D000318:Adrenergic beta-Agonists; D019390:Arrestins; D002352:Carrier Proteins; C476254:N4BP2 protein, human; D018343:Receptors, Adrenergic, beta-2; D012097:Repressor Proteins; D000071557:beta-Arrestins; C432966:ITCH protein, human; D051736:Proto-Oncogene Proteins c-mdm2; D044767:Ubiquitin-Protein Ligases; D045643:DNA Repair Enzymes; D007545:Isoproterenol