Ezrin directly interacts with the alpha1b-adrenergic receptor and plays a role in receptor recycling.
Using the yeast two-hybrid system, we identified ezrin as a protein interacting with the C-tail of the alpha1b-adrenergic receptor (AR). The interaction was shown to occur in vitro between the receptor C-tail and the N-terminal portion of ezrin, or Four-point-one ERM (FERM) domain. The alpha1b-AR/ezrin interaction occurred inside the cells as shown by the finding that the transfected alpha1b-AR and FERM domain or ezrin could be coimmunoprecipitated from human embryonic kidney 293 cell extracts. Mutational analysis of the alpha1b-AR revealed that the binding site for ezrin involves a stretch of at least four arginines on the receptor C-tail. The results from both receptor biotinylation and immunofluorescence experiments indicated that the FERM domain impaired alpha1b-AR recycling to the plasma membrane without affecting receptor internalization. The dominant negative effect of the FERM domain, which relies on its ability to mask the ezrin binding site for actin, was mimicked by treatment of cells with cytochalasin D, an actin depolymerizing agent. A receptor mutant (DeltaR8) lacking its binding site in the C-tail for ezrin displayed delayed receptor recycling. These findings identify ezrin as a new protein directly interacting with a G protein-coupled receptor and demonstrate the direct implication of ezrin in GPCR trafficking via an actin-dependent mechanism.
Stanasila
Laura
L
Département de Pharmacologie et de Toxicologie, Faculté de Biologie et de Médecine, Lausanne, Switzerland.
Abuin
Liliane
L
Diviani
Dario
D
Cotecchia
Susanna
S
D000199:Actins / Q000502:physiology; D001665:Binding Sites; D002460:Cell Line; D015638:Cytochalasin D / Q000494:pharmacology; D003598:Cytoskeletal Proteins / Q000737:chemistry / Q000502:physiology*; D006801:Humans; D018613:Microscopy, Confocal; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D018340:Receptors, Adrenergic, alpha-1 / Q000737:chemistry / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D000099149:Ezrin
D000199:Actins; D015638:Cytochalasin D; D003598:Cytoskeletal Proteins; D018340:Receptors, Adrenergic, alpha-1; D043562:Receptors, G-Protein-Coupled; D000099149:Ezrin; C547716:ADRA1B protein, human