The adaptor complex 2 directly interacts with the alpha 1b-adrenergic receptor and plays a role in receptor endocytosis.
Using the yeast two-hybrid system, we identified the mu 2 subunit of the clathrin adaptor complex 2 as a protein interacting with the C-tail of the alpha 1b-adrenergic receptor (AR). Direct association between the alpha 1b-AR and mu 2 was demonstrated using a solid phase overlay assay. The alpha 1b-AR/mu 2 interaction occurred inside the cells, as shown by the finding that the transfected alpha 1b-AR and the endogenous mu 2 could be coimmunoprecipitated from HEK-293 cell extracts. Mutational analysis of the alpha 1b-AR revealed that the binding site for mu 2 does not involve canonical YXX Phi or dileucine motifs but a stretch of eight arginines on the receptor C-tail. The binding domain of mu 2 for the receptor C-tail involves both its N terminus and the subdomain B of its C-terminal portion. The alpha 1b-AR specifically interacted with mu 2, but not with the mu 1, mu 3, or mu 4 subunits belonging to other AP complexes. The deletion of the mu 2 binding site in the C-tail markedly decreased agonist-induced receptor internalization as demonstrated by confocal microscopy as well as by the results of a surface receptor biotinylation assay. The direct association of the adaptor complex 2 with a G protein-coupled receptor has not been reported so far and might represent a common mechanism underlying clathrin-mediated receptor endocytosis.
Diviani
Dario
D
Institut de Pharmacologie et de Toxicologie, Faculté de Médecine, 1005 Lausanne, Switzerland.
Lattion
Anne-Laure
AL
Abuin
Liliane
L
Staub
Olivier
O
Cotecchia
Susanna
S
D033962:Adaptor Protein Complex 2 / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D033983:Adaptor Protein Complex mu Subunits / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D019921:Biotinylation; D015153:Blotting, Western; D002460:Cell Line; D002966:Clathrin / Q000502:physiology; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis / Q000187:drug effects; D004837:Epinephrine / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D017353:Gene Deletion; D015870:Gene Expression; D005982:Glutathione Transferase / Q000235:genetics; D049452:Green Fluorescent Proteins; D006801:Humans; D007163:Immunosorbent Techniques; D008164:Luminescent Proteins / Q000235:genetics; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D018340:Receptors, Adrenergic, alpha-1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D014162:Transfection; D020798:Two-Hybrid System Techniques
D033962:Adaptor Protein Complex 2; D033983:Adaptor Protein Complex mu Subunits; D002966:Clathrin; D008164:Luminescent Proteins; D018340:Receptors, Adrenergic, alpha-1; D011993:Recombinant Fusion Proteins; C438886:adaptor protein complex 2, mu 2 subunit; D049452:Green Fluorescent Proteins; D005982:Glutathione Transferase; D004837:Epinephrine