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.
Mesh Terms:
Adaptor Protein Complex 2, Adaptor Protein Complex mu Subunits, Amino Acid Sequence, Animals, Binding Sites, Biotinylation, Blotting, Western, Cell Line, Clathrin, Cricetinae, Electrophoresis, Polyacrylamide Gel, Endocytosis, Epinephrine, Escherichia coli, Gene Deletion, Gene Expression, Glutathione Transferase, Green Fluorescent Proteins, Humans, Immunosorbent Techniques, Luminescent Proteins, Microscopy, Confocal, Molecular Sequence Data, Mutagenesis, Polymerase Chain Reaction, Receptors, Adrenergic, alpha-1, Recombinant Fusion Proteins, Transfection, Two-Hybrid System Techniques
Adaptor Protein Complex 2, Adaptor Protein Complex mu Subunits, Amino Acid Sequence, Animals, Binding Sites, Biotinylation, Blotting, Western, Cell Line, Clathrin, Cricetinae, Electrophoresis, Polyacrylamide Gel, Endocytosis, Epinephrine, Escherichia coli, Gene Deletion, Gene Expression, Glutathione Transferase, Green Fluorescent Proteins, Humans, Immunosorbent Techniques, Luminescent Proteins, Microscopy, Confocal, Molecular Sequence Data, Mutagenesis, Polymerase Chain Reaction, Receptors, Adrenergic, alpha-1, Recombinant Fusion Proteins, Transfection, Two-Hybrid System Techniques
J. Biol. Chem.
Date: May. 23, 2003
PubMed ID: 12644451
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