Abstract
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.
Highlights
It is recognized by the G protein-coupled receptor kinases and becomes phosphorylated [2]
This study provides the evidence that the adaptor complex 2 (AP2) complex can associate with a G protein-coupled receptor, the ␣1b-adrenergic receptor (AR), through the direct interaction of its 2 subunit with the receptor
Previous studies have shown that the AP2 complex is implicated in the agonist-induced endocytosis of the 2-AR [5,6,7]
Summary
Expression Constructs—A cDNA fragment encoding the last 165 amino acids of the hamster ␣1b-AR (amino acids 351–515) (Fig. 2) was PCR-amplified and subcloned at EcoRI/SalI in pGEX4T1, pET30a, and pLexA plasmids to construct fusion proteins with GST, His, and LexA at the N terminus of the C-tail of the receptor, respectively. For constructing GST fusion proteins with different fragments of the C-tail, cDNA fragments encoding amino acids 351–380, 351–395, 351– 425, 351– 449, and 351– 477 of the ␣1b-AR were PCR-amplified and subcloned at EcoRI/SalI in pGEX4T1. GST Pull-down and Immunoprecipitation Experiments—For GST pull-down, HEK-293 cells expressing the various constructs grown in 100-mm dishes were lysed in 1 ml of buffer C (20 mM Tris, pH 7.4, 100 mM NaCl, 5 mM EDTA, 1% (w/v) Triton X-100, 5 g/ml aprotinin, 10 g/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride) and centrifugation at 100,000 ϫ g for 30 min at 4 °C. We determined that the biotinylation reagent did not cross the plasma membrane, as shown by the fact that the catalytic subunit of protein kinase A, which is cytoplasmic, could not be detected in streptavidin-Sepharose precipitates (results not shown)
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