Syntrophins Regulate 1 D - Adrenergic Receptors through a PDZ Domain - mediated Interaction

To findnovel cytoplasmic binding partners of the 1D-adrenergic receptor (AR), a yeast two-hybrid screen using the 1D-ARC terminus as bait was performed on a human brain cDNA library. -Syntrophin, a protein containing one PDZ domain and two pleckstrin homology domains, was isolated in this screen as an 1D-AR-interacting protein. -Syntrophin specifically recognized theC terminus of 1Dbut not 1Aor 1B-ARs. In blot overlay assays, the PDZ domains of syntrophin isoforms , 1, and 2 but not 1 or 2 showed strong selective interactions with the 1D-AR C-tail fusion protein. In transfected human embryonic kidney 293 cells, fulllength 1Dbut not 1Aor 1B-ARs co-immunoprecipitated with syntrophins, and the importance of the receptor C terminus for the 1D-AR/syntrophin interaction was confirmed using chimeric receptors. Mutation of the PDZ-interacting motif at the 1D-AR C terminus markedly decreased inositol phosphate formation stimulated by norepinephrine but not carbachol in transfected HEK293 cells. This mutation also dramatically decreased 1D-AR binding and protein expression. In addition, stable overexpression of -syntrophin significantly increased 1D-AR protein expression and binding but did not affect those with a mutated PDZ-interacting motif, suggesting that syntrophin plays an important role in maintaining receptor stability by directly interacting with the receptor PDZ-interacting motif. This direct interaction may provide new information about the regulation of 1D-AR signaling and the role of syntrophins in modulating G protein-coupled receptor function.