Syntrophins regulate α1D-adrenergic receptors through a PDZ domain-mediated interaction

To find novel cytoplasmic binding partners of the alpha(1D)-adrenergic receptor (AR), a yeast two-hybrid screen using the alpha(1D)-AR C terminus as bait was performed on a human brain cDNA library. alpha-Syntrophin, a protein containing one PDZ domain and two pleckstrin homology domains, was isolated in this screen as an alpha(1D)-AR-interacting protein. alpha-Syntrophin specifically recognized the C terminus of alpha(1D)- but not alpha(1A)- or alpha(1B)-ARs. In blot overlay assays, the PDZ domains of syntrophin isoforms alpha, beta 1, and beta 2 but not gamma 1 or gamma 2 showed strong selective interactions with the alpha(1D)-AR C-tail fusion protein. In transfected human embryonic kidney 293 cells, full-length alpha(1D)- but not alpha(1A)- or alpha(1B)-ARs co-immunoprecipitated with syntrophins, and the importance of the receptor C terminus for the alpha(1D)-AR/syntrophin interaction was confirmed using chimeric receptors. Mutation of the PDZ-interacting motif at the alpha(1D)-AR C terminus markedly decreased inositol phosphate formation stimulated by norepinephrine but not carbachol in transfected HEK293 cells. This mutation also dramatically decreased alpha(1D)-AR binding and protein expression. In addition, stable overexpression of alpha-syntrophin significantly increased alpha(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 alpha(1D)-AR signaling and the role of syntrophins in modulating G protein-coupled receptor function.