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

Abstract

To find novel cytoplasmic binding partners of the α1D- adrenergic receptor (AR), a yeast two-hybrid screen using the α1D-AR C terminus as bait was performed on a human brain cDNA library. α-Syntrophin, a protein containing one PDZ domain and two pleckstrin homology domains, was isolated in this screen as an α1D-AR-interacting protein. α-Syntrophin specifically recognized the C terminus of α1D- but not α1A- or α1B-ARs. In blot overlay assays, the PDZ domains of syntrophin isoforms α, β1, and β2 but not γ1 or γ2 showed strong selective interactions with the α1D-AR C-tail fusion protein. In transfected human embryonic kidney 293 cells, full-length α1D- but not α1A- or α1B-ARs co-immunoprecipitated with syntrophins, and the importance of the receptor C terminus for the α1D-AR/syntrophin interaction was confirmed using chimeric receptors. Mutation of the PDZ-interacting motif at the α1D-AR C terminus markedly decreased inositol phosphate formation stimulated by norepinephrine but not carbachol in transfected HEK293 cells. This mutation also dramatically decreased α1D-AR binding and protein expression. In addition, stable overexpression of α-syntrophin significantly increased α1D-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 α1D-AR signaling and the role of syntrophins in modulating G protein-coupled receptor function. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.