Abstract
To find novel cytoplasmic binding partners of the alpha1D-adrenergic receptor (AR), a yeast two-hybrid screen using the alpha1D-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 alpha1D-AR-interacting protein. Alpha-syntrophin specifically recognized the C terminus of alpha1D- but not alpha1A- or alpha1B-ARs. In blot overlay assays, the PDZ domains of syntrophin isoforms alpha, beta1, and beta2 but not gamma1 or gamma2 showed strong selective interactions with the alpha1D-AR C-tail fusion protein. In transfected human embryonic kidney 293 cells, full-length alpha1D- but not alpha1A- or alpha1B-ARs co-immunoprecipitated with syntrophins, and the importance of the receptor C terminus for the alpha1D-AR/syntrophin interaction was confirmed using chimeric receptors. Mutation of the PDZ-interacting motif at the alpha1D-AR C terminus markedly decreased inositol phosphate formation stimulated by norepinephrine but not carbachol in transfected HEK293 cells. This mutation also dramatically decreased alpha1D-AR binding and protein expression. In addition, stable overexpression of alpha-syntrophin significantly increased alpha1D-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 alpha1D-AR signaling and the role of syntrophins in modulating G protein-coupled receptor function.
Highlights
␣1-adrenergic receptor (AR) subtypes are important for the regulation of blood pressure [2, 3]
We have identified several closely related syntrophin family isoforms (␣, 1, and 2) as novel ␣1D-AR binding partners using a combination of yeast two-hybrid screening and biochemical techniques
We report that syntrophins directly interact with ␣1D-ARs through a PDZ domain-mediated interaction
Summary
Materials and Reagents—Materials used in this study were obtained from the following sources. 2 g of purified His6-tagged syntrophin PDZ domain fusion proteins were run on a 4 –20% Tris-glycine SDS-PAGE gel for 1.5–2 h at 125 V and transferred to ProTran nitrocellulose. 8 g of indicated plasmid DNA was mixed with Lipofectamine2000 and serum-free medium at RT for 20 min and added to HEK293 cells growing in a 150-mm tissue culture plate. Membrane Preparation—For radioligand binding, cells grown on 150-mm tissue culture plates were harvested in phosphate-buffered saline (10 mM phosphate buffer, 2.7 mM KCl, 137 mM NaCl, pH 7.4), and membrane preparations were prepared as previously described [18]. Cells were collected by centrifugation at 30,000 ϫ g for 20 min, and membrane preparations were prepared as previously described [13]. KD and Bmax were calculated by nonlinear regression using Prism (GraphPad)
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