Syntrophins regulate alpha1D-adrenergic receptors through a PDZ domain-mediated interaction.
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.
Chen
Zhongjian
Z
Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Hague
Chris
C
Hall
Randy A
RA
Minneman
Kenneth P
KP
D000818:Animals; D001921:Brain / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism; D049029:Dystrophin-Associated Proteins / Q000737:chemistry / Q000502:physiology*; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry; D051379:Mice; D009124:Muscle Proteins / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018340:Receptors, Adrenergic, alpha-1 / Q000502:physiology*; D020798:Two-Hybrid System Techniques
C547719:ADRA1D protein, human; C547720:Adra1d protein, mouse; D002135:Calcium-Binding Proteins; D049029:Dystrophin-Associated Proteins; D008565:Membrane Proteins; D009124:Muscle Proteins; D018340:Receptors, Adrenergic, alpha-1; C091824:syntrophin; C091825:syntrophin alpha1