Syntrophins Regulate α1D-Adrenergic Receptors through a PDZ Domain-mediated Interaction

Abstract

To find novel cytoplasmic binding partners of the ␣ 1D -adrenergic receptor (AR), a yeast two-hybrid screen using the ␣ 1D -AR C 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 the C terminus of ␣ 1D -but not ␣ 1A -or ␣ 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 ␣ 1D -but not ␣ 1A -or ␣ 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.2 are G protein-coupled receptors that mediate various important physiological functions of norepinephrine (NE) and epinephrine, particularly in the cardiovascular system where they are responsible for regulating vascular tone and peripheral resistance. Three ␣ 1 -AR subtypes have been cloned (␣ 1A -, ␣ 1B -, and ␣ 1D -AR) and display differences in sequence homology and affinities for subtype selective ligands (1). Upon agonist stimulation, all three ␣ 1 -AR subtypes signal through G␣ q/11 to increase phospholipase C activity and intracellular Ca 2ϩ mobilization (1, 2). In addition, recent studies using ␣ 1 -AR transgenic and knock-out mice have now revealed that all three ␣ 1 -AR subtypes are important for the regulation of blood pressure (2, 3). Therefore, it remains unclear if specific functional differences exist between the ␣ 1 -AR subtypes.Increasing evidence now suggests that ␣ 1 -AR subtypes display differences in their ability to interact with specific protein binding partners. The first ␣ 1 -AR subtype-selective binding partner identified was tissue transglutaminase II, which selectively associates with ␣ 1B -and ␣ 1D -ARs (4, 5). Since then, other proteins found to selectively associate with ␣ 1 -ARs include gC1qR (6), adaptor protein complex-2 (7), regulators of G protein signaling-2 (8), and spinophilin (9). These interactions were shown to be important for the signaling (8, 9), trafficking (6),...

The PDZ Protein Mupp1 Promotes Gi Coupling and Signaling of the Mt1 Melatonin Receptor

“…Effect of PDZ10 on MT 1 Internalization and DegradationInteraction of GPCRs with PDZ proteins has been shown to stabilize receptors by inhibiting either their constitutive or agonist-promoted internalization (8,34) or by interfering with receptor degradation (4,5). To study the role of MUPP1 in MT 1 internalization and degradation, we used the isolated PDZ10 domain of MUPP1 as a dominant negative to disrupt the MUPP1/MT 1 interaction.…”

“…Although G␤ subunits typically code for the "LWL" or "IWN" sequence at their C-tails, several G␥ subunits (e.g. G␥ 4 , G␥ 12 , and G␥ 13 ) have the COOH-terminal "TIL" sequence, which corresponds to the class I ((S/T)XL) PDZ domain recognition sequence. Alternatively, MUPP1 and G i may be physically linked through a third protein.…”

“…Binding of PDZ proteins to GPCRs has been reported to primarily regulate subcellular localization, trafficking, and stability of receptors (3). For instance, binding of MUPP1 and syntrophins to the ␥-aminobutyric acid type B (GABA B ) receptor and the ␣ 1D -adrenergic receptor, respectively, significantly increases receptor stability (4,5). In other cases, PDZ scaffolds determine the subcellular localization of GPCRs (6) and receptor endocytosis as shown for PSD-95 and the 5-HT 2A serotonin and ␤ 1 -adrenergic receptors (7,8).…”

GABAB Receptor Association with the PDZ Scaffold Mupp1 Alters Receptor Stability and Function

“…For example, disruption of the PDZ-interacting motif of aquaporin-4 increases the rate of aquaporin-4 degradation (30). Similarly, phosphatase and tensin homolog mutants deficient in PDZ binding have been reported to have reduced stability (31,32), and PDZ proteins of the syntrophin family have been shown to enhance the stability of both ␣ 1D -adrenergic receptors (33) and the transporter ABCA1 (34). The mechanisms underlying PDZ domain-mediated regulation of target protein stability are unclear.…”

α-Dystrobrevin-1 recruits α-catulin to the α 1D -adrenergic receptor/dystrophin-associated protein complex signalosome

“…Recently, we used yeast two-hybrid and proteomic screens to identify GIPs for a clinically important GPCR, the α 1D -adrenergic receptor (AR) (5). A member of the adrenergic family (α 1 , α 2 , β), α 1D -ARs are ubiquitously expressed on blood vessels and are responsible for increasing blood pressure during exercise, injury, stress, or cardiovascular disease (6).…”