Blood pressure is regulated by an alpha1D-adrenergic receptor/dystrophin signalosome

Abstract

Hypertension is a cardiovascular disease associated with increased plasma catecholamines, overactivation of the sympathetic nervous system, and increased vascular tone and total peripheral resistance. A key regulator of sympathetic nervous system function is the alpha(1D)-adrenergic receptor (AR), which belongs to the adrenergic family of G-protein-coupled receptors (GPCRs). Endogenous catecholamines norepinephrine and epinephrine activate alpha(1D)-ARs on vascular smooth muscle to stimulate vasoconstriction, which increases total peripheral resistance and mean arterial pressure. Indeed, alpha(1D)-AR KO mice display a hypotensive phenotype and are resistant to salt-induced hypertension. Unfortunately, little information exists about how this important GPCR functions because of an inability to obtain functional expression in vitro. Here, we identified the dystrophin proteins, syntrophin, dystrobrevin, and utrophin as essential GPCR-interacting proteins for alpha(1D)-ARs. We found that dystrophins complex with alpha(1D)-AR both in vitro and in vivo to ensure proper functional expression. More importantly, we demonstrate that knock-out of multiple syntrophin isoforms results in the complete loss of alpha(1D)-AR function in mouse aortic smooth muscle cells and abrogation of alpha(1D)-AR-mediated increases in blood pressure. Our findings demonstrate that syntrophin and utrophin associate with alpha(1D)-ARs to create a functional signalosome, which is essential for alpha(1D)-AR regulation of vascular tone and blood pressure.

FIGURE 1.

α_1D-ARs require syntrophin for functional expression in vitro. A, α_1D-AR-GFP localization (green) was examined in HEK293 cells co-stained with markers (expressing dsRed) for mitochondria (MT), peroxisome (PO), or endoplasmic reticulum (ER). B, Myc-α_1D-AR (green) localization with and withoutα-syntrophin-V5. C and D, α-syntrophin specifically increasesα_1D-AR-binding site density. [³H]prazosin radioligand binding was measured in WT orα-syntrophin-overexpressing HEK293 cells co-transfected with α_1D-AR (C) or α_1A-AR (D). E–H, α-syntrophin increases agonist stimulated α_1D-AR coupling to PI hydrolysis (E) and ERK1/2 activation (F) but has no effect on α_1A-AR functional responses (G and H). The results are the means ± S.E. of two to four experiments performed in triplicate.

FIGURE 2.

Characterization of the α_1D-AR/α-syntrophin linker constructs. A, schematic of the α_1D-AR-6G-α-syntrophin fusion construct (α_1D-6G). B–D, the ability of the α_1D-6G and 12G fusion proteins to form functional binding sites was determined by [³H]prazosin saturation radioligand binding (B) and the ability to couple to agonist-stimulated PI hydrolysis (C) and ERK1/2 activation (D) in HEK293 cells. The data were normalized to maximal α_1D-6G linker responses and are the means ± S.E. of two or three experiments performed in triplicate.

FIGURE 3.

The SU domain of α-syntrophin is required for α_1D-AR function. A, schematic of α_1D-6G deletion constructs. Stop codons were introduced before the SU domain (SU N-stop), after the PH2 domain (PH2 C-stop) and before the PH2 domain (PH2 N-stop). B, quantification of agonist-stimulated PI hydrolysis by α_1D-6G, the ΔPDZ-binding motif in α_1D-12G (RETDI → AAAAA), and α_1D-6G deletion constructs. The responses are normalized to maximal responses stimulated by α_1D-6G and are the means ± S.E. of three experiments performed in triplicate.

FIGURE 4.

Characterization of the α_1D-AR/α-syntrophin signalosome. A, HEK293 cells express β₁- and β₂-syntrophin isoforms. HEK293 cell lysate was immunoprecipitated with a pan-syntrophin antibody (p-syn) and was probed with anti-syntrophin isoform specific antibodies. B, syntrophins recruit utrophin into the α_1D-AR signalosome. HEK293 cell lysates expressing TAP-α_1D-AR were immunoprecipitated with streptavidin and blotted for hemagglutinin (located within the TAP-tag), syntrophin isoforms and utrophin. TAP-α_1A-AR is unable to associate with syntrophin/utrophin (middle lane). TAP-α_1D-ARs associate with syntrophins/utrophins in WT cells (left lane), and the interaction is increased in α-syntrophin-overexpressing cells (right lane). IB, immunoblot.

FIGURE 5.

The α_1D-AR/α-syntrophin signalosome forms in vivo. A, mouse α_1D-AR-GFP was immunoprecipitated (IP)/immunoblotted (IB) with antibodies directed against GFP. α_1D-AR-GFP was precipitated from HEK293 cells expressing α_1D-AR-GFP (lane 2) but not in untransfected HEK293 cells (lane 1). B and C, α_1D-AR-GFP was immunoprecipitated from HEK293 cells (lane 2) and blotted for with our in-house rabbit anti-mouse-α_1D-AR antibody (6976P). 6976P recognizes α_1D-AR-GFP in transfected HEK293 cells (B, lane 2) but not in untransfected HEK293 cells (B, lane 1). The ability of 6976P to detect α_1D-AR-GFP was blocked by pretreating the antibody with immunizing peptide (C), confirming its specificity. D, aortic smooth muscle lysates were immunoprecipitated with a pansyntrophin antibody (p-syn) and probed for α-syntrophin (top row), α_1D-AR (middle row) or utrophin (bottom row). L, lysate load on beads; FT, flow through collected; EL, eluate collected from beads.

FIGURE 6.

α- and β₂-Syntrophin are both required for α_1D-AR mediated blood pressure responses. A, resting blood pressure is abrogated in α/β₂-syntrophin KO mice. B, systolic blood pressure and heart rates (HR) were measured by tail cuff in conscious mice. ^* indicates significant difference as determined by unpaired t test (p < 0.05). C, PE-stimulated increases in SBP are eliminated in α/β₂-syntrophin KO mice. The mice were pretreated with saline (S), BMY 7378 (B), or prazosin (Pz) prior to PE injection. All of the recordings are the means ± S.E. of six to nine mice/group.

FIGURE 7.

α_1D-AR mediated Ca²⁺ mobilization requires syntrophins. A, aortic smooth muscle cells express the α- and β₂-syntrophin isoforms. Cell lysates from WT, α-syntrophin, and α/β₂-syntrophin KO mice were probed with anti-syntrophin isoform-specific antibodies. B and C, α_1D-AR functional responses are eliminated in ASMCs from α/β₂-syntrophin KO mice. Freshly dissociated ASMCs were isolated from WT (B) and α/β₂-syntrophin (C) mice, and Ca²⁺ mobilization was measured in response to 100 μm PE and 100 μm UTP (inset). The data were normalized to maximal WT responses. IB, immunoblot.

FIGURE 8.

Proposed model of the α_1D-AR/syntrophin signalosome. Syntrophins anchor α_1D-AR at the plasma membrane through interactions with dystrophin-utrophin and dystrobrevin. The dystrophin-utrophin complex can bind up to four syntrophins, allowing other syntrophins in the complex to scaffold additional regulators of signal transduction in close proximity to α_1D-AR (i.e. PMCA, nNOS, TRPC, PLCβ3, and/or RGS11).