Alpha-dystrobrevin-1 recruits alpha-catulin to the alpha1D-adrenergic receptor/dystrophin-associated protein complex signalosome

Abstract

α(1D)-Adrenergic receptors (ARs) are key regulators of cardiovascular system function that increase blood pressure and promote vascular remodeling. Unfortunately, little information exists about the signaling pathways used by this important G protein-coupled receptor (GPCR). We recently discovered that α(1D)-ARs form a "signalosome" with multiple members of the dystrophin-associated protein complex (DAPC) to become functionally expressed at the plasma membrane and bind ligands. However, the molecular mechanism by which the DAPC imparts functionality to the α(1D)-AR signalosome remains a mystery. To test the hypothesis that previously unidentified molecules are recruited to the α(1D)-AR signalosome, we performed an extensive proteomic analysis on each member of the DAPC. Bioinformatic analysis of our proteomic data sets detected a common interacting protein of relatively unknown function, α-catulin. Coimmunoprecipitation and blot overlay assays indicate that α-catulin is directly recruited to the α(1D)-AR signalosome by the C-terminal domain of α-dystrobrevin-1 and not the closely related splice variant α-dystrobrevin-2. Proteomic and biochemical analysis revealed that α-catulin supersensitizes α(1D)-AR functional responses by recruiting effector molecules to the signalosome. Taken together, our study implicates α-catulin as a unique regulator of GPCR signaling and represents a unique expansion of the intricate and continually evolving array of GPCR signaling networks.

Fig. 1.

α-Catulin is a unique member of the α_1D-AR/DAPC signalosome. (A) TAP-tagged α_1D-AR, α/β₁/β₂-syntrophin, and α-DB1/2 were expressed in HEK293 cells, purified, and subjected to LC/MS. Bait proteins are indicated by yellow circles. Identified proteins known to be members of the DAPC are shown in green circles (DMD, dystrophin; UTR, utrophin; DTNA, α-DB; DTNB, β-DB; SNTA, α-syntrophin; SNTB1, β₁-syntrophin; SNTB2, β₂-syntrophin). α-Catulin (CTNAAL1, in red) was identified by our TAP/MS screening as a unique member of the signalosome. (B) TAP-α_1D-AR coimmunoprecipitated endogenous α-catulin, α-catulin-flag, α-DB1, and syntrophins in HEK293 cells.

Fig. 2.

The N terminus of α-catulin interacts directly with the C terminus of α-DB1. (A) TAP-α-DB1 or TAP-α-DB2 were expressed with (+) or without (–) α-catulin-flag in HEK293 cells. TAP-proteins were immunoprecipitated and analyzed by Western blotting as indicated. (B) α-Catulin-flag coimmunoprecipitates α-DB1, dystrophin (DMD), syntrophin, and utrophin in HEK293 cells. (C) Untransfected, TAP-α-DB1, or TAP-α-DB2–expressing HEK293 cell lysate was run on SDS/PAGE, transferred to nitrocellulose, blocked, and incubated with GST-α-catulin expressed and purified from E. coli. Blots were washed and simultaneously Western blotted for GST and either α-DB1 (Top), α-DB2 (Middle), or β-DB (Bottom). Overlays of the GST and dystrobrevin images are shown in the far right column, with GST signals in red and dystrobrevin signals in green. (D) Upper: TAP-α-catulin deletion constructs. Deletion constructs were expressed in HEK293 cells, purified using streptavidin Sepharose, washed, and analyzed by SDS/PAGE. Lower: Blots were Western blotted for both TAP-α-catulin deletions and α-DB1.

Fig. 3.

α-Catulin colocalizes with α-dystrobrevin. HEK293 and mouse quadriceps muscle slices were stained for α-catulin and α-dystrobrevin-1. Blood vessel (BV), nerve bundle (NB), and muscle (M) sections of quadriceps are indicated.

Fig. 4.

α-Catulin supersensitizes α_1D-AR signaling. Effects of α-catulin knockdown or overexpression on H/F-α_1D-AR-6G-syntrophin coupling to IP3 production (A) and ERK1/2 phosphorylation (B) in HEK293 cells. Representative well staining for phospho-ERK is shown. Statistical analysis was performed using an unpaired t test. ***P < 0.001; **P < 0.05. (C) TAP/MS screen of α-catulin interacting proteins purified from HEK293 cells. DAPC proteins are indicated by green circles, and unique proteins are indicated by red circles. (D) TAP-α-catulin coimmunoprecipitated endogenous PLCβ2. (E) Blot overlay of GST-α-catulin indicates that α-catulin binds directly to PLCβ2, although with low affinity. (F) TAP-α-DB1 or TAP-α-DB2 were immunoprecipitated from HEK293 cells, run on SDS/PAGE, and probed for copurification of α-catulin, PLCβ2, and other members of the DAPC. (G) Proposed model of the α_1D-AR/DAPC signalosome, including α-catulin, liprins, and PLC-β2.