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ABSTRACT: α(1D)-Adrenergic receptors, key regulators of cardiovascular system function, are organized as a multi-protein complex in the plasma membrane. Using a Type-I PDZ-binding motif in their distal C-terminal domain, α(1D)-ARs associate with syntrophins and dystrophin-associated protein complex (DAPC) members utrophin, dystrobrevin and α-catulin. Three of the five syntrophin isoforms (α, β(1) and β(2)) interact with α(1D)-ARs and our previous studies suggest multiple isoforms are required for proper α(1D)-AR function in vivo. This study determined the contribution of each specific syntrophin isoform to α(1D)-AR function. Radioligand binding experiments reveal α-syntrophin enhances α(1D)-AR binding site density, while phosphoinositol and ERK1/2 signaling assays indicate β(2)-syntrophin augments full and partial agonist efficacy for coupling to downstream signaling mechanisms. The results of this study provide clear evidence that the cytosolic components within the α(1D)-AR/DAPC signalosome significantly alter the pharmacological properties of α(1)-AR ligands in vitro.
Biochemical and Biophysical Research Communications 08/2011; 412(4):596-601. · 2.48 Impact Factor
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ABSTRACT: Muscleblind-like 3 (MBNL3) belongs to a family of RNA binding proteins that regulate alternative splicing. We have generated a set of monoclonal antibodies (MAbs) against mouse MBNL3, three of which do not cross-react with the other muscleblind-like (MBNL) proteins, MBNL1 and MBNL2. Epitope mapping revealed that MAbs P1C7, P1E7, SP1C2, and P2E6 recognize distinct, non-overlapping segments of the MBNL3 polypeptide sequence. Immunohistochemical staining of proliferating muscle precursor cells localized MBNL3 to the nucleus in a punctate pattern, characteristic of subcellular structures in the nucleus enriched in pre-messenger RNA splicing factors. Although MBNL3 did not co-localize with SC35 and PSP1 (widely used markers of splicing speckles and paraspeckles), the punctate localization pattern of MBNL3 within interchromatin regions of the nucleus is highly predictive of proteins involved in pre-mRNA processing. Monoclonal antibodies specific for mouse MBNL3 will facilitate further investigation of the expression pattern and unique functions of this splicing factor during development and in different adult mouse tissues.
Hybridoma (2005) 04/2011; 30(2):181-8. · 0.42 Impact Factor
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John S. Lyssand,
Jennifer L. Whiting, Kyung-Soon Lee,
Ryan Kastl,
Jennifer L. Wacker,
Michael R. Bruchas,
Mayumi Miyatake,
Lorene K. Langeberg,
Charles Chavkin,
John D. Scott,
Richard G. Gardner,
Marvin E. Adams,
Chris Hague
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ABSTRACT: α1D-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 α1D-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 α1D-AR signalosome remains a mystery. To test the hypothesis that previously unidentified molecules are recruited to the α1D-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 α1D-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 α1D-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.
Proceedings of the National Academy of Sciences 12/2010; 107(50):21854-21859. · 9.68 Impact Factor
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John S Lyssand,
Jennifer L Whiting, Kyung-Soon Lee,
Ryan Kastl,
Jennifer L Wacker,
Michael R Bruchas,
Mayumi Miyatake,
Lorene K Langeberg,
Charles Chavkin,
John D Scott,
Richard G Gardner,
Marvin E Adams,
Chris Hague
[show abstract]
[hide abstract]
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.
Proceedings of the National Academy of Sciences 11/2010; 107(50):21854-9. · 9.68 Impact Factor
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ABSTRACT: Mammalian MBNL (muscleblind-like) proteins are regulators of alternative splicing and have been implicated in myotonic dystrophy, the most common form of adult onset muscular dystrophy. MBNL3 functions as an inhibitor of muscle differentiation and is expressed in proliferating muscle precursor cells but not in differentiated skeletal muscle. Here we demonstrate that MBNL3 regulates the splicing pattern of the muscle transcription factor myocyte enhancer factor 2 (Mef2) by promoting exclusion of the alternatively spliced β-exon. Expression of the transcriptionally more active (+)β isoform of Mef2D was sufficient to overcome the inhibitory effects of MBNL3 on muscle differentiation. These data suggest that MBNL3 antagonizes muscle differentiation by disrupting Mef2 β-exon splicing. MBNL3 regulates Mef2D splicing by directly binding to intron 7 downstream of the alternatively spliced exon in the pre-mRNA. The RNA binding activity of MBNL3 requires the CX(7)CX(4-6)CX(3)H zinc finger domains. Using a cell culture model of myotonic dystrophy and myotonic dystrophy patient tissue, we have evidence that expression of CUG expanded RNAs can lead to an increase in MBNL3 expression and a decrease in Mef2D β-exon splicing. These studies suggest that elevating MBNL3 activity in myogenic cells could lead to muscle degeneration disorders such as myotonic dystrophy.
Journal of Biological Chemistry 10/2010; 285(44):33779-87. · 4.77 Impact Factor
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ABSTRACT: Muscle differentiation is controlled by positive and negative signals. While much attention has been placed on proteins that promote muscle formation, the importance of negative regulators has been underemphasized. MBNL3/CHCR belongs to the muscleblind family of Cys3His zinc finger proteins implicated in myotonic dystrophy. MBNL3 is expressed in myoblasts, muscle precursor cells, and during the early stages of myogenesis, but is detected at very low levels in terminally differentiated myotubes. Constitutive expression of MBNL3 inhibits myotube formation and antagonizes myogenin and myosin heavy chain expression. To identify MBNL3 target genes, we compared the expression profile of C2C12 mouse myoblasts that constitutively express MBNL3 with control cells. From the 15,247 genes represented on the DNA microarray, classification by biological function indicated that genes involved in muscle development/contraction and cell adhesion were down-regulated by MBNL3 expression. mRNA and protein levels for the muscle transcription factor MyoD and E-box regulated transcription were reduced in C2C12-MBNL3 expressing cells. We hypothesize that MBNL3 serves to antagonize muscle differentiation by suppressing MyoD expression levels to prevent unwanted myogenic gene transcription. These findings are the first indication that a mammalian muscleblind-like (MBNL) protein plays a regulatory role in muscle differentiation under nonpathogenic conditions.
Differentiation 04/2008; 76(3):299-309. · 2.81 Impact Factor
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ABSTRACT: Muscleblind-like (MBNL) proteins are believed to be regulators of myogenesis and are implicated in myotonic dystrophy. While Drosophila melanogaster muscleblind is required for terminal muscle differentiation, mammalian MBNL3 functions as an inhibitor of myogenesis. In this study, we analyzed the expression pattern of MBNL3 in different adult mouse tissues and tissue culture cells. MBNL3 transcript is enriched in the lung, spleen, and testis and not in heart and skeletal muscle. By Western blotting, we found that MBNL3 was expressed in C2C12 myoblasts and ts13 myofibroblasts, but was detected at significantly lower levels in fibroblasts. MBNL3 protein levels decreased when cells were shifted to muscle differentiation conditions, but the closely related MBNL1 protein was unaffected. These results suggest that myoblasts and fibroblasts respond to differentiation conditions by activating signaling pathways that repress MBNL3 but not MBNL1 expression.
Biochemical and Biophysical Research Communications 10/2007; 361(1):151-5. · 2.48 Impact Factor
