Purification notesPurified from rabbit serum by sequential epitope-specific chromatography. The antibody has been negatively preadsorbed using a non-phosphopeptide corresponding to the site of phosphorylation to remove antibody that is reactive with non-phosphorylated beta-arrestin-1 protein. The final product is generated by affinity chromatography using a beta Arrestin 1 derived peptide that is phosphorylated at serine 412.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 0.1 - 1 µg/ml. Detects a band of approximately 55 kDa.
FunctionFunctions in regulating agonist-mediated G-protein coupled receptor (GPCR) signaling by mediating both receptor desensitization and resensitization processes. During homologous desensitization, beta-arrestins bind to the GPRK-phosphorylated receptor and sterically preclude its coupling to the cognate G-protein; the binding appears to require additional receptor determinants exposed only in the active receptor conformation. The beta-arrestins target many receptors for internalization by acting as endocytic adapters (CLASPs, clathrin-associated sorting proteins) and recruiting the GPRCs to the adapter protein 2 complex 2 (AP-2) in clathrin-coated pits (CCPs). However, the extent of beta-arrestin involvement appears to vary significantly depending on the receptor, agonist and cell type. Internalized arrestin-receptor complexes traffic to intracellular endosomes, where they remain uncoupled from G-proteins. Two different modes of arrestin-mediated internalization occur. Class A receptors, like ADRB2, OPRM1, ENDRA, D1AR and ADRA1B dissociate from beta-arrestin at or near the plasma membrane and undergo rapid recycling. Class B receptors, like AVPR2, AGTR1, NTSR1, TRHR and TACR1 internalize as a complex with arrestin and traffic with it to endosomal vesicles, presumably as desensitized receptors, for extended periods of time. Receptor resensitization then requires that receptor-bound arrestin is removed so that the receptor can be dephosphorylated and returned to the plasma membrane. Involved in internalization of P2RY4 and UTP-stimulated internalization of P2RY2. Involved in phopshorylation-dependent internalization of OPRD1 ands subsequent recycling. Involved in the degradation of cAMP by recruiting cAMP phosphodiesterases to ligand-activated receptors. Beta-arrestins function as multivalent adapter proteins that can switch the GPCR from a G-protein signaling mode that transmits short-lived signals from the plasma membrane via small molecule second messengers and ion channels to a beta-arrestin signaling mode that transmits a distinct set of signals that are initiated as the receptor internalizes and transits the intracellular compartment. Acts as signaling scaffold for MAPK pathways such as MAPK1/3 (ERK1/2). ERK1/2 activated by the beta-arrestin scaffold is largely excluded from the nucleus and confined to cytoplasmic locations such as endocytic vesicles, also called beta-arrestin signalosomes. Recruits c-Src/SRC to ADRB2 resulting in ERK activation. GPCRs for which the beta-arrestin-mediated signaling relies on both ARRB1 and ARRB2 (codependent regulation) include ADRB2, F2RL1 and PTH1R. For some GPCRs the beta-arrestin-mediated signaling relies on either ARRB1 or ARRB2 and is inhibited by the other respective beta-arrestin form (reciprocal regulation). Inhibits ERK1/2 signaling in AGTR1- and AVPR2-mediated activation (reciprocal regulation). Is required for SP-stimulated endocytosis of NK1R and recruits c-Src/SRC to internalized NK1R resulting in ERK1/2 activation, which is required for the antiapoptotic effects of SP. Is involved in proteinase-activated F2RL1-mediated ERK activity. Acts as signaling scaffold for the AKT1 pathway. Is involved in alpha-thrombin-stimulated AKT1 signaling. Is involved in IGF1-stimulated AKT1 signaling leading to increased protection from apoptosis. Involved in activation of the p38 MAPK signaling pathway and in actin bundle formation. Involved in F2RL1-mediated cytoskeletal rearrangement and chemotaxis. Involved in AGTR1-mediated stress fiber formation by acting together with GNAQ to activate RHOA. Appears to function as signaling scaffold involved in regulation of MIP-1-beta-stimulated CCR5-dependent chemotaxis. Involved in attenuation of NF-kappa-B-dependent transcription in response to GPCR or cytokine stimulation by interacting with and stabilizing CHUK. May serve as nuclear messenger for GPCRs. Involved in OPRD1-stimulated transcriptional regulation by translocating to CDKN1B and FOS promoter regions and recruiting EP300 resulting in acetylation of histone H4. Involved in regulation of LEF1 transcriptional activity via interaction with DVL1 and/or DVL2 Also involved in regulation of receptors others than GPCRs. Involved in Toll-like receptor and IL-1 receptor signaling through the interaction with TRAF6 which prevents TRAF6 autoubiquitination and oligomerization required for activation of NF-kappa-B and JUN. Binds phosphoinositides. Binds inositolhexakisphosphate (InsP6).
Sequence similaritiesBelongs to the arrestin family.
DomainThe [DE]-X(1,2)-F-X-X-[FL]-X-X-X-R motif mediates interaction the AP-2 complex subunit AP2B1 (By similarity). Binding to phosphorylated GPCRs induces a conformationanl change that exposes the motif to the surface. The N-terminus binds InsP6 with low affinity. The C-terminus binds InsP6 with high affinity.
Post-translational modificationsConstitutively phosphorylated at Ser-412 in the cytoplasm. At the plasma membrane, is rapidly dephosphorylated, a process that is required for clathrin binding and ADRB2 endocytosis but not for ADRB2 binding and desensitization. Once internalized, is rephosphorylated. The ubiquitination status appears to regulate the formation and trafficking of beta-arrestin-GPCR complexes and signaling. Ubiquitination appears to occur GPCR-specific. Ubiquitinated by MDM2; the ubiquitination is required for rapid internalization of ADRB2. Deubiquitinated by USP33; the deubiquitination leads to a dissociation of the beta-arrestin-GPCR complex. Stimulation of a class A GPCR, such as ADRB2, induces transient ubiquitination and subsequently promotes association with USP33.
Cellular localizationCytoplasm. Nucleus. Cell membrane. Membrane > clathrin-coated pit. Cell projection > pseudopodium. Cytoplasmic vesicle. Translocates to the plasma membrane and colocalizes with antagonist-stimulated GPCRs. The monomeric form is predominantly located in the nucleus. The oligomeric form is located in the cytoplasm. Translocates to the nucleus upon stimulation of OPRD1.
Western blot - beta Arrestin 1 (phospho S412) antibody (ab4472)
Extracts prepared from PC12 cells were resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF. Membranes were blocked with a 5% BSA-TBST buffer overnight at 4°C, then were incubated with
0.75 µg/mL phospho beta Arrestin 1 (Ser 412) antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1), the non-phosphopeptide corresponding to the immunogen (2), a generic phospho-serine containing peptide (3), or, the phosphopeptide immunogen (4). After washing, membranes were incubated with goat F(ab’)2 anti-rabbit IgG alkaline phosphatase and signals were detected using the Tropix WesternStar method.
Extracts and membranes were prepared as above. The membrane was either not treated (5, 7) or treated (6, 8) with PP2A phosphatase, then incubated with beta Arrestin 1 pan antibody (5, 6), or 0.75 µg/mL phospho beta Arrestin 1 (Ser 412 antibody (7, 8) for two hours at room temperature. After washing, membranes were incubated with goat F(ab’)2 anti-rabbit IgG alkaline phosphatase and signals were detected using the Tropix WesternStar method. The data show that only the peptide corresponding to phospho beta Arrestin 1 (Ser 412) blocks the antibody signal, demonstrating the specificity of the antibody. The data also show that phosphatase stripping eliminates the signal produced by the phospho antibody, but not the pan antibody, verifying that it is phosphospecific.
References for Anti-beta Arrestin 1 (phospho S412) antibody (ab4472)
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