S-Antigen: preparation and characterization of site-specific monoclonal antibodies

Background:The ability of arrestin-3 to facilitate activation of JNK1 and JNK2 has never been reported. Results: Arrestin-3 binds JNK1␣1 and JNK2␣2 and promotes their phosphorylation by MKK4 and MKK7 in vitro and in intact cells. Conclusion: Arrestin-3 promotes the activation of ubiquitous JNK1 and JNK2 isoforms. Significance: Arrestin-3 scaffolds MKK4/7-JNK1/2/3 signaling modules and facilitates activation of ubiquitous JNK isoforms.

Section: Methodsmentioning

confidence: 99%

Section: Arrestin-3 Binds Jnk1␣1/jnk2␣2 In Vitro and In Intactmentioning

confidence: 99%

See 1 more Smart Citation

Arrestin-3 Binds c-Jun N-terminal Kinase 1 (JNK1) and JNK2 and Facilitates the Activation of These Ubiquitous JNK Isoforms in Cells via Scaffolding

Kook

Zhan

Kaoud

et al. 2013

“…In a recently proposed model, the inactive conformation of arrestins is stabilized by an intact polar core, which includes residues Asp 26 , and Arg 394 of rat ␤-arrestin 2), as well as a three element hydrophobic interaction involving ␤-strand I of the N terminus, the last ␤-strand XX of the C terminus, and ␣-helix I (15). Upon activation-dependent phosphorylation of a 7MSR, multiple phosphate moieties at the C terminus of the receptor would disrupt the delicate charge network within the polar core, thus releasing the constraints that keep arrestin in the basal state.…”

Section: Requirement For Phosphorylation On Residues In Syntheticmentioning

confidence: 99%

Activation-dependent Conformational Changes in β-Arrestin 2

Xiao

Shenoy

Nobles

et al. 2004

139

122

␤-Arrestins are multifunctional adaptor proteins, which mediate desensitization, endocytosis, and alternate signaling pathways of seven membrane-spanning receptors (7MSRs). Crystal structures of the basal inactive state of visual arrestin (arrestin 1) and ␤-arrestin 1 (arrestin 2) have been resolved. However, little is known about the conformational changes that occur in ␤-arrestins upon binding to the activated phosphorylated receptor. Here we characterize the conformational changes in ␤-arrestin 2 (arrestin 3) by comparing the limited tryptic proteolysis patterns and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) profiles of ␤-arrestin 2 in the presence of a phosphopeptide (V 2 R-pp) derived from the C terminus of the vasopressin type II receptor (V 2 R) or the corresponding nonphosphopeptide (V 2 R-np). V 2 R-pp binds to ␤-arrestin 2 specifically, whereas V 2 R-np does not. Activation of ␤-arrestin 2 upon V 2 R-pp binding involves the release of its C terminus, as indicated by exposure of a previously inaccessible cleavage site, one of the polar core residues Arg 394 , and rearrangement of its N terminus, as indicated by the shielding of a previously accessible cleavage site, residue Arg 8 . Interestingly, binding of the polyanion heparin also leads to release of the C terminus of ␤-arrestin 2; however, heparin and V 2 R-pp have different binding site(s) and/or induce different conformational changes in ␤-arrestin 2. Release of the C terminus from the rest of ␤-arrestin 2 has functional consequences in that it increases the accessibility of a clathrin binding site (previously demonstrated to lie between residues 371 and 379) thereby enhancing clathrin binding to ␤-arrestin 2 by 10-fold. Thus, the V 2 R-pp can activate ␤-arrestin 2 in vitro, most likely mimicking the effects of an activated phosphorylated 7MSR. These results provide the first direct evidence of conformational changes associated with the transition of ␤-arrestin 2 from its basal inactive conformation to its biologically active conformation and establish a system in which receptor-␤-arrestin interactions can be modeled in vitro.Seven membrane-spanning receptors (7MSRs), 1 also referred to as G protein-coupled receptors (GPCRs), constitute the largest known family of cell surface receptors (1, 2). The human genome encodes ϳ1,000 7MSRs, which function primarily in the transmission of diverse signals (including light, odorants, chemoattractants, neurotransmitters, and hormones) from the extracellular environment to the interior of the cell (1, 2). The dynamic sensitivity of 7MSR function is in large part a function of their regulation by the G proteincoupled receptor kinase (GRK)/␤-arrestin system (1, 3). This regulation is accomplished by a two-step process involving the phosphorylation of the receptor, usually at its C terminus, by GRKs, and the subsequent binding of ␤-arrestins, which prevents further receptor activation of G proteins (desensitization) (1, 4). ␤-Arrestin binding to the receptor also f...

“…Standard two-electrode voltage clamp recordings were performed to register Kir3 currents activated by agonist perfusion, as described (26). The expression levels of all forms of arrestin in oocytes were determined by quantitative Western blotting with F4C1 monoclonal anti-arrestin antibody (27) using the corresponding purified arrestins as standards as described (12). Arrestin expression levels were between 1.6 and 4 pmol/g of total protein.…”

Section: Materials-[␥-mentioning

confidence: 99%

Conservation of the Phosphate-sensitive Elements in the Arrestin Family of Proteins

Celver¹,

Vishnivetskiy²,

Chavkin³

et al. 2002

130

188

Arrestins play a key role in the homologous desensitization of G protein-coupled receptors (GPCRs). These cytosolic proteins selectively bind to the agonist-activated and GPCR kinase-phosphorylated forms of the GPCR, precluding its further interaction with the G protein. Certain mutations in visual arrestin yield "constitutively active" proteins that bind with high affinity to the light-activated form of rhodopsin without requiring phosphorylation. The crystal structure of visual arrestin shows that these activating mutations perturb two groups of intramolecular interactions that keep arrestin in its basal (inactive) state. Here we introduced homologous mutations into arrestin2 and arrestin3 and found that the resulting mutants bind to the ␤ 2 -adrenoreceptor in vitro in a phosphorylation-independent fashion. The same mutants effectively desensitize both the ␤ 2 -adrenergic and ␦-opioid receptors in the absence of receptor phosphorylation in Xenopus oocytes. Moreover, the arrestin mutants also desensitize the truncated ␦-opioid receptor from which the C terminus, containing critical phosphorylation sites, has been removed. Conservation of the phosphate-sensitive hot spots in nonvisual arrestins suggests that the overall fold is similar to that of visual arrestin and that the mechanisms whereby receptor-attached phosphates drive arrestin transition into the active binding competent state are conserved throughout the arrestin family of proteins.Signaling by various members of the superfamily of G protein-coupled receptors (GPCRs) 1 is attenuated by a uniform two-step mechanism (1). First, the agonist-activated receptor that catalyzes GDP/GTP exchange on G proteins is specifically phosphorylated by a G protein-coupled receptor kinase (GRK). An arrestin protein then binds to the active phosphoreceptor, which makes further G protein interaction impossible by simple steric exclusion (2). Because of the high affinity of nonvisual arrestins 2 for various components of the trafficking machinery (3-5), the arrestin-receptor complex is then internalized. Arrestin binding also serves to switch GPCR signaling from G proteins to various mitogen-activated protein kinases (6 -8). The loss of active receptor conformation in the endosomes (presumably due to ligand dissociation) facilitates arrestin dissociation, rendering the phosphoreceptor accessible to protein phosphatases. The dephosphorylated receptor can then be recycled back to the plasma membrane (9).Perfectly timed binding and dissociation of arrestins, which are ensured by their remarkable selectivity for the phosphorylated/activated form of their cognate receptors, are equally important for high fidelity of this quenching mechanism. Destabilization of certain intramolecular interactions that support the basal (inactive) arrestin conformation by the activated phosphoreceptor is the mechanistic basis of arrestin selectivity (10,15,19,20). The visual arrestin residues involved in the stabilization of its basal state have been identified by extensive mutagenesis (10 -15,...