Crystal Structure of Arrestin-3 Reveals the Basis of the Difference in Receptor Binding Between Two Non-visual Subtypes

Department of Pharmacology, Vanderbilt University, Nashville, TN 37232, USA.
Journal of Molecular Biology (Impact Factor: 4.33). 02/2011; 406(3):467-78. DOI: 10.1016/j.jmb.2010.12.034
Source: PubMed


Arrestins are multi-functional proteins that regulate signaling and trafficking of the majority of G protein-coupled receptors (GPCRs), as well as sub-cellular localization and activity of many other signaling proteins. We report the first crystal structure of arrestin-3, solved at 3.0 Å resolution. Arrestin-3 is an elongated two-domain molecule with overall fold and key inter-domain interactions that hold the free protein in the basal conformation similar to the other subtypes. Arrestin-3 is the least selective member of the family, binding a wide variety of GPCRs with high affinity and demonstrating lower preference for active phosphorylated forms of the receptors. In contrast to the other three arrestins, part of the receptor-binding surface in the arrestin-3 C-domain does not form a contiguous β-sheet, which is consistent with increased flexibility. By swapping the corresponding elements between arrestin-2 and arrestin-3 we show that the presence of this loose structure is correlated with reduced arrestin selectivity for activated receptors, consistent with a conformational change in this β-sheet upon receptor binding.

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Available from: Vsevolod V Gurevich
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    • "Purified arrestin-3, GST-MKK4, GST-MKK7, His-JNK1α1, His-JNK2α2, and His- JNK3α2. The protocol for purifying arrestin-3 has been described previously (Zhan et al., 2011a; Vishnivetskiy et al., 2014). Protocols for the purification of the other proteins are described in the Support Protocols in the present unit, as are those for studying the activation of MKK4 (p-MKK4) and MKK7 (p-MKK7). "
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    ABSTRACT: Only one out of four mammalian arrestin subtypes, arrestin-3, facilitates the activation of JNK family kinases. Here we describe two different protocols used for elucidating the mechanisms involved. One is based on reconstitution of signaling modules from purified proteins: arrestin-3, MKK4, MKK7, JNK1, JNK2, and JNK3. The main advantage of this method is that it unambiguously establishes which effects are direct because only intended purified proteins are present in these assays. The key drawback is that the upstream-most kinases of these cascades, ASK1 or other MAPKKKs, are not available in purified form, limiting reconstitution to incomplete two-kinase modules. The other approach is used for analyzing the effects of arrestin-3 on JNK activation in intact cells. In this case, signaling modules include ASK1 and/or other MAPKKKs. However, as every cell expresses thousands of different proteins their possible effects on the readout cannot be excluded. Nonetheless, the combination of in vitro reconstitution from purified proteins and cell-based assays makes it possible to elucidate the mechanisms of arrestin-3-dependent activation of JNK family kinases. © 2015 by John Wiley & Sons, Inc. Copyright © 2015 John Wiley & Sons, Inc.
    Full-text · Article · Mar 2015 · Current protocols in pharmacology
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    • "Arrestin-2 and -3 were purified as previously described (Gurevich and Benovic 2000; Zhan et al. 2011). GST fusion proteins on sepharose beads were made up to a total volume of 500 lL in assay buffer (20 mM Tris, pH 7.5, 200 mM NaCl), and different amounts of purified arrestin-2 or arrestin-3 (final concentrations of 40, 200, 500, 1000, 1500 and 2000 ng/mL; 2000 ng/mL is approximately equal to 44 nM) were added, and mixed by rotation at 4°C for different lengths of time (30 min, 1 h or 2.5 h). "
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    ABSTRACT: Phosphorylation is considered a key event in the signalling and regulation of the μ opioid receptor (MOPr). Here we used mass spectroscopy to determine the phosphorylation status of the C-terminal tail of the rat MOPr expressed in HEK-293 cells. Under basal conditions, MOPr is phosphorylated on Ser(363) and Thr(370) , while in the presence of morphine or [D-Ala2, NMe-Phe4, Gly-ol5]-enkephalin (DAMGO), the COOH-terminus is phosphorylated at three additional residues, Ser(356) , Thr(357) , and Ser(375) . Using N-terminal Glutathione S Transferase (GST) fusion proteins of the cytoplasmic, C-terminal tail of MOPr and point mutations of the same, we show that, in vitro, purified G protein-coupled receptor kinase 2 (GRK2) phosphorylates Ser(375) , PKC phosphorylates Ser(363) whilst CaMKII phosphorylates Thr(370) . Phosphorylation of the GST fusion protein of the C-terminal tail of MOPr enhanced its ability to bind arrestin-2 and -3. Hence, our study identifies both the basal and agonist-stimulated phospho-acceptor sites in the C-terminal tail of MOPr, and suggests that the receptor is subject to phosphorylation and hence regulation by multiple protein kinases. © 2012 International Society for Neurochemistry, J. Neurochem. (2012) 10.1111/jnc.12071.
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    • "Pre-activated mutants of both arrestins with C-terminal deletions, arrestin-2-(1–393) and arrestin-3-(1–392), are the least selective in this regard, comparably binding active and inactive ERK2 (Fig. 1C). Arrestin-3 is the most promiscuous in terms of GPCRs it binds, the least selective for active phosphorylated forms of the receptors [21], and appears to be more flexible that arrestin-2 [8]. Truncated mutants are even less selective in receptor binding [32], [33], [36], [38]. "
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    ABSTRACT: Arrestins are multifunctional signaling adaptors originally discovered as proteins that "arrest" G protein activation by G protein-coupled receptors (GPCRs). Recently GPCR complexes with arrestins have been proposed to activate G protein-independent signaling pathways. In particular, arrestin-dependent activation of extracellular signal-regulated kinase 1/2 (ERK1/2) has been demonstrated. Here we have performed in vitro binding assays with pure proteins to demonstrate for the first time that ERK2 directly binds free arrestin-2 and -3, as well as receptor-associated arrestins-1, -2, and -3. In addition, we showed that in COS-7 cells arrestin-2 and -3 association with β(2)-adrenergic receptor (β2AR) significantly enhanced ERK2 binding, but showed little effect on arrestin interactions with the upstream kinases c-Raf1 and MEK1. Arrestins exist in three conformational states: free, receptor-bound, and microtubule-associated. Using conformationally biased arrestin mutants we found that ERK2 preferentially binds two of these: the "constitutively inactive" arrestin-Δ7 mimicking microtubule-bound state and arrestin-3A, a mimic of the receptor-bound conformation. Both rescue arrestin-mediated ERK1/2/activation in arrestin-2/3 double knockout fibroblasts. We also found that arrestin-2-c-Raf1 interaction is enhanced by receptor binding, whereas arrestin-3-c-Raf1 interaction is not.
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