A β-Arrestin Binding Determinant Common to the Second Intracellular Loops of Rhodopsin Family G Protein-coupled Receptors

Duke University, Durham, North Carolina, United States
Journal of Biological Chemistry (Impact Factor: 4.57). 03/2006; 281(5):2932-8. DOI: 10.1074/jbc.M508074200
Source: PubMed

ABSTRACT beta-Arrestins have been shown to inhibit competitively G protein-dependent signaling and to mediate endocytosis for many of the hundreds of nonvisual rhodopsin family G protein-coupled receptors (GPCR). An open question of fundamental importance concerning the regulation of signal transduction of several hundred rhodopsin-like GPCRs is how these receptors of limited sequence homology, when considered in toto, can all recruit and activate the two highly conserved beta-arrestin proteins as part of their signaling/desensitization process. Although the serine and threonine residues that form GPCR kinase phosphorylation sites are common beta-arrestin-associated receptor determinants regulating receptor desensitization and internalization, the agonist-activated conformation of a GPCR probably reveals the most fundamental determinant mediating the GPCR and arrestin interaction. Here we identified a beta-arrestin binding determinant common to the rhodopsin family GPCRs formed from the proximal 10 residues of the second intracellular loop. We demonstrated by both gain and loss of function studies for the serotonin 2C, beta2-adrenergic, alpha2a)adrenergic, and neuropeptide Y type 2 receptors that the highly conserved amino acids, proline and alanine, naturally occurring in rhodopsin family receptors six residues distal to the highly conserved second loop DRY motif regulate beta-arrestin binding and beta-arrestin-mediated internalization. In particular, as demonstrated for the beta2 AR, this occurs independently of changes in GPCR kinase phosphorylation. These results suggest that a GPCR conformation directed by the second intracellular loop, likely using the loop itself as a binding patch, may function as a switch for transitioning beta-arrestin from its inactive form to its active receptor-binding state.

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    • "While internalization of the edited receptors can be induced by agonist stimulation, a study by the Caron laboratory demonstrated that the unedited version of the 5-HT2CR is constitutively internalized due to its interactions with β-arrestins and that this receptor is highly associated with βarr2-GFP even in the absence of agonist (Marion et al., 2004). Interestingly, mutation of proline 158 in the second intracellular loop of the receptor to an alanine relocalizes the receptor to the cellular membrane and dramatically decreases its constitutive interactions with β-arrestin2 (Marion et al., 2006). These studies demonstrate that the 5-HT2CR is capable of binding to β-arrestins in vitro and implicate that β-arrestins are responsible for the differences in constitutive activity observed for the various 5-HT2CR edited products. "
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    ABSTRACT: Serotonin receptors are the product of 15 distinct genes, 14 of which are G protein-coupled receptors. These receptors are expressed in a wide range of cell types, including distinct neuronal populations, and promote diverse functional responses in multiple organ systems. These receptors are important for mediating the in vivo effects of their cognate neurotransmitter, serotonin, as well as the endogenous tryptamines. In addition, the actions of many drugs are mediated, either directly or indirectly, through serotonin receptors, including antidepressants, antipsychotics, anxiolytics, sleep aids, migraine therapies, gastrointestinal therapeutics and hallucinogenic drugs. It is becoming increasingly evident that serotonin receptors can engage in differential signaling that is determined by the chemical nature of the ligand and that ligands that demonstrate a predilection for inducing a particular signaling cascade are considered to have "functional selectivity". The elucidation of the cellular signaling pathways that mediate the physiological responses to serotonin and other agonists is an active area of investigation and will be an onward-looking focal point for determining how to effectively and selectively promote beneficial serotonergic mimicry while avoiding unwanted clinical side effects. This review highlights the modulation of serotonin 2A, 2C, and four receptors by β-arrestins, which may represent a fulcrum for biasing receptor responsiveness in vivo.
    Critical Reviews in Biochemistry and Molecular Biology 10/2010; 45(6):555-66. DOI:10.3109/10409238.2010.516741 · 7.71 Impact Factor
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    • "M. Labasque et al. Molecular Biology of the Cell 4648 tor has identified a critical ␤-arrestin binding determinant common to the rhodopsin family GPCRs formed by the proximal 10 residues of the second intracellular loop (Marion et al., 2006), whereas the C-terminal tail was proposed to govern the stability of the receptor–␤-arrestin complex (Tohgo et al., 2003). Accordingly, CaM bound to the juxtamembrane region of the 5-HT 2C receptor C-terminus might function to stabilize 5-HT 2C receptor–␤-arrestin interaction . "
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    ABSTRACT: The serotonin (5-hydroxytryptamine; 5-HT)(2C) receptor is a G protein-coupled receptor (GPCR) exclusively expressed in CNS that has been implicated in numerous brain disorders, including anxio-depressive states. Like many GPCRs, 5-HT(2C) receptors physically interact with a variety of intracellular proteins in addition to G proteins. Here, we show that calmodulin (CaM) binds to a prototypic Ca(2+)-dependent "1-10" CaM-binding motif located in the proximal region of the 5-HT(2C) receptor C-terminus upon receptor activation by 5-HT. Mutation of this motif inhibited both beta-arrestin recruitment by 5-HT(2C) receptor and receptor-operated extracellular signal-regulated kinase (ERK) 1,2 signaling in human embryonic kidney-293 cells, which was independent of G proteins and dependent on beta-arrestins. A similar inhibition was observed in cells expressing a dominant-negative CaM or depleted of CaM by RNA interference. Expression of the CaM mutant also prevented receptor-mediated ERK1,2 phosphorylation in cultured cortical neurons and choroid plexus epithelial cells that endogenously express 5-HT(2C) receptors. Collectively, these findings demonstrate that physical interaction of CaM with recombinant and native 5-HT(2C) receptors is critical for G protein-independent, arrestin-dependent receptor signaling. This signaling pathway might be involved in neurogenesis induced by chronic treatment with 5-HT(2C) receptor agonists and their antidepressant-like activity.
    Molecular biology of the cell 10/2008; 19(11):4640-50. DOI:10.1091/mbc.E08-04-0422 · 4.47 Impact Factor
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    • "Although both phosphorylated and nonphosphorylated residues contribute to the arrestin binding site (Gurevich and Gurevich, 2006), and differential phosphorylation of D 2 and D 3 receptors by GPCR kinases is likely to contribute to the differential affinity of the subtypes for arrestins (Kim et al., 2001; Cho et al., 2006), the finding that nonphosphorylated GST fusion proteins also display the preference of arrestin for the D 2 receptor over the D 3 receptor suggests that arrestin binding to phosphorylated residues does not entirely explain the preference for the D 2 receptor, and that nonphosphorylated residues that are revealed by receptor activation or by phosphorylation of other sites on the receptor also contribute to subtype selectivity. The D 2 -IC2 fragment evaluated in this study can be roughly divided into three segments: the proximal half that includes the cytoplasmic end of the third transmembrane helix and is highly conserved, with only two residues that are not identical or similar to the D 3 -IC2, as well as a shared proline residue that is an important determinant of arrestin binding in rhodopsinclass GPCRs (Raman et al., 1999; Marion et al., 2006), four residues with no homology to a stretch of six residues in the same location of the D 3 receptor, and the distal segment that includes the beginning of the fourth transmembrane helix, with only two nonhomologous residues and a potential site of phosphorylation by protein kinase C. Using chimeric GST-IC2 constructs , we first determined that the two segments comprising the distal half of IC2 contain determinants of the selective binding of arrestin3 to D 2 -IC2. Although some substitutions within the first of these two segments dramatically decreased the binding of arrestin to D 2 -IC2, we did not identify any substitution that enhanced the binding of arrestin to D 3 -IC2. "
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    ABSTRACT: Dopamine D(2) and D(3) receptors are similar subtypes with distinct interactions with arrestins; the D(3) receptor mediates less agonist-induced translocation of arrestins than the D(2) receptor. The goals of this study were to compare nonphosphorylated arrestin-binding determinants in the second intracellular domain (IC2) of the D(2) and D(3) receptors to identify residues that contribute to the differential binding of arrestin to the subtypes. Arrestin 3 bound to glutathione transferase (GST) fusion proteins of the D(2) receptor IC2 more avidly than to the D(3) receptor IC2. Mutagenesis of the fusion proteins identified a residue at the C terminus of IC2, Lys149, that was important for the preferential binding of arrestin 3 to D(2)-IC2; arrestin binding to D(2)-IC2-K149C was greatly decreased compared with wild-type D(2)-IC2, whereas binding to the reciprocal mutant D(3)-IC2-C147K was enhanced compared with wild-type D(3)-IC2. Mutating this lysine in the full-length D(2) receptor to cysteine decreased the ability of the D(2) receptor to mediate agonist-induced arrestin 3 translocation to the membrane and decreased agonist-induced receptor internalization in human embryonic kidney 293 cells. The reciprocal mutation in the D(3) receptor increased receptor-mediated translocation of arrestin 3 without affecting agonist-induced receptor internalization. G protein-coupled receptor crystal structures suggest that Lys149, at the junction of IC2 and the fourth membrane-spanning helix, has intramolecular interactions that contribute to maintaining an inactive receptor state. It is suggested that the preferential agonist-induced binding of arrestin3 to the D(2) receptor over the D(3) receptor is due in part to Lys149, which could be exposed as a result of receptor activation.
    Molecular pharmacology 10/2008; 75(1):19-26. DOI:10.1124/mol.108.050542 · 4.13 Impact Factor
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