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


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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    • "Since small molecule modulators of Lgr5 function have not been reported, we used a prototypical GPCR with better characterized trafficking and signaling modes to validate this system. Most class 1 GPCRs possess a canonical 'DRY' motif at the end of transmembrane-domain three which can stabilize the receptor in an inactive state and is also important in G protein-coupling (Fig. 2a)[21,22]. Mutations to this domain contribute to disease by resulting in constitutive activation and internalization of the receptor[20,23]in addition to misfolded receptor that accumulates in the ER[24]. "
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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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    • "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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