Elsevier

Cellular Signalling

Volume 26, Issue 7, July 2014, Pages 1523-1531
Cellular Signalling

Mutations in arrestin-3 differentially affect binding to neuropeptide Y receptor subtypes

https://doi.org/10.1016/j.cellsig.2014.03.019Get rights and content

Highlights

  • Receptor specificity of arrestins can be enhanced by targeted mutagenesis.

  • Arrestins selective for neuropeptide Y1 over Y2 receptor were developed.

  • Mutations differentially affect agonist-dependent and -independent recruitment.

  • Receptor-specific non-visual arrestins are novel tools for research and therapy.

Abstract

Based on the identification of residues that determine receptor selectivity in arrestins and the phylogenetic analysis of the arrestin (arr) family, we introduced fifteen mutations of receptor-discriminator residues in arr-3, which were identified previously using mutagenesis, in vitro binding, and BRET-based recruitment assay in intact cells. The effects of these mutations were tested using neuropeptide Y receptors Y1R and Y2R. NPY-elicited arr-3 recruitment to Y1R was not affected by these mutations, or even alanine substitution of all ten residues (arr-3-NCA), which prevented arr-3 binding to other receptors tested so far. However, NCA and two other mutations prevented agonist-independent arr-3 pre-docking to Y1R. In contrast, eight out of 15 mutations significantly reduced agonist-dependent arr-3 recruitment to Y2R. NCA eliminated arr-3 binding to active Y2R, whereas Tyr239Thr reduced it ~ 7-fold. Thus, manipulation of key residues on the receptor-binding surface generates arr-3 with high preference for Y1R over Y2R. Several mutations differentially affect arr-3 pre-docking and agonist-induced recruitment. Thus, arr-3 recruitment to the receptor involves several mechanistically distinct steps. Targeted mutagenesis can fine-tune arrestins directing them to specific receptors and particular activation states of the same receptor.

Introduction

G protein-coupled receptors (GPCRs) are high value drug targets, with almost half of the marketed drugs acting on these cell surface proteins [1], [2]. The demand for specific tools for molecular intervention keeps growing, with the emphasis on minimizing side effects and expanding the repertoire of therapeutically addressable GPCRs. The identification of arrestins as key regulators in GPCR desensitization and internalization and mediators of G protein-independent signaling opened new possibilities for the manipulation of receptor function. Arrestins bind activated and phosphorylated GPCRs and block receptor–G-protein interaction, while serving as adaptors for key components of the endocytic machinery and numerous signaling proteins [3], [4]. The two visual subtypes, arrestin-11 (arr-1) and arrestin-4 (arr-4), are expressed exclusively in photoreceptors and pinealocytes, and bind rhodopsin and cone opsins [5], [6]. In contrast, two ubiquitously expressed non-visual subtypes, arrestin-2 (arr-2) and arrestin-3 (arr-3), are fairly promiscuous and interact with a huge range of GPCRs [3]. Recent advances in our understanding of the molecular basis of receptor specificity of arrestin proteins pave the way to targeted construction of reengineered receptor-specific arrestins [7], [8].

Here we tested the interactions of mutants of the non-visual arr-3 and two members of the neuropeptide Y (NPY) receptor family. The NPY system plays a central role in the regulation of energy homeostasis and is involved in the pathophysiology of cancer progression, obesity, mood disorders, and epilepsy [9]. This makes the neuropeptide Y system a desirable drug target for future therapies. NPY receptors respond to three endogenous peptidic agonists, NPY, peptide YY (PYY) and the pancreatic polypeptide (PP). Neuropeptide Y receptor family includes four subtypes in humans: Y1R, Y2R, Y4R, and Y5R [10]. Y receptors belong to class A of the GPCR superfamily, coupled to Gi/o proteins [11], and are expressed in numerous tissues including the brain, blood vessels, heart, and gastrointestinal tract [12], [13], [14], [15], [16]. They have overlapping binding profiles for their endogenous ligands with Y1R, Y2R, and Y5R binding NPY and PYY with high affinity, whereas Y4R prefers PP [17].

In this complex multiligand–multireceptor system Y1R and Y2R occupy a distinct position. They are frequently expressed in the same tissues [18], where they can induce either synergistic or antagonizing effects on food intake, anxiety, and depression [19], [20]. Antagonism is thought to be due to the pre-synaptic expression of Y2R in NPY-ergic neurons, where it functions as an inhibitory autoreceptor blocking NPY release and thus Y1R- and Y5R-mediated effects [21], as revealed by using Y1R- and Y2R-selective agonists and antagonists. However, as Y receptor knockout models showed inconclusive results [20], [22], subtype selective arrestins would be valuable tools to unravel phenotypes assigned to either Y1R- or Y2R-mediated effects.

Unlike the Y5R, both Y1R and Y2R undergo arr-3-dependent internalization, which terminates their G-protein-mediated signaling. Arr-3 binding motifs in these receptors have been recently identified. Like in many GPCRs, these motifs include C-terminal serine/threonine clusters which serve as phosphorylation substrates [23], [24], [25] for G protein-coupled receptor kinases (GRKs [26]). Even though phosphorylated serines and threonines favor the interaction of non-visual arrestins with their cognate receptors, previous studies showed that phosphorylation does not contribute to the arrestin receptor specificity and is not even mandatory for all arrestin–GPCR interactions [27], [28], [29]. Although arrestins engage an extensive surface of the receptor, only a few residues on the concave sides of the arrestin N- and C-domains determine its receptor specificity [28]. This finding made it possible to construct the mutants of naturally promiscuous arr-3 that are specific for GPCR groups or single receptor subtypes [30]. Importantly, these studies on D1 and D2 dopamine, β2-adrenergic (β2AR) and M2 muscarinic receptors showed that mutations of arr-3 similarly affected the interactions with activated phosphorylated receptors and agonist-independent arrestin pre-docking [30]. This supports the idea that arrestins pre-select their target receptors before they become active and phosphorylated, enabling the use of reengineered arrestins for the manipulation of GPCR functions. Here we report the identification of key arr-3 residues that discriminate between Y1 and Y2 receptors and their functional states.

Section snippets

Materials

Porcine NPY (pNPY) was produced by automated solid phase peptide synthesis using the Fmoc/tBu (9-fluorenylmethoxycarbonyl-tert-butyl) strategy, as described [31]. [3H] myo-inositol was from GE Healthcare Europe GmbH (Braunschweig, Germany). Restriction endonucleases and other DNA modifying enzymes were from New England Biolabs (Ipswich, MA). Cell culture reagents and media were from Mediatech-Corning (Manassas, VA), Life-technologies (Carlsbad, CA), or PAA Laboratories GmbH (Pasching, Austria).

Renilla luciferase (RLuc8) tagged neuropeptide Y receptors are functional

Agonist-driven arrestin recruitment studies require fluorophore or luminogen-tagged receptors, which must be functional. The functionality of fused receptors is characterized by ligand affinity, expression at the cell membrane, and agonist potency. To test the functionality of Y1R and Y2R RLuc constructs, we measured the accumulation of [3H]-inositol phosphates in cells co-transfected with a chimeric Gαq that couples to receptors that interact with Gi proteins [49]. This procedure bypasses the

Discussion

True arrestins specifically binding active phosphorylated GPCRs were so far described in metazoans [52], [53], [54]. Vertebrates express four arrestin subtypes, two of which (arr-1 and arr-4) are highly specialized and function in the visual system. The remaining two non-visual arrestins (arr-2 and arr-3) interact with hundreds of receptors belonging to the GPCR superfamily [3]. Although all arrestins preferentially bind their cognate receptors in the active phosphorylated state, relative

Conclusions

Here we described two mutants of the most promiscuous non-visual subtype, arr-3, that show high preference for Y1R over Y2R. One of these, Tyr239Thr, pre-docks normally, whereas the other, NCA, demonstrates virtually exclusively agonist-induced binding. Moreover, two other mutants, REGCP and NEGCP, completely lost the ability to pre-dock to Y1R, while retaining perfectly normal agonist-induced binding. These newly described mutants expand the arsenal of molecular tools for the study of the

Author contribution

LEG, SB, and LW contributed new reagents, performed experiments, and analyzed data. LEG, SB, AGB-S and VVG designed the study and wrote the manuscript.

Conflict of interests

The authors declare no conflict of interest.

Acknowledgments

We thank Mr. Denis Hüvel for the help in making some of the arrestin-3 mutants. We are also grateful to Dr. J. A. Javitch (Columbia University, New York) for providing the plasmid encoding Venus and Dr. N. A. Lambert (Medical College of Georgia, Augusta, GA) for the plasmid encoding Renilla luciferase variant 8. This work was supported by NIH grants GM077561, GM081756, and EY011500 (VVG) and by the DFG (SFB1052/A3), the European Union and the Free State of Saxony ESF (AGB-S). SB is grateful for

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