Identification and profiling of novel α1A-adrenoceptor-CXC chemokine receptor 2 heteromer

We have provided the first evidence for specific heteromerization between the α(1A)-adrenoceptor (α(1A)AR) and CXC chemokine receptor 2 (CXCR2) in live cells. α(1A)AR and CXCR2 are both expressed in areas such as the stromal smooth muscle layer of the prostate. By utilizing the G protein-coupled receptor (GPCR) heteromer identification technology on the live cell-based bioluminescence resonance energy transfer (BRET) assay platform, our studies in human embryonic kidney 293 cells have identified norepinephrine-dependent β-arrestin recruitment that was in turn dependent upon co-expression of α(1A)AR with CXCR2. These findings have been supported by co-localization observed using confocal microscopy. This norepinephrine-dependent β-arrestin recruitment was inhibited not only by the α(1)AR antagonist Terazosin but also by the CXCR2-specific allosteric inverse agonist SB265610. Furthermore, Labetalol, which is marketed for hypertension as a nonselective β-adrenoceptor antagonist with α(1)AR antagonist properties, was identified as a heteromer-specific-biased agonist exhibiting partial agonism for inositol phosphate production but essentially full agonism for β-arrestin recruitment at the α(1A)AR-CXCR2 heteromer. Finally, bioluminescence resonance energy transfer studies with both receptors tagged suggest that α(1A)AR-CXCR2 heteromerization occurs constitutively and is not modulated by ligand. These findings support the concept of GPCR heteromer complexes exhibiting distinct pharmacology, thereby providing additional mechanisms through which GPCRs can potentially achieve their diverse biological functions. This has important implications for the use and future development of pharmaceuticals targeting these receptors.     

Structural basis of M3 muscarinic receptor dimer/oligomer formation

Class A G protein-coupled receptors (GPCRs) are known to form dimers and/or oligomeric arrays in vitro and in vivo. These complexes are thought to play important roles in modulating class A GPCR function. Many studies suggest that residues located on the "outer" (lipid-facing) surface of the transmembrane (TM) receptor core are critically involved in the formation of class A receptor dimers (oligomers). However, no clear consensus has emerged regarding the identity of the TM helices or TM subsegments involved in this process. To shed light on this issue, we have used the M(3) muscarinic acetylcholine receptor (M3R), a prototypic class A GPCR, as a model system. Using a comprehensive and unbiased approach, we subjected all outward-facing residues (70 amino acids total) of the TM helical bundle (TM1-7) of the M3R to systematic alanine substitution mutagenesis. We then characterized the resulting mutant receptors in radioligand binding and functional studies and determined their ability to form dimers (oligomers) in bioluminescence resonance energy transfer saturation assays. We found that M3R/M3R interactions are not dependent on the presence of one specific structural motif but involve the outer surfaces of multiple TM subsegments (TM1-5 and -7) located within the central and endofacial portions of the TM receptor core. Moreover, we demonstrated that the outward-facing surfaces of most TM helices play critical roles in proper receptor folding and/or function. Guided by the bioluminescence resonance energy transfer data, molecular modeling studies suggested the existence of multiple dimeric/oligomeric M3R arrangements, which may exist in a dynamic equilibrium. Given the high structural homology found among all class A GPCRs, our results should be of considerable general relevance.     

Allosteric and orthosteric sites in CC chemokine receptor (CCR5), a chimeric receptor approach

Chemokine receptors play a major role in immune system regulation and have consequently been targets for drug development leading to the discovery of several small molecule antagonists. Given the large size and predominantly extracellular receptor interaction of endogenous chemokines, small molecules often act more deeply in an allosteric mode. However, opposed to the well described molecular interaction of allosteric modulators in class C 7-transmembrane helix (7TM) receptors, the interaction in class A, to which the chemokine receptors belong, is more sparsely described. Using the CCR5 chemokine receptor as a model system, we studied the molecular interaction and conformational interchange required for proper action of various orthosteric chemokines and allosteric small molecules, including the well known CCR5 antagonists TAK-779, SCH-C, and aplaviroc, and four novel CCR5 ago-allosteric molecules. A chimera was successfully constructed between CCR5 and the closely related CCR2 by transferring all extracellular regions of CCR2 to CCR5, i.e. a Trojan horse that resembles CCR2 extracellularly but signals through a CCR5 transmembrane unit. The chimera bound CCR2 (CCL2 and CCL7), but not CCR5 chemokines (CCL3 and CCL5), with CCR2-like high affinities and potencies throughout the CCR5 signaling unit. Concomitantly, high affinity binding of small molecule CCR5 agonists and antagonists was retained in the transmembrane region. Importantly, whereas the agonistic and antagonistic properties were preserved, the allosteric enhancement of chemokine binding was disrupted. In summary, the Trojan horse chimera revealed that orthosteric and allosteric sites could be structurally separated and still act together with transmission of agonism and antagonism across the different receptor units.     

Molecular Characterization of Oxysterol Binding to the Epstein-Barr Virus-induced Gene 2 (GPR183)

Oxysterols are oxygenated cholesterol derivates that are emerging as a physiologically important group of molecules. Although they regulate a range of cellular processes, only few oxysterol-binding effector proteins have been identified, and the knowledge of their binding mode is limited. Recently, the family of G protein-coupled seven transmembrane-spanning receptors (7TM receptors) was added to this group. Specifically, the Epstein-Barr virus-induced gene 2 (EBI2 or GPR183) was shown to be activated by several oxysterols, most potently by 7α,25-dihydroxycholesterol (7α,25-OHC). Nothing is known about the binding mode, however. Using mutational analysis, we identify here four key residues for 7α,25-OHC binding: Arg-87 in TM-II (position II:20/2.60), Tyr-112 and Tyr-116 (positions III:09/3.33 and III:13/3.37) in TM-III, and Tyr-260 in TM-VI (position VI:16/6.51). Substituting these residues with Ala and/or Phe results in a severe decrease in agonist binding and receptor activation. Docking simulations suggest that Tyr-116 interacts with the 3β-OH group in the agonist, Tyr-260 with the 7α-OH group, and Arg-87, either directly or indirectly, with the 25-OH group, although nearby residues likely also contribute. In addition, Tyr-112 is involved in 7α,25-OHC binding but via hydrophobic interactions. Finally, we show that II:20/2.60 constitutes an important residue for ligand binding in receptors carrying a positively charged residue at this position. This group is dominated by lipid- and nucleotide-activated receptors, here exemplified by the CysLTs, P2Y12, and P2Y14. In conclusion, we present the first molecular characterization of oxysterol binding to a 7TM receptor and identify position II:20/2.60 as a generally important residue for ligand binding in certain 7TM receptors.     

Annexin A1 interaction with the FPR2/ALX receptor: identification of distinct domains and downstream associated signaling

Understanding how proresolving agonists selectively activate FPR2/ALX is a crucial step in the clarification of proresolution molecular networks that can be harnessed for the design of novel therapeutics for inflammatory disease. FPR2/ALX, a G protein-coupled receptor belonging to the formyl peptide receptor (FPR) family, conveys the biological functions of a variety of ligands, including the proresolution mediators annexin A1 (AnxA1) and lipoxin A(4), as well as the activating and proinflammatory protein serum amyloid A. FPR2/ALX is the focus of intense screening for novel anti-inflammatory therapeutics, and the small molecule compound 43 was identified as a receptor ligand. Here, we used chimeric FPR1 and FPR2/ALX clones (stably transfected in HEK293 cells) to identify the N-terminal region and extracellular loop II as the FPR2/ALX domain required for AnxA1-mediated signaling. Genomic responses were also assessed with domain-specific effects emerging, so the N-terminal region is required for AnxA1 induction of JAG1 and JAM3, whereas it is dispensable for modulation of SGPP2. By comparison, serum amyloid A non-genomic responses were reliant on extracellular loops I and II, whereas the small molecule compound 43 activated extracellular loop I with downstream signaling dependent on transmembrane region II. In desensitization experiments, the N-terminal region was dispensable for AnxA1-induced FPR2/ALX down-regulation in both the homologous and heterologous desensitization modes.     

Mutational Analysis of Cysteine Residues of the Insect Odorant Co-receptor (Orco) from Drosophila melanogaster Reveals Differential Effects on Agonist- and Odorant-tuning Receptor-dependent Activation

Insect odorant receptors are heteromeric odorant-gated cation channels comprising a conventional odorant-sensitive tuning receptor (ORx) and a highly conserved co-receptor known as Orco. Orco is found only in insects, and very little is known about its structure and the mechanism leading to channel activation. In the absence of an ORx, Orco forms homomeric channels that can be activated by a synthetic agonist, VUAA1. Drosophila melanogaster Orco (DmelOrco) contains eight cysteine amino acid residues, six of which are highly conserved. In this study, we replaced individual cysteine residues with serine or alanine and expressed Orco mutants in Flp-In 293 T-Rex cells. Changes in intracellular Ca²⁺ levels were used to determine responses to VUAA1. Replacement of two cysteines (Cys-429 and Cys-449) in a predicted intracellular loop (ICL3), individually or together, gave variants that all showed similar increases in the rate of response and sensitivity to VUAA1 compared with wild-type DmelOrco. Kinetic modeling indicated that the response of the Orco mutants to VUAA1 was faster than wild-type Orco. The enhanced sensitivity and faster response of the Cys mutants was confirmed by whole-cell voltage clamp electrophysiology. In contrast to the results from direct agonist activation of Orco, the two cysteine replacement mutants when co-expressed with a tuning receptor (DmelOR22a) showed an ∼10-fold decrease in potency for activation by 2-methyl hexanoate. Our work has shown that intracellular loop 3 is important for Orco channel activation. Importantly, this study also suggests differences in the structural requirements for the activation of homomeric and heteromeric Orco channel complexes.     

Mutually opposite signal modulation by hypothalamic heterodimerization of ghrelin and melanocortin-3 receptors

Interaction and cross-talk of G-protein-coupled receptors (GPCRs) are of considerable interest because an increasing number of examples implicate a profound functional and physiological relevance of homo- or hetero-oligomeric GPCRs. The ghrelin (growth hormone secretagogue receptor (GHSR)) and melanocortin-3 (MC3R) receptors are both known to have orexigenic effects on the hypothalamic control of body weight. Because in vitro studies indicate heterodimerization of GHSR and MC3R, we investigated their functional interplay. Combined in situ hybridization and immunohistochemistry indicated that the vast majority of GHSR-expressing neurons in the arcuate nucleus also express MC3R. In vitro coexpression of MC3R and GHSR promoted enhanced melanocortin-induced intracellular cAMP accumulation compared with activation of MC3R in the absence of GHSR. In contrast, agonist-independent basal signaling activity and ghrelin-induced signaling of GHSR were impaired, most likely due to interaction with MC3R. By taking advantage of naturally occurring GHSR mutations and an inverse agonist for GHSR, we demonstrate that the observed enhanced MC3R signaling capability depends directly on the basal activity of GHSR. In conclusion, we demonstrate a paradigm-shifting example of GPCR heterodimerization allowing for mutually opposite functional influence of two hypothalamic receptors controlling body weight. We found that the agonist-independent active conformation of one GPCR can determine the signaling modalities of another receptor in a heterodimer. Our discovery also implies that mutations within one of two interacting receptors might affect both receptors and different pathways simultaneously. These findings uncover mechanisms of important relevance for pharmacological targeting of GPCR in general and hypothalamic body weight regulation in particular.     

Regulation of β2-adrenergic receptor maturation and anterograde trafficking by an interaction with Rab geranylgeranyltransferase: modulation of Rab geranylgeranylation by the receptor

Previous reports by us and others demonstrated that G protein-coupled receptors interact functionally with Rab GTPases. Here, we show that the β(2)-adrenergic receptor (β(2)AR) interacts with the Rab geranylgeranyltransferase α-subunit (RGGTA). Confocal microscopy showed that β(2)AR co-localizes with RGGTA in intracellular compartments and at the plasma membrane. Site-directed mutagenesis revealed that RGGTA binds to the L(339)L(340) motif in the β(2)AR C terminus known to be involved in the transport of the receptor from the endoplasmic reticulum to the cell surface. Modulation of the cellular levels of RGGTA protein by overexpression or siRNA-mediated knockdown of the endogenous protein demonstrated that RGGTA has a positive role in the maturation and anterograde trafficking of the β(2)AR, which requires the interaction of RGGTA with the β(2)AR L(339)L(340) motif. Furthermore, the β(2)AR modulates the geranylgeranylation of Rab6a, Rab8a, and Rab11a, but not of other Rab proteins tested in this study. Regulation of Rab geranylgeranylation by the β(2)AR was dependent on the RGGTA-interacting L(339)L(340) motif. Interestingly, a RGGTA-Y107F mutant was unable to regulate Rab geranylgeranylation but still promoted β(2)AR maturation, suggesting that RGGTA may have functions independent of Rab geranylgeranylation. We demonstrate for the first time an interaction between a transmembrane receptor and RGGTA which regulates the maturation and anterograde transport of the receptor, as well as geranylgeranylation of Rab GTPases.     

The Arginine of the DRY Motif in Transmembrane Segment III Functions as a Balancing Micro-switch in the Activation of the β2-Adrenergic Receptor

Recent high resolution x-ray structures of the β2-adrenergic receptor confirmed a close salt-bridge interaction between the suspected micro-switch residue ArgIII:26 (Arg3.50) and the neighboring AspIII:25 (Asp3.49). However, neither the expected "ionic lock" interactions between ArgIII:26 and GluVI:-06 (Glu6.30) in the inactive conformation nor the interaction with TyrV:24 (Tyr5.58) in the active conformation were observed in the x-ray structures. Here we find through molecular dynamics simulations, after removal of the stabilizing T4 lysozyme, that the expected salt bridge between ArgIII:26 and GluVI:-06 does form relatively easily in the inactive receptor conformation. Moreover, mutational analysis of GluVI:-06 in TM-VI and the neighboring AspIII:25 in TM-III demonstrated that these two residues do function as locks for the inactive receptor conformation as we observed increased G(s) signaling, arrestin mobilization, and internalization upon alanine substitutions. Conversely, TyrV:24 appears to play a role in stabilizing the active receptor conformation as loss of function of G(s) signaling, arrestin mobilization, and receptor internalization was observed upon alanine substitution of TyrV:24. The loss of function of the TyrV:24 mutant could partly be rescued by alanine substitution of either AspIII:25 or GluVI:-06 in the double mutants. Surprisingly, removal of the side chain of the ArgIII:26 micro-switch itself had no effect on G(s) signaling and internalization and only reduced arrestin mobilization slightly. It is suggested that ArgIII:26 is equally important for stabilizing the inactive and the active conformation through interaction with key residues in TM-III, -V, and -VI, but that the ArgIII:26 micro-switch residue itself apparently is not essential for the actual G protein activation.     

Ubiquitination in the first cytoplasmic loop of μ-opioid receptors reveals a hierarchical mechanism of lysosomal down-regulation

μ-Type opioid receptors (MORs) are members of the large seven-transmembrane receptor family which transduce the effects of both endogenous neuropeptides and clinically important opioid drugs. Prolonged activation of MORs promotes their proteolytic degradation by endocytic trafficking to lysosomes. This down-regulation process is known to contribute to homeostatic regulation of cellular opioid responsiveness, but mechanisms that mediate and control MOR down-regulation have not been defined. We show here that lysosomal down-regulation of MORs is ESCRT (endosomal sorting complex required for transport)-dependent and involves ubiquitin-promoted transfer of internalized MORs from the limiting endosome membrane to lumen. We also show that MOR down-regulation measured by conventional radioligand binding assay is determined specifically by ubiquitination in the first cytoplasmic loop. Surprisingly, we were unable to find any role of ubiquitination in determining whether internalized receptors recycle or are delivered to lysosomes. Instead, this decision is dictated specifically by the MOR C-tail and occurs irrespectively of the presence or absence of receptor ubiquitination. Our results support a hierarchical organization of discrete ubiquitin-independent and -dependent sorting operations, which function non-redundantly in the conserved down-regulation pathway to mediate precise endocytic control. Furthermore, they show that this hierarchical mechanism discriminates the endocytic regulation of naturally occurring MOR isoforms. Moreover, they are the first to reveal, we believe, for any seven-transmembrane receptor, a functional role of ubiquitination in the first cytoplasmic loop.     

GRK2 protein-mediated transphosphorylation contributes to loss of function of μ-opioid receptors induced by neuropeptide FF (NPFF2) receptors

Neuropeptide FF (NPFF) interacts with specific receptors to modulate opioid functions in the central nervous system. On dissociated neurons and neuroblastoma cells (SH-SY5Y) transfected with NPFF receptors, NPFF acts as a functional antagonist of μ-opioid (MOP) receptors by attenuating the opioid-induced inhibition of calcium conductance. In the SH-SY5Y model, MOP and NPFF(2) receptors have been shown to heteromerize. To understand the molecular mechanism involved in the anti-opioid activity of NPFF, we have investigated the phosphorylation status of the MOP receptor using phospho-specific antibody and mass spectrometry. Similarly to direct opioid receptor stimulation, activation of the NPFF(2) receptor by [D-Tyr-1-(NMe)Phe-3]NPFF (1DMe), an analog of NPFF, induced the phosphorylation of Ser-377 of the human MOP receptor. This heterologous phosphorylation was unaffected by inhibition of second messenger-dependent kinases and, contrarily to homologous phosphorylation, was prevented by inactivation of G(i/o) proteins by pertussis toxin. Using siRNA knockdown we could demonstrate that 1DMe-induced Ser-377 cross-phosphorylation and MOP receptor loss of function were mediated by the G protein receptor kinase GRK2. In addition, mass spectrometric analysis revealed that the phosphorylation pattern of MOP receptors was qualitatively similar after treatment with the MOP agonist Tyr-D-Ala-Gly (NMe)-Phe-Gly-ol (DAMGO) or after treatment with the NPFF agonist 1DMe, but the level of multiple phosphorylation was more intense after DAMGO. Finally, NPFF(2) receptor activation was sufficient to recruit β-arrestin2 to the MOP receptor but not to induce its internalization. These data show that NPFF-induced heterologous desensitization of MOP receptor signaling is mediated by GRK2 and could involve transphosphorylation within the heteromeric receptor complex.     

Activation state of alpha4beta1 integrin on sickle red blood cells is linked to the duffy antigen receptor for chemokines (DARC) expression

In sickle cell anemia, reticulocytes express enhanced levels of α4β1 integrin that interact mainly with vascular cell adhesion molecule-1 and fibronectin, promoting vaso-occlusion. These interactions are known to be highly sensitive to the inflammatory chemokine IL-8. The Duffy antigen receptor for chemokines (DARC) modulates the function of inflammatory processes. However, the link between α4β1 activation by chemokines and DARC erythroid expression is not or poorly explored. Therefore, the capacity of α4β1 to mediate Duffy-negative and Duffy-positive sickle reticulocyte (SRe) adhesion to immobilized vascular cell adhesion molecule-1 and fibronectin was evaluated. Using static adhesion assays, we found that, under basal conditions, Duffy-positive SRe adhesion was 2-fold higher than that of Duffy-negative SRes. Incubating the cells with IL-8 or RANTES (regulated on activation normal T cell expressed and secreted) increased Duffy-positive SRe adhesion only, whereas Mn(2+) increased cell adhesion independently of the Duffy phenotype. Flow cytometry analyses performed with anti-β1 and anti-α4 antibodies, including a conformation-sensitive one, in the presence or absence of IL-8, revealed that Duffy-positive and Duffy-negative SRes displayed similar erythroid α4β1 expression levels, but with distinct activation states. IL-8 did not affect α4β1 affinity in Duffy-positive SRes but induced its clustering as corroborated by immunofluorescence microscopy. Our results indicate that in Duffy-negative SRes α4β1 integrin is constitutively expressed in a low affinity state, whereas in Duffy-positive SRes α4β1 is expressed in a higher chemokine-sensitive affinity state. This activation state associated with DARC RBC expression may influence the intensity of the inflammatory responses encountered in sickle cell anemia and participate in its interindividual clinical expression variability.     

Palmitoylation of Protease-activated Receptor-1 Regulates Adaptor Protein Complex-2 and -3 Interaction with Tyrosine-based Motifs and Endocytic Sorting

Protease-activated receptor-1 (PAR1) is a G protein-coupled receptor for the coagulant protease thrombin. Thrombin binds to and cleaves the N terminus of PAR1, generating a new N terminus that functions as a tethered ligand that cannot diffuse away. In addition to rapid desensitization, PAR1 trafficking is critical for the regulation of cellular responses. PAR1 displays constitutive and agonist-induced internalization. Constitutive internalization of unactivated PAR1 is mediated by the clathrin adaptor protein complex-2 (AP-2), which binds to a distal tyrosine-based motif localized within the C-terminal tail (C-tail) domain. Once internalized, PAR1 is sorted from endosomes to lysosomes via AP-3 interaction with a second C-tail tyrosine motif proximal to the transmembrane domain. However, the regulatory processes that control adaptor protein recognition of PAR1 C-tail tyrosine-based motifs are not known. Here, we report that palmitoylation of PAR1 is critical for regulating proper utilization of tyrosine-based motifs and endocytic sorting. We show that PAR1 is basally palmitoylated at highly conserved C-tail cysteines. A palmitoylation-deficient PAR1 mutant is competent to signal and exhibits a marked increase in constitutive internalization and lysosomal degradation compared with wild type receptor. Intriguingly, enhanced constitutive internalization of PAR1 is mediated by AP-2 and requires the proximal tyrosine-based motif rather than the distal tyrosine motif used by wild type receptor. Moreover, palmitoylation-deficient PAR1 displays increased degradation that is mediated by AP-3. These findings suggest that palmitoylation of PAR1 regulates appropriate utilization of tyrosine-based motifs by adaptor proteins and endocytic trafficking, processes that are critical for maintaining appropriate expression of PAR1 at the cell surface.     

The Cytomegalovirus UL146 Gene Product vCXCL1 Targets Both CXCR1 and CXCR2 as an Agonist

Large DNA viruses, such as herpesvirus and poxvirus, encode proteins that target and exploit the chemokine system of their host. UL146 and UL147 in the cytomegalovirus (CMV) genome encode the two CXC chemokines vCXCL1 and vCXCL2. In this study, vCXCL1 was probed against a panel of the 18 classified human chemokine receptors. In calcium mobilization assays vCXCL1 acted as an agonist on both CXCR1 and CXCR2 but did not activate or block any of the other 16 chemokine receptors. vCXCL1 was characterized and compared with CXCL1/GROα, CXCL2/GROβ, CXCL3/GROγ, CXCL5/ENA-78, CXCL6/GCP-2, CXCL7/NAP-2 and CXCL8/IL-8 in competition binding, calcium mobilization, inositol triphosphate turnover, and chemotaxis assays using CXCR1- and CXCR2-expressing Chinese hamster ovary, 300.19, COS7, and L1.2 cells. The affinities of vCXCL1 for the CXCR1 and CXCR2 receptors were 44 and 5.6 nm, respectively, as determined in competition binding against radioactively labeled CXCL8. In calcium mobilization, phosphatidylinositol turnover, and chemotaxis assays, vCXCL1 acted as a highly efficacious activator of both receptors, with a rather low potency for the CXCR1 receptor but comparable with CXCL5 and CXCL7. It is suggested that CMV uses the UL146 gene product expressed in infected endothelial cells to attract neutrophils by activating their CXCR1 and CXCR2 receptors, whereby neutrophils can act as carriers of the virus to uninfected endothelial cells. In that way a lasting pool of CMV-infected endothelial cells could be maintained.     

Melanopsin is highly resistant to light and chemical bleaching in vivo

Melanopsin is the photopigment of mammalian intrinsically photosensitive retinal ganglion cells, where it contributes to light entrainment of circadian rhythms, and to the pupillary light response. Previous work has shown that the melanopsin photocycle is independent of that used by rhodopsin (Tu, D. C., Owens, L. A., Anderson, L., Golczak, M., Doyle, S. E., McCall, M., Menaker, M., Palczewski, K., and Van Gelder, R. N. (2006) Inner retinal photoreception independent of the visual retinoid cycle. Proc. Natl. Acad. Sci. U.S.A. 103, 10426-10431). Here we determined the ability of apo-melanopsin, formed by ex vivo UV light bleaching, to use selected chromophores. We found that 9-cis-retinal, but not all-trans-retinal or 9-cis-retinol, is able to restore light-dependent ipRGC activity after bleaching. Melanopsin was highly resistant to both visible-spectrum photic bleaching and chemical bleaching with hydroxylamine under conditions that fully bleach rod and cone photoreceptor cells. These results suggest that the melanopsin photocycle can function independently of both rod and cone photocycles, and that apo-melanopsin has a strong preference for binding cis-retinal to generate functional pigment. The data support a model in which retinal is continuously covalently bound to melanopsin and may function through a reversible, bistable mechanism.     

Manipulation of Very Few Receptor Discriminator Residues Greatly Enhances Receptor Specificity of Non-visual Arrestins

Based on the identification of residues that determine receptor selectivity of arrestins and the analysis of the evolution in the arrestin family, we introduced 10 mutations of "receptor discriminator" residues in arrestin-3. The recruitment of these mutants to M2 muscarinic (M2R), D1 (D1R) and D2 (D2R) dopamine, and β(2)-adrenergic receptors (β(2)AR) was assessed using bioluminescence resonance energy transfer-based assays in cells. Seven of 10 mutations differentially affected arrestin-3 binding to individual receptors. D260K and Q262P reduced the binding to β(2)AR, much more than to other receptors. The combination D260K/Q262P virtually eliminated β(2)AR binding while preserving the interactions with M2R, D1R, and D2R. Conversely, Y239T enhanced arrestin-3 binding to β(2)AR and reduced the binding to M2R, D1R, and D2R, whereas Q256Y selectively reduced recruitment to D2R. The Y239T/Q256Y combination virtually eliminated the binding to D2R and reduced the binding to β(2)AR and M2R, yielding a mutant with high selectivity for D1R. Eleven of 12 mutations significantly changed the binding to light-activated phosphorhodopsin. Thus, manipulation of key residues on the receptor-binding surface modifies receptor preference, enabling the construction of non-visual arrestins specific for particular receptor subtypes. These findings pave the way to the construction of signaling-biased arrestins targeting the receptor of choice for research or therapeutic purposes.