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      Identification and Profiling of Novel α1A-Adrenoceptor-CXC Chemokine Receptor 2 Heteromer*

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          Abstract

          Background: Receptor heteromers are macromolecular complexes containing at least two different receptor subunits, resulting in distinct pharmacology.

          Results: The observed α 1AAR-CXCR2 heteromer recruits β-arrestin strongly upon activation with norepinephrine, in contrast to α 1AAR alone.

          Conclusion: Heteromerization with CXCR2 dramatically changes α 1AAR pharmacology, revealing the potential for heteromer-specific biased agonism.

          Significance: Such heteromer-specific novel pharmacology has important implications for drug discovery.

          Abstract

          We have provided the first evidence for specific heteromerization between the α 1A-adrenoceptor (α 1AAR) and CXC chemokine receptor 2 (CXCR2) in live cells. α 1AAR 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 1AAR 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 1AR 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 1AR 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 1AAR-CXCR2 heteromer. Finally, bioluminescence resonance energy transfer studies with both receptors tagged suggest that α 1AAR-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.

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          Most cited references 67

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          Chemokine signaling via the CXCR2 receptor reinforces senescence.

          Cells enter senescence, a state of stable proliferative arrest, in response to a variety of cellular stresses, including telomere erosion, DNA damage, and oncogenic signaling, which acts as a barrier against malignant transformation in vivo. To identify genes controlling senescence, we conducted an unbiased screen for small hairpin RNAs that extend the life span of primary human fibroblasts. Here, we report that knocking down the chemokine receptor CXCR2 (IL8RB) alleviates both replicative and oncogene-induced senescence (OIS) and diminishes the DNA-damage response. Conversely, ectopic expression of CXCR2 results in premature senescence via a p53-dependent mechanism. Cells undergoing OIS secrete multiple CXCR2-binding chemokines in a program that is regulated by the NF-kappaB and C/EBPbeta transcription factors and coordinately induce CXCR2 expression. CXCR2 upregulation is also observed in preneoplastic lesions in vivo. These results suggest that senescent cells activate a self-amplifying secretory network in which CXCR2-binding chemokines reinforce growth arrest.
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            Teaching old receptors new tricks: biasing seven-transmembrane receptors.

            Seven-transmembrane receptors (7TMRs; also known as G protein-coupled receptors) are the largest class of receptors in the human genome and are common targets for therapeutics. Originally identified as mediators of 7TMR desensitization, beta-arrestins (arrestin 2 and arrestin 3) are now recognized as true adaptor proteins that transduce signals to multiple effector pathways. Signalling that is mediated by beta-arrestins has distinct biochemical and functional consequences from those mediated by G proteins, and several biased ligands and receptors have been identified that preferentially signal through either G protein- or beta-arrestin-mediated pathways. These ligands are not only useful tools for investigating the biochemistry of 7TMR signalling, they also have the potential to be developed into new classes of therapeutics.
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              A monomeric G protein-coupled receptor isolated in a high-density lipoprotein particle efficiently activates its G protein.

              G protein-coupled receptors (GPCRs) respond to a diverse array of ligands, mediating cellular responses to hormones and neurotransmitters, as well as the senses of smell and taste. The structures of the GPCR rhodopsin and several G proteins have been determined by x-ray crystallography, yet the organization of the signaling complex between GPCRs and G proteins is poorly understood. The observations that some GPCRs are obligate heterodimers, and that many GPCRs form both homo- and heterodimers, has led to speculation that GPCR dimers may be required for efficient activation of G proteins. However, technical limitations have precluded a definitive analysis of G protein coupling to monomeric GPCRs in a biochemically defined and membrane-bound system. Here we demonstrate that a prototypical GPCR, the beta2-adrenergic receptor (beta2AR), can be incorporated into a reconstituted high-density lipoprotein (rHDL) phospholipid bilayer particle together with the stimulatory heterotrimeric G protein, Gs. Single-molecule fluorescence imaging and FRET analysis demonstrate that a single beta2AR is incorporated per rHDL particle. The monomeric beta2AR efficiently activates Gs and displays GTP-sensitive allosteric ligand-binding properties. These data suggest that a monomeric receptor in a lipid bilayer is the minimal functional unit necessary for signaling, and that the cooperativity of agonist binding is due to G protein association with a receptor monomer and not receptor oligomerization.
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                Author and article information

                Journal
                J Biol Chem
                J. Biol. Chem
                jbc
                jbc
                JBC
                The Journal of Biological Chemistry
                American Society for Biochemistry and Molecular Biology (9650 Rockville Pike, Bethesda, MD 20814, U.S.A. )
                0021-9258
                1083-351X
                13 April 2012
                27 February 2012
                27 February 2012
                : 287
                : 16
                : 12952-12965
                Affiliations
                From the []Laboratory for Molecular Endocrinology-G Protein-Coupled Receptors, Western Australian Institute for Medical Research and Centre for Medical Research, The University of Western Australia, Nedlands, Perth, Western Australia 6009,
                [§ ]Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052 and
                []Dimerix Bioscience Pty Ltd, Nedlands, Perth, Western Australia 6009, Australia
                Author notes
                [2 ] To whom correspondence should be addressed: Western Australian Institute for Medical Research, B Block, QEII Medical Centre, Nedlands, Perth, Western Australia 6009, Australia. Tel.: 618-9346-1980; Fax: 618-9346-1818; E-mail: kevin.pfleger@ 123456waimr.uwa.edu.au .
                [1]

                Both authors contributed equally to this work.

                M111.322834
                10.1074/jbc.M111.322834
                3340001
                22371491
                © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

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