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

  • Sanam Mustafa
    Footnotes
    Affiliations
    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
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  • Heng B. See
    Footnotes
    Affiliations
    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
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  • Ruth M. Seeber
    Affiliations
    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
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  • Stephen P. Armstrong
    Affiliations
    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
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  • Carl W. White
    Affiliations
    Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052
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  • Sabatino Ventura
    Affiliations
    Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052
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  • Mohammed Akli Ayoub
    Affiliations
    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
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  • Kevin D.G. Pfleger
    Correspondence
    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;
    Affiliations
    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

    Dimerix Bioscience Pty Ltd, Nedlands, Perth, Western Australia 6009, Australia
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  • Author Footnotes
    * This research was funded by National Health and Medical Research Council of Australia Project Grant 566736 and Dimerix Bioscience Pty Ltd. K. D. G. P. is an Australian Research Council Future Fellow (FT100100271) as well as Chief Scientific Officer of Dimerix Bioscience Pty Ltd, a spin-out company of the University of Western Australia that has been assigned the rights to the GPCR-HIT technology. K. D. G. P. has a minor shareholding in Dimerix.
    This article contains supplemental Figs. 1–3.
    1 Both authors contributed equally to this work.
Open AccessPublished:February 27, 2012DOI:https://doi.org/10.1074/jbc.M111.322834
      We have provided the first evidence for specific heteromerization between the α1A-adrenoceptor (α1AAR) and CXC chemokine receptor 2 (CXCR2) in live cells. α1AAR 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 α1AAR 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 α1AR 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 α1AR 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 α1AAR-CXCR2 heteromer. Finally, bioluminescence resonance energy transfer studies with both receptors tagged suggest that α1AAR-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.

      Introduction

      G protein-coupled receptors (GPCRs)
      The abbreviations used are: GPCR
      G protein-coupled receptor
      HIT
      heteromer identification technology
      α1AAR
      α1A-adrenoceptor
      AR
      adrenoceptor
      BRET
      bioluminescence resonance energy transfer
      eBRET
      extended BRET
      HEK
      human embryonic kidney
      NE
      norepinephrine
      OxR1
      orexin receptor 1
      Rluc
      Renilla luciferase
      RT
      room temperature
      V2R
      vasopressin receptor 2
      CXCR2
      CXC chemokine receptor 2
      GLP-1
      glucagon-like peptide-1.
      are expressed in almost every human cell and have diverse physiological and pathophysiological roles. Our understanding of GPCR signaling is now evolving from considering these seven transmembrane receptors (7TMRs) simply as “on-off” switches consisting of only one functional unit to accepting that they probably act more like “microprocessors” (
      • Kenakin T.P.
      Cellular assays as portals to seven-transmembrane receptor-based drug discovery.
      ), signaling complexes containing potentially multiple GPCRs along with various other interacting proteins. Termed heteromerization, the accepted definition states that a receptor heteromer is a “macromolecular complex composed of at least two (functional) receptor units with biochemical properties that are demonstrably different from those of its individual components” (
      • Ferré S.
      • Baler R.
      • Bouvier M.
      • Caron M.G.
      • Devi L.A.
      • Durroux T.
      • Fuxe K.
      • George S.R.
      • Javitch J.A.
      • Lohse M.J.
      • Mackie K.
      • Milligan G.
      • Pfleger K.D.
      • Pin J.P.
      • Volkow N.D.
      • Waldhoer M.
      • Woods A.S.
      • Franco R.
      Building a new conceptual framework for receptor heteromers.
      ). Recent evidence has suggested that GPCRs can function as monomers (
      • Whorton M.R.
      • Bokoch M.P.
      • Rasmussen S.G.
      • Huang B.
      • Zare R.N.
      • Kobilka B.
      • Sunahara R.K.
      A monomeric G protein-coupled receptor isolated in a high density lipoprotein particle efficiently activates its G protein.
      ,
      • Ernst O.P.
      • Gramse V.
      • Kolbe M.
      • Hofmann K.P.
      • Heck M.
      Monomeric G protein-coupled receptor rhodopsin in solution activates its G protein transducin at the diffusion limit.
      ,
      • Whorton M.R.
      • Jastrzebska B.
      • Park P.S.
      • Fotiadis D.
      • Engel A.
      • Palczewski K.
      • Sunahara R.K.
      Efficient coupling of transducin to monomeric rhodopsin in a phospholipid bilayer.
      ,
      • Kuszak A.J.
      • Pitchiaya S.
      • Anand J.P.
      • Mosberg H.I.
      • Walter N.G.
      • Sunahara R.K.
      Purification and functional reconstitution of monomeric μ-opioid receptors. Allosteric modulation of agonist binding by Gi2.
      ) as well as forming complexes containing only one type of GPCR (termed “homomers” (
      • Ferré S.
      • Baler R.
      • Bouvier M.
      • Caron M.G.
      • Devi L.A.
      • Durroux T.
      • Fuxe K.
      • George S.R.
      • Javitch J.A.
      • Lohse M.J.
      • Mackie K.
      • Milligan G.
      • Pfleger K.D.
      • Pin J.P.
      • Volkow N.D.
      • Waldhoer M.
      • Woods A.S.
      • Franco R.
      Building a new conceptual framework for receptor heteromers.
      )). Consequently, for most GPCRs, heteromerization is probably not obligatory. However, it does have the potential to provide additional mechanisms for signal integration and allosteric modulation when it occurs, and with these come exciting new opportunities for novel pharmaceutical development.
      The α1AAR is coupled to the Gq/11 protein/phospholipase C signaling pathway. Subsequent activation of phospholipase C causes the production of inositol trisphosphate and diacylglycerol, particularly resulting in contraction of smooth muscle (
      • Eckert R.E.
      • Schreier U.
      • Drescher P.
      • Madsen P.O.
      • Derouet H.
      • Becht E.
      • Steffens J.
      • Ziegler M.
      Regulation of prostatic smooth muscle contractility by intracellular second messengers. Implications for the conservative treatment of benign prostatic hyperplasia.
      ).
      Currently, α1AARs can be functionally subclassified into two pharmacologically distinct phenotypes; the “standard” α1AAR phenotype is predominantly found in brain, heart, and blood vessels, whereas a functional isoform of α1AAR, the so-called “α1LAR,” is more prominent in prostate and bladder neck (
      • Muramatsu I.
      • Oshita M.
      • Ohmura T.
      • Kigoshi S.
      • Akino H.
      • Gobara M.
      • Okada K.
      Pharmacological characterization of α1-adrenoceptor subtypes in the human prostate. Functional and binding studies.
      ,
      • Ford A.P.
      • Arredondo N.F.
      • Blue Jr., D.R.
      • Bonhaus D.W.
      • Jasper J.
      • Kava M.S.
      • Lesnick J.
      • Pfister J.R.
      • Shieh I.A.
      • Vimont R.L.
      • Williams T.J.
      • McNeal J.E.
      • Stamey T.A.
      • Clarke D.E.
      RS-17053 (N-[2-(2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro-α,α-dimethyl-1H-indole-3-ethanamine hydrochloride), a selective α 1A-adrenoceptor antagonist, displays low affinity for functional α 1-adrenoceptors in human prostate. implications for adrenoceptor classification.
      ). Using genetically modified knock-out mice, it has recently been shown that both functional phenotypes arise from the same α1AAR gene (
      • Gray K.
      • Short J.
      • Ventura S.
      The α1A-adrenoceptor gene is required for the α1L-adrenoceptor-mediated response in isolated preparations of the mouse prostate.
      ) even though studies have indicated that a splice variant or polymorphism is not responsible for the difference in pharmacology (
      • Shibata K.
      • Hirasawa A.
      • Moriyama N.
      • Kawabe K.
      • Ogawa S.
      • Tsujimoto G.
      α1a-adrenoceptor polymorphism. Pharmacological characterization and association with benign prostatic hypertrophy.
      ,
      • Suzuki F.
      • Taniguchi T.
      • Takauji R.
      • Murata S.
      • Muramatsu I.
      Splice isoforms of α(1a)-adrenoceptor in rabbit.
      ,
      • Ramsay D.
      • Carr I.C.
      • Pediani J.
      • Lopez-Gimenez J.F.
      • Thurlow R.
      • Fidock M.
      • Milligan G.
      High affinity interactions between human α1A-adrenoceptor C-terminal splice variants produce homo- and heterodimers but do not generate the α1L-adrenoceptor.
      ). Furthermore, the α1LAR phenotype is not observed in radioligand binding experiments using homogenized prostate tissue but is seen in functional studies using whole tissue or cells (
      • Muramatsu I.
      • Tanaka T.
      • Suzuki F.
      • Li Z.
      • Hiraizumi-Hiraoka Y.
      • Anisuzzaman A.S.
      • Yamamoto H.
      • Horinouchi T.
      • Morishima S.
      Quantifying receptor properties. The tissue segment binding method; a powerful tool for the pharmacome analysis of native receptors.
      ,
      • Nishimune A.
      • Suzuki F.
      • Yoshiki H.
      • Morishima S.
      • Muramatsu I.
      α1-Adrenoceptor pharmacome. α1L-adrenoceptor and α1A-adrenoceptor in the lower urinary tract.
      ). This has led to the suggestion that an interacting protein may be associated with the α1AAR in tissues exhibiting α1LAR pharmacology (
      • Muramatsu I.
      • Tanaka T.
      • Suzuki F.
      • Li Z.
      • Hiraizumi-Hiraoka Y.
      • Anisuzzaman A.S.
      • Yamamoto H.
      • Horinouchi T.
      • Morishima S.
      Quantifying receptor properties. The tissue segment binding method; a powerful tool for the pharmacome analysis of native receptors.
      ,
      • Nishimune A.
      • Suzuki F.
      • Yoshiki H.
      • Morishima S.
      • Muramatsu I.
      α1-Adrenoceptor pharmacome. α1L-adrenoceptor and α1A-adrenoceptor in the lower urinary tract.
      ). Indeed, a recent publication has provided evidence for cysteine-rich with epidermal growth factor-like domain 1α (CRELD1α) interacting with α1AAR to modulate function, largely in terms of receptor expression levels (
      • Nishimune A.
      • Suzuki F.
      • Yoshiki H.
      • Morishima S.
      • Muramatsu I.
      Identification of cysteine-rich epidermal growth factor-like domain 1α (CRELD1α) as a novel α1A-adrenoceptor-down-regulating protein and establishment of an α1L-adrenoceptor-expressing cell line.
      ). Evidence for α1AAR forming complexes with itself (
      • Ramsay D.
      • Carr I.C.
      • Pediani J.
      • Lopez-Gimenez J.F.
      • Thurlow R.
      • Fidock M.
      • Milligan G.
      High affinity interactions between human α1A-adrenoceptor C-terminal splice variants produce homo- and heterodimers but do not generate the α1L-adrenoceptor.
      ,
      • Uberti M.A.
      • Hall R.A.
      • Minneman K.P.
      Subtype-specific dimerization of α 1-adrenoceptors. Effects on receptor expression and pharmacological properties.
      ), with splice variants (
      • Ramsay D.
      • Carr I.C.
      • Pediani J.
      • Lopez-Gimenez J.F.
      • Thurlow R.
      • Fidock M.
      • Milligan G.
      High affinity interactions between human α1A-adrenoceptor C-terminal splice variants produce homo- and heterodimers but do not generate the α1L-adrenoceptor.
      ), and with α1BAR (
      • Uberti M.A.
      • Hall R.A.
      • Minneman K.P.
      Subtype-specific dimerization of α 1-adrenoceptors. Effects on receptor expression and pharmacological properties.
      ) has also been published. No effects on receptor pharmacological properties were observed, but co-expression of α1BAR did appear to increase α1AAR expression levels (
      • Uberti M.A.
      • Hall R.A.
      • Minneman K.P.
      Subtype-specific dimerization of α 1-adrenoceptors. Effects on receptor expression and pharmacological properties.
      ).
      Our hypothesis is that α1AAR function can also be modulated by heteromerization with GPCRs from outside the adrenoceptor family. Interestingly, in addition to the aforementioned precedents for α1AAR being modulated by interacting proteins, recent evidence for heteromerization of the β2-adrenoceptor (β2AR) and CXC chemokine receptor 4 playing a potential role in cardiac myocyte survival has provided a precedent for adrenoceptor-chemokine receptor heteromerization (
      • LaRocca T.J.
      • Schwarzkopf M.
      • Altman P.
      • Zhang S.
      • Gupta A.
      • Gomes I.
      • Alvin Z.
      • Champion H.C.
      • Haddad G.
      • Hajjar R.J.
      • Devi L.A.
      • Schecter A.D.
      • Tarzami S.T.
      β2-Adrenergic receptor signaling in the cardiac myocyte is modulated by interactions with CXCR4.
      ). Taken together with suggestions that the diverse functional effects mediated by the α1ARs may not be entirely explicable by Gq/11 signaling pathways (
      • Cotecchia S.
      The α1-adrenergic receptors. Diversity of signaling networks and regulation.
      ), we hypothesize that heteromerization may provide a mechanism for such diversity. This in turn provides a potential opportunity to develop therapies that target this receptor only when in a complex with another GPCR and, therefore, possibly also in a tissue-selective manner.
      Although the chemokine receptors clearly play a major role in the immune system, their widespread expression pattern suggests their role may extend beyond chemotaxis and cell migration. CXCR2 is activated by multiple chemokines, and signaling via this receptor has been shown to reinforce cell growth arrest (cellular senescence) (
      • Acosta J.C.
      • O'Loghlen A.
      • Banito A.
      • Guijarro M.V.
      • Augert A.
      • Raguz S.
      • Fumagalli M.
      • Da Costa M.
      • Brown C.
      • Popov N.
      • Takatsu Y.
      • Melamed J.
      • d'Adda di Fagagna F.
      • Bernard D.
      • Hernando E.
      • Gil J.
      Chemokine signaling via the CXCR2 receptor reinforces senescence.
      ). In contrast, depletion of CXCR2 results in escape from senescence. A role for CXCR2 in senescence has been elucidated in prostate intraepithelial neoplasia, with fibroblasts that are undergoing oncogene-induced senescence up-regulating CXCR2 and its ligands, suggestive of a positive feedback loop (
      • Acosta J.C.
      • O'Loghlen A.
      • Banito A.
      • Guijarro M.V.
      • Augert A.
      • Raguz S.
      • Fumagalli M.
      • Da Costa M.
      • Brown C.
      • Popov N.
      • Takatsu Y.
      • Melamed J.
      • d'Adda di Fagagna F.
      • Bernard D.
      • Hernando E.
      • Gil J.
      Chemokine signaling via the CXCR2 receptor reinforces senescence.
      ). Furthermore, CXCR2 is well established as a receptor that is capable of forming complexes with both itself (
      • Trettel F.
      • Di Bartolomeo S.
      • Lauro C.
      • Catalano M.
      • Ciotti M.T.
      • Limatola C.
      Ligand-independent CXCR2 dimerization.
      ,
      • Wilson S.
      • Wilkinson G.
      • Milligan G.
      The CXCR1 and CXCR2 receptors form constitutive homo- and heterodimers selectively and with equal apparent affinities.
      ) and other GPCRs (
      • Wilson S.
      • Wilkinson G.
      • Milligan G.
      The CXCR1 and CXCR2 receptors form constitutive homo- and heterodimers selectively and with equal apparent affinities.
      ,
      • Parenty G.
      • Appelbe S.
      • Milligan G.
      CXCR2 chemokine receptor antagonism enhances DOP opioid receptor function via allosteric regulation of the CXCR2-DOP receptor heterodimer.
      ).
      Although establishment of novel heteromer-specific pharmacology in native tissue is the ultimate goal, this is hampered by the general lack of experimental tools to study heteromerization of endogenously expressed receptors (
      • Mustafa S.
      • Ayoub M.A.
      • Pfleger K.D.
      Uncovering GPCR heteromer-biased ligands.
      ). Therefore, our first step has been to provide evidence at least of receptor co-localization, which we have done using immunohistochemistry that indicates CXCR2 expression specifically in a region of the prostate well established as expressing α1AAR (
      • Morishima S.
      • Suzuki F.
      • Nishimune A.
      • Yoshiki H.
      • Akino H.
      • Yokoyama O.
      • Muramatsu I.
      Visualization and tissue distribution of α1L-adrenoceptor in human prostate by the fluorescently labeled ligand Alexa-488-silodosin.
      ). Our next step has been to establish the existence and novel pharmacology of α1AAR heteromerization in a recombinant system, to identify the “biochemical fingerprint of a receptor heteromer.” By applying the GPCR heteromer identification technology (HIT) approach (
      • Mustafa S.
      • Ayoub M.A.
      • Pfleger K.D.
      Uncovering GPCR heteromer-biased ligands.
      ,
      • Ayoub M.A.
      • Pfleger K.D.
      Recent advances in bioluminescence resonance energy transfer technologies to study GPCR heteromerization.
      ,
      • See H.B.
      • Seeber R.M.
      • Kocan M.
      • Eidne K.A.
      • Pfleger K.D.
      Application of G protein-coupled receptor-heteromer identification technology to monitor β-arrestin recruitment to G protein-coupled receptor heteromers.
      ,
      • Mustafa S.
      • Pfleger K.D.
      G protein-coupled receptor heteromer identification technology. Identification and profiling of GPCR heteromers.
      ,
      • Porrello E.R.
      • Pfleger K.D.
      • Seeber R.M.
      • Qian H.
      • Oro C.
      • Abogadie F.
      • Delbridge L.M.
      • Thomas W.G.
      Heteromerization of angiotensin receptors changes trafficking and arrestin recruitment profiles.
      ) on a live cell bioluminescence resonance energy transfer (BRET) platform, supported by confocal microscopy experiments, we have provided evidence for novel and specific heteromerization between α1AAR and CXCR2 that results in norepinephrine (NE)-induced recruitment of β-arrestin2 to the receptor complex. This is in stark contrast to the very weak β-arrestin2 interaction observed with α1AAR monomers/homomers in transfected HEK293 cells (
      • Stanasila L.
      • Abuin L.
      • Dey J.
      • Cotecchia S.
      Different internalization properties of the α1a- and α1b-adrenergic receptor subtypes. The potential role of receptor interaction with β-arrestins and AP50.
      ) but, very interestingly, is consistent with the observation of α1AAR·β-arrestin2 complex formation specifically in prostate stroma (
      • Hennenberg M.
      • Schlenker B.
      • Roosen A.
      • Strittmatter F.
      • Walther S.
      • Stief C.
      • Gratzke C.
      β-Arrestin-2 is expressed in human prostate smooth muscle and a binding partner of α1A-adrenoceptors.
      ).

      EXPERIMENTAL PROCEDURES

      Materials

      α1AAR and CCR2 cDNAs were obtained from the Missouri S&T cDNA Resource Center. CXCR2 cDNA was kindly provided by Aron Chakera (Oxford University). CCR2/Rluc8 and CXCR2/Rluc8 cDNA constructs were generated from plasmids containing the respective receptor cDNA tagged with Renilla luciferase (Rluc), also kindly provided by Aron Chakera. Similarly, the α1AAR/Rluc8 and orexin receptor 1 (OxR1)/Rluc8 cDNA constructs were generated from α1AAR/Rluc and OxR1/Rluc, respectively (formerly produced by PCR amplification of receptor cDNA to remove the stop codon and ligation into pcDNA3 containing Rluc). OxR1/Rluc was generated previously in our laboratory by Matthew Dalrymple from OxR1 cDNA kindly provided by Masashi Yanagisawa (Howard Hughes Medical Institute). With all of these constructs, the Rluc coding region was replaced with Rluc8 cDNA from pcDNA3.1-Rluc8 kindly provided by Andreas Loening and Sanjiv Gambhir (Stanford University) (
      • Loening A.M.
      • Fenn T.D.
      • Wu A.M.
      • Gambhir S.S.
      Consensus guided mutagenesis of Renilla luciferase yields enhanced stability and light output.
      ) as described previously for other GPCR constructs (
      • Kocan M.
      • See H.B.
      • Seeber R.M.
      • Eidne K.A.
      • Pfleger K.D.
      Demonstration of improvements to the bioluminescence resonance energy transfer (BRET) technology for the monitoring of G protein-coupled receptors in live cells.
      ). Vasopressin receptor 2 (V2R)/Rluc8 was generated as described previously from V2R cDNA kindly provided by Brian Feldman (Stanford University) (
      • Kocan M.
      • See H.B.
      • Sampaio N.G.
      • Eidne K.A.
      • Feldman B.J.
      • Pfleger K.D.
      Agonist-independent interactions between β-arrestins and mutant vasopressin type II receptors associated with nephrogenic syndrome of inappropriate antidiuresis.
      ). The β-arrestin2/Venus cDNA construct was prepared previously from pcC2-Venus kindly provided by Atsushi Miyawaki (RIKEN Brain Science Institute, Wako-city, Japan) (
      • Kocan M.
      • See H.B.
      • Seeber R.M.
      • Eidne K.A.
      • Pfleger K.D.
      Demonstration of improvements to the bioluminescence resonance energy transfer (BRET) technology for the monitoring of G protein-coupled receptors in live cells.
      ). CXCR2/Venus and V2R/Venus were prepared by replacing Rluc8 cDNA with Venus cDNA in the CXCR2/Rluc8 and V2R/Rluc8 constructs, respectively, as described previously for V2R/Venus (
      • Kocan M.
      • See H.B.
      • Sampaio N.G.
      • Eidne K.A.
      • Feldman B.J.
      • Pfleger K.D.
      Agonist-independent interactions between β-arrestins and mutant vasopressin type II receptors associated with nephrogenic syndrome of inappropriate antidiuresis.
      ). Ligands used were NE, CXCL8 (Interleukin-8), CCL2 (MCP-1), arginine vasopressin, Terazosin, and Labetalol (Sigma) as well as SB265610 (Tocris) and Orexin A (American Peptide Co.).

      Cell Culture and Transfection

      HEK293FT cells were maintained at 37 °C in 5% CO2 and complete media (Dulbecco's modified Eagle's medium (DMEM) containing 0.3 mg/ml glutamine, 100 IU/ml penicillin and 100 μg/ml streptomycin (Invitrogen)) supplemented with 10% fetal calf serum (FCS) and 400 μg/ml Geneticin (Invitrogen). Transient transfections were carried out 24 h after seeding about 550,000 cells/well of a 6-well plate. Genejuice (Novagen) transfection reagent was used according to the manufacturer's instructions. Cells were harvested with 0.05% trypsin-EDTA (Invitrogen). For testing the CXCR2 antibody specificity, Chinese hamster ovary (CHO) cells were seeded at a density of 40,000 cells/well of a 12-well plate and transiently transfected using Lipofectamine (Invitrogen) as per the manufacturer's instructions. Cells were maintained at 37 °C in 5% CO2 and high glucose (25 mm) DMEM (supplemented with 4 mm Glutamax (Invitrogen), 5% FCS, 16 mm HEPES, 100 IU/ml penicillin, 100 μg/ml streptomycin, 1 mg/ml hygromycin B, and 1 mm sodium pyruvate).

      Assessment of CXCR2 Antibody Specificity

      CHO cells were immunostained 36 h after transfecting with 1 μg of CXCR2 or CCR2 cDNA per well of a 12-well plate. Cells were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde in PBS for 10 min at room temperature (RT). After three 10-min washes in PBS, the cells were blocked and permeabilized (30 min, RT) with 5% donkey serum in PBS containing 0.1% Triton X-100 and incubated overnight at 4 °C with rabbit polyclonal anti-CXCR2 antibodies (1:200; Abcam, ab14935) in PBS containing 5% donkey serum, 0.1% sodium azide, and 0.1% Triton X-100. Cells were then washed as above and incubated with Alexa Fluor 594 donkey anti-rabbit antibodies (1:200; Invitrogen; 1 h, RT). Cells were washed again as above, and the nucleus was stained with TO-PRO-3 Iodide (1:3000 in PBS, Invitrogen). Images were acquired using a Nikon A1R confocal microscope with 561 and 640 lasers and 595/50 and 700/75 emission filters, respectively, as well as a Nikon S Plan Fluor 40× objective lens. 12 bit (1024 × 1024) images were collected using the NIS Elements software.

      Prostate Immunohistochemistry

      Male C57Bl6/J mice were killed by cervical dislocation, and the ventral prostate was dissected out. Tissue was fixed in 4% formaldehyde in PBS (2 h, RT), washed 4 times with 7% sucrose in PBS containing 0.01% sodium azide (10 min each time), and stored in this solution (48 h, 4 °C). Prostates were then embedded in optimum cutting temperature compound (Sakura Finetek), snap-frozen in melting isopentane, and stored at −80 °C until use. Frozen 10-μm prostate sections were cut using a cryostat (Leica CM 1850), thawed onto Superfrost Plus slides (Menzel-Glaser), and left to air dry (1 h). Sections were washed with PBS (10 min) then blocked with 10% donkey serum and 1% bovine serum albumin (BSA) in PBS containing 0.1% lysine, 0.1% sodium azide, and 0.2% Tween 20 (30 min, RT). After blocking, slide-mounted sections were incubated (40–48 h, 4 °C) with rabbit polyclonal anti-CXCR2 antibodies (1:100; Abcam, ab14935) in PBS containing 0.01% BSA, 5% donkey serum, 0.1% lysine, 0.1% sodium azide, and 0.1% Tween 20. For negative control sections, rabbit polyclonal anti-CXCR2 antibodies were omitted. Sections were washed 4 times with PBS (10 min each time) and incubated with fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit antibodies (1:200; Jackson ImmunoResearch Laboratories; 1 h, RT). After secondary antibody incubation, sections were again washed 4 times with PBS (10 min each time), then stained (10 min, RT) with DAPI (300 ng/ml, Sigma) in PBS. Sections were then washed with PBS and mounted with Vectashield mounting media (Vector Laboratories). Sections were viewed using an Olympus BX61 fluorescence microscope fitted with an Olympus mercury burner light source attachment and Olympus UPlanSApo 20× and 40× objectives. CXCR2 staining (FITC, green) was viewed using an Olympus U-MNIBA3 filter cube consisting of a DM505 dichroic mirror, BP470-490 exciter filter, and BA515-550 barrier filter. Staining for DAPI (blue) was viewed using an Olympus U-MNUA2 filter cube consisting of a DM400 dichroic mirror, BP360-370 exciter filter, and BA420-460 barrier filter. An Olympus F-view II digital camera and analySIS LS Research software were used to acquire 16 bit photomicrographs. Prior approval for animal experimentation was granted by the Monash University Standing Committee on Animal Ethics (VCPA 2009/15).

      GPCR-HIT BRET Assays

      As described previously (
      • See H.B.
      • Seeber R.M.
      • Kocan M.
      • Eidne K.A.
      • Pfleger K.D.
      Application of G protein-coupled receptor-heteromer identification technology to monitor β-arrestin recruitment to G protein-coupled receptor heteromers.
      ), HEK293FT cells were transiently transfected with cDNA encoding a GPCR fused to Rluc8 (GPCR/Rluc8) and β-arrestin2 fused to Venus (β-arrestin2/Venus) along with a second GPCR that was untagged with respect to BRET signaling or empty vector. Initially DNA amounts of 0.7, 0.3, and 0.1 μg/well of a 6-well plate were used for each construct respectively, but this was changed to 0.1, 0.3, and 0.1 μg/well, respectively, after extensive cDNA titration assays (supplemental Fig. 1). 48 h post-transfection cells were incubated at 37 °C, 5% CO2 for 2 h with 30 μm EnduRen (Promega) to ensure substrate equilibrium was reached. BRET measurements were taken at 37 °C using the VICTOR Light plate reader with Wallac 1420 software (PerkinElmer Life Sciences). Filtered light emissions were sequentially measured at 400–475 and 520–540 nm. The BRET signal was calculated by subtracting the ratio of 520–540 nm emission over 400–475-nm emission for a vehicle-treated cell sample from the same ratio for a second aliquot of the same cells treated with agonist, as described previously (
      • Pfleger K.D.
      • Dromey J.R.
      • Dalrymple M.B.
      • Lim E.M.
      • Thomas W.G.
      • Eidne K.A.
      Extended bioluminescence resonance energy transfer (eBRET) for monitoring prolonged protein-protein interactions in live cells.
      ,
      • Pfleger K.D.
      • Seeber R.M.
      • Eidne K.A.
      Bioluminescence resonance energy transfer (BRET) for the real-time detection of protein-protein interactions.
      ). In this calculation the vehicle-treated cell sample represents the background, eliminating the requirement for measuring a donor-only control sample (
      • Pfleger K.D.
      • Dromey J.R.
      • Dalrymple M.B.
      • Lim E.M.
      • Thomas W.G.
      • Eidne K.A.
      Extended bioluminescence resonance energy transfer (eBRET) for monitoring prolonged protein-protein interactions in live cells.
      ,
      • Pfleger K.D.
      • Seeber R.M.
      • Eidne K.A.
      Bioluminescence resonance energy transfer (BRET) for the real-time detection of protein-protein interactions.
      ). For these BRET kinetic assays, the final pretreatment reading is presented at the zero time point (time of ligand/vehicle addition). The situation where the addition of ligand specific for the untagged GPCR results in a ligand-induced BRET signal indicates β-arrestin binding specifically to a heteromer complex (
      • See H.B.
      • Seeber R.M.
      • Kocan M.
      • Eidne K.A.
      • Pfleger K.D.
      Application of G protein-coupled receptor-heteromer identification technology to monitor β-arrestin recruitment to G protein-coupled receptor heteromers.
      ).

      Measurement of Fluorescence

      Fluorescence after light excitation was measured on an EnVision 2102 multilabel plate reader (PerkinElmer Life Sciences) using a 485/14 excitation filter, 535/25 emission filter, and D505 mirror.

      BRET Assays with Both Receptors Tagged

      BRET saturation assays were performed using HEK293FT cells transiently transfected with cDNA encoding α1AAR/Rluc8 and either CXCR2/Venus, V2R/Venus, or pcDNA3 empty vector only, the latter being donor-only control cells. 0.05 μg of α1AAR/Rluc8 cDNA along with between 0.01 and 0.85 μg of Venus-tagged receptor cDNA was transfected per well of a 6-well plate. In each case total cDNA was made up to 0.9 μg with pcDNA3 empty vector cDNA. For assessing the effect of ligands on the BRET between tagged receptors, cells were similarly transfected with cDNA encoding α1AAR/Rluc8 and either CXCR2/Venus or pcDNA3 empty vector. 0.25 μg of each construct was transfected per well of a 6-well plate (0.5 μg/well total). Triplicate wells of each transfection were aliquoted into both a white well microplate for BRET measurements and a parallel black microplate for fluorescence measurements. 48 h post-transfection, cells for BRET measurements were incubated at 37 °C, 5% CO2 for 2 h with 30 μm EnduRen (Promega). Meanwhile, fluorescence measurements were taken after light excitation as described above. BRET measurements were taken at 37 °C using the VICTOR Light plate reader with Wallac 1420 software (PerkinElmer Life Sciences). Filtered light emissions were sequentially measured at 400–475 and 520–540 nm. The BRET ratio was calculated by subtracting the ratio of 520–540 nm emission over 400–475 nm emission for donor-only control cells from the same ratio for cells transfected with both α1AAR/Rluc8 and Venus-tagged receptor, as described previously (
      • Pfleger K.D.
      • Dromey J.R.
      • Dalrymple M.B.
      • Lim E.M.
      • Thomas W.G.
      • Eidne K.A.
      Extended bioluminescence resonance energy transfer (eBRET) for monitoring prolonged protein-protein interactions in live cells.
      ). The fluorescence/luminescence ratio for the BRET saturation assays was generated by dividing the fluorescence values in arbitrary units (obtained in parallel after laser excitation) by the luminescence values also in arbitrary units (obtained as part of the BRET assay).

      Measurement of Total Inositol Phosphate Production Using [3H]myo-Inositol

      HEK293FT cells were seeded in 6-well plates and transiently transfected the next day. 24 h post-transfection cells were split into 24-well plates in inositol-free Complete Medium (MP Biomedicals) containing 1% dialyzed FCS. 6–8 h later medium was replaced with inositol-free medium containing 1% dialyzed FCS and 1 μCi/ml [3H]myo-inositol (Amersham Biosciences) followed by overnight incubation. Total inositol phosphate production was then measured as described previously (
      • Pfleger K.D.
      • Dromey J.R.
      • Dalrymple M.B.
      • Lim E.M.
      • Thomas W.G.
      • Eidne K.A.
      Extended bioluminescence resonance energy transfer (eBRET) for monitoring prolonged protein-protein interactions in live cells.
      ). This method was used to generate the data for Fig. 2B.

      Measurement of Inositol 1-Phosphate Production Using Time-resolved FRET

      The determination of inositol 1-phosphate accumulation was performed in 96-well microplates using the IP-One HTRF assay (CisBio Bioassays, Bagnol sur Ceze, France) (
      • Trinquet E.
      • Fink M.
      • Bazin H.
      • Grillet F.
      • Maurin F.
      • Bourrier E.
      • Ansanay H.
      • Leroy C.
      • Michaud A.
      • Durroux T.
      • Maurel D.
      • Malhaire F.
      • Goudet C.
      • Pin J.P.
      • Naval M.
      • Hernout O.
      • Chrétien F.
      • Chapleur Y.
      • Mathis G.
      D-myo-Inositol 1-phosphate as a surrogate of D-myo-inositol 1,4,5-tris phosphate to monitor G protein-coupled receptor activation.
      ). For most assays, cells were incubated for 1 h at 37 °C in the stimulation buffer (10 mm HEPES (pH 7.4), 1 mm CaCl2, 0.5 mm MgCl2, 4 mm KCl, 146 mm NaCl, 5.5 mm glucose, and 50 mm LiCl) with or without the indicated ligands. For the dose-response curves, cells were incubated in the stimulation buffer with NE or Labetalol at increasing doses for 40 min after preincubation with 10 μm Labetalol or vehicle for 20 min. Cells were then lysed using the conjugate-lysis buffer mixed with the terbium cryptate-labeled anti-inositol 1-phosphate antibody and the d2-labeled inositol 1-phosphate analog according to the manufacturer's instructions. The assay was incubated for 1 h at RT and terbium cryptate fluorescence and time-resolved FRET signal were measured at 620 and 665 nm, respectively, 50 μs after excitation at 337 nm using the PHERAstar FS (BMG Labtech, Mornington, Victoria, Australia) or EnVision 2102 (PerkinElmer Life Sciences) multilabel plate reader. This method was used to generate the data for Figs. 5F, 7, and 8, B and D.
      Figure thumbnail gr5
      FIGURE 5Specificity of β-arrestin recruitment to the α1AAR-CXCR2 heteromer. eBRET kinetic profiles were generated with HEK293FT cells expressing β-arrestin2/Venus and α1AAR with CXCR2/Rluc8 (A), CCR2/Rluc8 (B), V2R/Rluc8 (C), or OxR1/Rluc8 (D). Cells were treated with the agonist for the Rluc8-tagged GPCR (100 nm) or NE (100 nm). Data are representative of at least three independent experiments. AVP, arginine vasopressin. OxA, Orexin A. Luminescence and fluorescence values were recorded for the different combinations (E) as well as NE-induced inositol phosphate production (F). Data in F are shown as a percentage of the signal generated by NE-treated CXCR2/Rluc8, β-arrestin2/Venus, and α1AAR-expressing cells (control, first column). For E and F, data are shown as the mean ± S.E. of at least three independent experiments carried out with the same transfections as used to generate the data for A–D.
      Figure thumbnail gr2
      FIGURE 2Profiling of the novel α1AAR-CXCR2 heteromer. HEK293FT cells co-expressing β-arrestin2/Venus with either α1AAR/Rluc8 only or CXCR2/Rluc8 and α1AAR were monitored at 37 °C using BRET to generate dose-response curves (A). The mean BRET EC50 value ± S.E. for the CXCR2/Rluc8 and α1AAR combination was 29.7 ± 2.1 nm, and the mean Hill slope ± S.E. was 1.4 ± 0.1. Total inositol phosphate signaling dose-response curves were also generated to demonstrate the functionality of α1AAR/Rluc8 compared with untagged α1AAR and to compare the signaling of α1AAR in the presence and absence of CXCR2 (B). The mean EC50 values ± S.E. were not significantly different for α1AAR/Rluc8 and α1AAR co-expressed with CXCR2 compared with α1AAR (p > 0.05) and were 30.1 ± 11.6, 86.3 ± 45.6, and 52.2 ± 14.9 nm, respectively (statistics carried out using logEC50 values). Extended BRET (eBRET) kinetic profiles (
      • Pfleger K.D.
      • Dromey J.R.
      • Dalrymple M.B.
      • Lim E.M.
      • Thomas W.G.
      • Eidne K.A.
      Extended bioluminescence resonance energy transfer (eBRET) for monitoring prolonged protein-protein interactions in live cells.
      ,
      • Dalrymple M.B.
      • Jaeger W.C.
      • Eidne K.A.
      • Pfleger K.D.
      Temporal profiling of orexin receptor-arrestin-ubiquitin complexes reveals differences between receptor subtypes.
      ) were generated with cells co-expressing CXCR2/Rluc8 and β-arrestin2/Venus with or without α1AAR and activated with 100 nm CXCL8 and/or 100 μm NE (C). Profiles were also generated with cells co-expressing α1AAR/Rluc8 and β-arrestin2/Venus with or without CXCR2 (D). Data are representative of at least three independent experiments.
      Figure thumbnail gr4
      FIGURE 4Use of Terazosin (α1AR antagonist) and SB265610 (CXCR2 inverse agonist) to interrogate β-arrestin2/Venus recruitment. eBRET kinetic profiles were generated for the CXCR2/Rluc8, β-arrestin2/Venus and α1AAR combination in HEK293FT cells by treating with CXCL8 or vehicle (A) or NE or vehicle (B) ∼30 min before a second treatment with vehicle, 10 μm Terazosin, and/or 10 μm SB265610. Data are representative of three independent experiments.
      Figure thumbnail gr7
      FIGURE 7Effect of Terazosin and SB265610 on NE-induced inositol phosphate production from cells expressing α1AAR and CXCR2. Data are shown as the mean ± S.E. of three independent experiments generated from the same transfections of HEK293FT cells co-expressing CXCR2/Rluc8, β-arrestin2/Venus, and α1AAR used to generate data shown in . Data are shown as a percentage of the signal generated by NE-treated cells pretreated with vehicle (control, first column).
      Figure thumbnail gr8
      FIGURE 8Comparison of Labetalol and NE treatment on β-arrestin2/Venus recruitment and inositol phosphate production. HEK293FT cells co-expressing β-arrestin2/Venus with either α1AAR/Rluc8 or CXCR2/Rluc8 and α1AAR were monitored at 37 °C for BRET (A) and inositol phosphate production (B) to generate dose-response curves. In a separate set of experiments, dose-response curves for BRET (C) and inositol phosphate production (D) were generated with the same transfection of HEK293FT cells transiently co-expressing CXCR2/Rluc8, β-arrestin2/Venus, and α1AAR. For both, preincubation with 10 μm Labetalol or vehicle for 20 min was followed by activation with increasing doses of NE or Labetalol as indicated for 40 min. For comparison, both C and D have been presented as percentage of response to NE in the absence of Labetalol treatment (NE control). The mean BRET EC50 values ± S.E. for NE and Labetalol (with vehicle preincubation) were 16.2 ± 1.6 and 96.2 ± 10.1 nm, respectively. The mean inositol phosphate production EC50 values ± S.E. for NE with vehicle preincubation, Labetalol with vehicle preincubation, and NE with Labetalol preincubation were 7.1 ± 1.6, 19.9 ± 6.6, and 9634 ± 2495 nm, respectively. eBRET kinetic profiles were generated for the CXCR2/Rluc8, β-arrestin2/Venus, and α1AAR combination (E), showing the effect of 10 μm Labetalol compared with 10 μm Terazosin (Tera) when added ∼30 min after activation by a submaximal dose of NE (100 nm). The effect of a submaximal dose of Labetalol (300 nm) with or without subsequent 10 μm Terazosin treatment is also shown along with the effect of 10 μm Terazosin alone. Data are representative of three independent experiments.

      Confocal Microscopy Assessing Localization

      550,000 HEK293FT cells/well were seeded in a 6-well plate and transiently transfected the next day with 0.1 μg of HA-α1AAR, 0.3 μg of β-arrestin2/Venus, and 0.1 μg of CXCR2 cDNA. Alternatively, cells were transfected with 0.3 μg of β-arrestin2/Venus cDNA with or without 0.1 μg of CXCR2 cDNA as controls. In each case total cDNA was made up to 0.5 μg with pcDNA3 empty vector cDNA. These cells were then seeded in 12-well plates containing sterile poly-d-lysine-coated coverslips at a density of 550,000 cells/well 24 h post-transfection and allowed to attach overnight at 37 °C in 5% CO2. Treatments were carried out the next day; transfected cells were labeled with an anti-HA antibody (Sigma; 40 min) followed by ligand/vehicle treatment (30 min), both at 37 °C. Cells were washed with PBS at 4 °C and fixed with 4% (w/v) paraformaldehyde/5% (w/v) sucrose (10 min, RT) and washed again. Nuclei were stained with Hoechst 33258 (Invitrogen; 10 min, RT), and cells were permeabilized with 3% (w/v) milk, 0.15% (v/v) Triton X-100 (10 min, RT). Cells were then incubated with goat anti-rabbit Alexa 546 (Invitrogen; 1 h, RT). Cells were washed as before, and the coverslips mounted onto microscope slides with polyvinyl alcohol/glycerol mounting media. Coverslips were then sealed and stored at 4 °C in the dark until required. Cells were imaged with a Nikon A1Si confocal microscope with 405-, 488-, and 561-nm lasers utilizing a Nikon Apo 60×, NA 1.49 TIRF oil immersion lens. 12 bit, 1024 × 1024 pixel images were collected as Z-stacks with 4-line averaging and deconvoluted using the 3D Iterative Blind Deconvolution algorithm in the Lab Imaging package of NIS Elements software (Nikon).

      Data Presentation and Statistical Analysis

      Data were analyzed using Prism 5 graphing software (GraphPad) with α designated as 0.05. Sigmoidal curves were fitted to the dose-response data using non-linear regression. Statistical analysis of logEC50 values was carried out using one-way analysis of variance. BRET saturation curves were generated using non-linear regression assuming one site binding.

      RESULTS

      Prostate Immunohistochemistry

      To assess CXCR2 expression in the prostate, we utilized an antibody that has been used extensively to label this receptor (
      • Wang Y.
      • Luo W.
      • Reiser G.
      Activation of protease-activated receptors in astrocytes evokes a novel neuroprotective pathway through release of chemokines of the growth-regulated oncogene/cytokine-induced neutrophil chemoattractant family.
      ,
      • Sugimoto N.
      • Rui T.
      • Yang M.
      • Bharwani S.
      • Handa O.
      • Yoshida N.
      • Yoshikawa T.
      • Kvietys P.R.
      Points of control exerted along the macrophage-endothelial cell-polymorphonuclear neutrophil axis by PECAM-1 in the innate immune response of acute colonic inflammation.
      ,
      • Martínez Muñoz L.
      • Lucas P.
      • Navarro G.
      • Checa A.I.
      • Franco R.
      • Martínez-A C.
      • Rodríguez-Frade J.M.
      • Mellado M.
      Dynamic regulation of CXCR1 and CXCR2 homo- and heterodimers.
      ,
      • Moldobaeva A.
      • Wagner E.M.
      Difference in proangiogenic potential of systemic and pulmonary endothelium. role of CXCR2.
      ). This antibody labeled CXCR2-transfected CHO cells (supplemental Fig. 2A) but not CCR2-transfected (supplemental Fig. 2B) or mock-transfected (supplemental Fig. 2C) CHO cells assessed in parallel. Consequently, specific expression of CXCR2 was identified in the thin stromal smooth muscle layer of mouse prostate tissue (Fig. 1). Expression of α1AAR in the prostate stroma is well established in the literature (
      • Kojima Y.
      • Sasaki S.
      • Hayashi Y.
      • Tsujimoto G.
      • Kohri K.
      Subtypes of α1-adrenoceptors in BPH. Future prospects for personalized medicine.
      ). Indeed, in the human prostatic α1AARs resulting in the α1LAR phenotype are found exclusively in the stromal layer (
      • Morishima S.
      • Suzuki F.
      • Nishimune A.
      • Yoshiki H.
      • Akino H.
      • Yokoyama O.
      • Muramatsu I.
      Visualization and tissue distribution of α1L-adrenoceptor in human prostate by the fluorescently labeled ligand Alexa-488-silodosin.
      ). Given this overlapping expression pattern in prostate tissue, specifically in prostate stroma, the functional interaction between α1AAR and CXCR2 was further investigated in a recombinant system.
      Figure thumbnail gr1
      FIGURE 1Immunofluorescent localization of CXCR2 in the mouse prostate stroma. Representative photomicrographs are shown (n = 5) of fixed, frozen cross-sections of the mouse prostate immunostained with rabbit polyclonal antibodies to CXCR2 (green) with DAPI (blue) counterstaining. The control section incubated without CXCR2 antibodies being present shows an absence of specific staining (A). In contrast, positive CXCR2 immunostaining can be seen specifically in the stroma, as indicated by white arrows (B). Panels C and D (40× objective) are higher magnification of panels A and B (20× objective), respectively. Scale bars, 50 μm.

      Profiling of Functional Interaction between α1AAR and CXCR2

      A very strong dose-dependent BRET signal, indicative of an α1AAR-CXCR2 heteromer, was observed after NE treatment of HEK293FT cells expressing β-arrestin2/Venus, CXCR2/Rluc8, and untagged α1AAR (Fig. 2A). This result was despite a distinct lack of BRET signal at any dose of NE with cells expressing β-arrestin2/Venus and α1AAR/Rluc8 (Fig. 2A), consistent with previous evidence for α1AAR interacting extremely weakly with β-arrestin in transfected HEK293 cells (
      • Stanasila L.
      • Abuin L.
      • Dey J.
      • Cotecchia S.
      Different internalization properties of the α1a- and α1b-adrenergic receptor subtypes. The potential role of receptor interaction with β-arrestins and AP50.
      ). The fusion with Rluc8 had no effect on NE-induced inositol phosphate signaling from the α1AAR (Fig. 2B), providing evidence for the functionality of the α1AAR/Rluc8 construct. Furthermore, co-expression of CXCR2 did not appear to influence NE-induced coupling to the inositol phosphate signaling pathway (Fig. 2B). The NE-induced BRET signal between CXCR2/Rluc8 and β-arrestin2/Venus was dependent upon the co-expression of α1AAR, although α1AAR was not required for strong CXCL8 (interleukin-8)-induced recruitment of β-arrestin2/Venus to CXCR2/Rluc8 (Fig. 2C). Although the signal obtained after co-stimulation with NE and CXCL8 was greater, it was clearly less than the sum of separate NE and CXCL8 treatments (Fig. 2C). BRET kinetic profiles for the reciprocal arrangement of α1AAR/Rluc8 and β-arrestin2/Venus with or without CXCR2 were notably of much smaller magnitude (Fig. 2D), indicating substantially less resonance energy transfer. These profiles confirmed that both the NE- and CXCL8-induced BRET signals require the presence of CXCR2 and that NE induces little or no proximity between α1AAR/Rluc8 and β-arrestin2/Venus in the absence of CXCR2 (Fig. 2D). Furthermore, co-treatment with both agonists results in a substantially greater signal with this orientation. These observations are consistent with CXCR2 playing a key role in the interaction of the heteromer with β-arrestin2.

      BRET Titration Assays

      Extensive titration assays were carried out to show the effect of varying the concentration and ratio of CXCR2/Rluc8, β-arrestin2/Venus, and α1AAR cDNA transfected (supplemental Fig. 1). These experiments showed that specific ligand-induced BRET signals were obtained with a wide range of cDNA concentrations and ratios. Furthermore, these signals were similar when CXCL8-induced (supplemental Fig. 1, A, B, E, F, and I) compared with when NE-induced (supplemental Fig. 1, C, D, G, H, and J). Both CXCL8-induced (supplemental Fig. 1F) and NE-induced (supplemental Fig. 1H) BRET signals were obtained with very low concentrations of cDNA, providing further support for these signals not being artifacts of protein overexpression. Finally, when the donor cDNA amount was kept constant and acceptor cDNA amount increased, signal saturation was observed (supplemental Fig. 1, I and J), again providing evidence for signal specificity.

      Confocal Microscopy Assessing Localization

      To further support our findings, agonist-treated HEK293FT cells co-expressing HA-α1AAR and β-arrestin2/Venus with or without untagged CXCR2 were visualized (Fig. 3). In the absence of ligand treatment, β-arrestin2/Venus was diffuse throughout the cytoplasm (Fig. 3, F and H), and HA-α1AAR was primarily localized to the plasma membrane (Fig. 3, K and M) of both cell populations. This is particularly clear in the merged images (Fig. 3, P and R). NE treatment did not change this expression pattern in the absence of CXCR2 (Fig. 3, G, L, and Q). As mentioned previously, α1AAR has been shown to interact very weakly, if at all, with β-arrestin2 (
      • Stanasila L.
      • Abuin L.
      • Dey J.
      • Cotecchia S.
      Different internalization properties of the α1a- and α1b-adrenergic receptor subtypes. The potential role of receptor interaction with β-arrestins and AP50.
      ); therefore, it is not surprising that NE treatment did not result in a change in the localization of β-arrestin2/Venus or noticeable α1AAR internalization. However, in the presence of CXCR2, NE-induced HA-α1AAR internalization into punctate vesicles was observed (Fig. 3N) along with some β-arrestin2/Venus translocation (Fig. 3I). CXCL8 treatment of cells expressing both receptors also resulted in β-arrestin2/Venus translocation (Fig. 3J) and HA-α1AAR internalization (Fig. 3O), with colocalization of β-arrestin2/Venus and HA-α1AAR clearly evident (Fig. 3T). These observations are consistent with the BRET data shown in Fig. 2 and support the role of CXCR2 in α1AAR internalization.
      Figure thumbnail gr3
      FIGURE 3β-Arrestin2/Venus translocation and HA-α1AAR internalization studies on cells with or without untagged CXCR2. HEK293FT cells were visualized using confocal microscopy to observe the nuclei with Hoechst staining (blue, A–E), β-arrestin2/Venus localization (green, F–J), HA-α1AAR localization (red, K–O), and merged images of all three (P–T). Cells expressing β-arrestin2/Venus and HA-α1AAR were treated with vehicle (A, F, K, and P) or 100 nm NE (B, G, L, and Q) for 30 min. Similarly, cells expressing β-arrestin2/Venus, HA-α1AAR, and untagged CXCR2 were treated with vehicle (C, H, M, and R) or 100 nm NE (D, I, N, and S) or 100 nm CXCL8 (E, J, O, and T) for 30 min. Scale bars, 10 μm.
      Further controls are provided in the form of cells assessed in parallel that were transfected with β-arrestin2/Venus only or β-arrestin2/Venus and untagged CXCR2 in the absence of HA-α1AAR (supplemental Fig. 3). No translocation was observed in β-arrestin2/Venus-transfected cells upon treatment with vehicle (supplemental Fig. 3A), NE (supplemental Fig. 3B), or CXCL8 (supplemental Fig. 3C). Similarly, no β-arrestin translocation was observed in cells transfected with β-arrestin2/Venus and CXCR2 upon treatment with vehicle (supplemental Fig. 3D) or NE (supplemental Fig. 3E); however, a punctate distribution was observed upon treatment with CXCL8 (supplemental Fig. 3F).

      Use of Selective Inhibitors to Interrogate β-Arrestin2 Recruitment to Heteromer

      Terazosin is a selective α1AR antagonist (
      • Hieble J.P.
      • Ruffolo Jr., R.R.
      The use of α-adrenoceptor antagonists in the pharmacological management of benign prostatic hypertrophy. An overview.
      ), and SB265610 has recently been characterized as a cell-permeable selective allosteric inverse agonist of CXCR2 believed to bind to the intracellular C-terminal tail (
      • Bradley M.E.
      • Bond M.E.
      • Manini J.
      • Brown Z.
      • Charlton S.J.
      SB265610 is an allosteric, inverse agonist at the human CXCR2 receptor.
      ,
      • Salchow K.
      • Bond M.E.
      • Evans S.C.
      • Press N.J.
      • Charlton S.J.
      • Hunt P.A.
      • Bradley M.E.
      A common intracellular allosteric binding site for antagonists of the CXCR2 receptor.
      ).
      With cells co-expressing CXCR2/Rluc8, β-arrestin2/Venus, and α1AAR, as expected, Terazosin did not inhibit the CXCL8-induced BRET signal (Fig. 4A), but did inhibit the NE-induced BRET signal (Fig. 4B). Whether the former result indicates that CXCL8-induced β-arrestin recruitment to the α1AAR-CXCR2 heteromer can occur in the presence of an α1AR antagonist, or whether this BRET signal is predominantly from β-arrestin2/Venus recruitment to CXCR2/Rluc8 monomers/homomers is unclear. This ambiguity illustrates the value of profiling with the ligand specific for the untagged receptor. However, the data clearly demonstrate that NE-induced β-arrestin2 recruitment to the α1AAR-CXCR2 heteromer requires the activation of α1AAR.
      A particularly important finding was that, in addition to blocking the CXCL8-induced BRET signal (Fig. 4A), SB265610 also blocked the NE-induced BRET signal about as effectively as terazosin (Fig. 4B). Terazosin and SB265610 in combination had the same effect on blocking CXCL8-induced BRET as SB265610 alone (Fig. 4A), whereas the combination had a slightly greater effect on blocking NE-induced BRET compared with either Terazosin or SB265610 alone (Fig. 4B). The categorization of SB265610 as an inverse agonist (
      • Bradley M.E.
      • Bond M.E.
      • Manini J.
      • Brown Z.
      • Charlton S.J.
      SB265610 is an allosteric, inverse agonist at the human CXCR2 receptor.
      ) is supported by the reduction in basal ligand-induced BRET ratio over time (Fig. 4, A and B).
      The inhibition of both CXCL8- and NE-induced BRET signals by SB265610 indicates a critical role for CXCR2 in β-arrestin2 recruitment to the α1AAR-CXCR2 heteromer regardless of which receptor is initially activated by agonist.

      Specificity of β-Arrestin Recruitment to α1AAR-CXCR2 Heteromer

      NE-induced β-arrestin recruitment shows specificity for the α1AAR-CXCR2 heteromer (Fig. 5A), as it is not observed when untagged α1AAR is co-expressed with various other Rluc8-tagged GPCRs, including chemokine receptor CCR2 (Fig. 5B), vasopressin receptor 2 (V2R, Fig. 5C) and orexin receptor 1 (OxR1, Fig. 5D). Luminescence and fluorescence values were similar for the different combinations, indicating similar expression of Rluc8 and Venus-tagged proteins (Fig. 5E). NE-induced inositol phosphate production was also similar for each combination, indicating similar expression of α1AAR (Fig. 5F). The proximity/orientation dependence of BRET is such that a lack of BRET signal does not necessarily mean a lack of proximity between proteins of interest (
      • Levoye A.
      • Dam J.
      • Ayoub M.A.
      • Guillaume J.L.
      • Couturier C.
      • Delagrange P.
      • Jockers R.
      The orphan GPR50 receptor specifically inhibits MT1 melatonin receptor function through heterodimerization.
      ), but it is at least consistent with this situation. It is also important to note that a receptor could be in close proximity to α1AAR without altering its pharmacology (in this case without enabling NE-induced β-arrestin recruitment).

      Assessment of BRET between Receptors

      BRET saturation assays were carried out to compare with the findings from the GPCR-HIT assay, assessing the α1AAR and CXCR2 combination as well as the α1AAR and V2R combination as a control. A saturation curve with a BRETmax of 0.96 was fitted to the data with α1AAR/Rluc8 and CXCR2/Venus, consistent with these receptors being in close proximity in a constitutive heteromer (Fig. 6A). Surprisingly, despite no NE-induced β-arrestin recruitment being observed with GPCR-HIT with V2R/Rluc8, β-arrestin2/Venus, and α1AAR (Fig. 5C), the α1AAR/Rluc8 and V2R/Venus combination resulted in a saturation curve with a BRETmax of 1.22 (Fig. 6A). This is consistent with V2R being in close proximity to α1AAR in these cells yet not modulating α1AAR pharmacology to enable NE to induce β-arrestin recruitment. This further illustrates the specificity of the GPCR-HIT assay due to its dependence upon a ligand-induced functional effect.
      Figure thumbnail gr6
      FIGURE 6Assessment of BRET between receptors. Saturation BRET data were generated with HEK293FT cells co-expressing α1AAR/Rluc8 with either CXCR2/Venus or V2R/Venus (A). The graph was generated by combining data from four independent experiments carried out in triplicate (error bars represent mean ± S.E. of triplicate data). Cells transfected with equal amounts of α1AAR/Rluc8 and CXCR2/Venus cDNA were treated for 20 min with vehicle, 10 μm Terazosin, 10 μm SB265610, or both (B). Similarly, cells were treated for 20 min with vehicle, 100 nm CXCL8, or 100 nm NE (C). Data for B and C shown as the mean ± S.E. of three independent experiments. Data are presented as BRET ratio (minus donor-only control) to assess the constitutive BRET signal between the receptors.
      One possible explanation for the inhibition of the ligand-induced BRET signals observed in Fig. 4 by Terazosin and/or SB265610 is that the heteromer is disrupted by treatment with these ligands. However, the constitutive BRET signal between α1AAR/Rluc8 and CXCR2/Venus was not altered by treatment with Terazosin, SB265610, or a combination of both (Fig. 6B). The effect of CXCL8 and NE was also assessed, and again no change in BRET signal was observed (Fig. 6C).

      Effect of Inhibitors on NE-induced Inositol Phosphate Production

      In contrast to BRET investigating β-arrestin recruitment, NE-induced inositol phosphate production was completely inhibited by Terazosin but was unaffected by SB265610 (Fig. 7). Note that, crucially, the NE-induced BRET signal observed is specific for the heteromer, whereas the NE-induced inositol phosphate signal could result from activation of either α1AAR-CXCR2 heteromers or α1AAR monomers/homomers. Therefore, the lack of inhibition by SB265610 could be explained by the ability of α1AAR to recruit G protein to the heteromer even when CXCR2 is in an inactive conformation, implying recruitment of the G protein directly to the activated α1AAR protomer. Alternatively or additionally, the inositol phosphate production observed may result from activation of α1AAR monomers/homomers, as the inositol phosphate assay cannot distinguish between these receptor populations.

      Evidence for Labetalol Exhibiting Heteromer-specific Biased Agonism

      Upon preliminary testing of a selection of adrenoceptor-selective compounds, agonist activity was observed with Labetalol, which is marketed as a nonselective β-AR antagonist with α1AR antagonist properties. As partial agonist activity at β-ARs had been observed previously with this compound (
      • Galandrin S.
      • Bouvier M.
      Distinct signaling profiles of β1 and β2 adrenergic receptor ligands toward adenylyl cyclase and mitogen-activated protein kinase reveals the pluridimensionality of efficacy.
      ,
      • Wisler J.W.
      • DeWire S.M.
      • Whalen E.J.
      • Violin J.D.
      • Drake M.T.
      • Ahn S.
      • Shenoy S.K.
      • Lefkowitz R.J.
      A unique mechanism of β-blocker action. Carvedilol stimulates β-arrestin signaling.
      ) but to our knowledge not at α-ARs, we profiled this particular compound further. Similarly to NE, a very strong dose-dependent BRET signal was observed after Labetalol treatment of cells expressing β-arrestin2/Venus, CXCR2/Rluc8, and untagged α1AAR, in contrast to the distinct lack of BRET signal with cells expressing β-arrestin2/Venus and α1AAR/Rluc8 (Fig. 8A). From the same transfections, Labetalol-induced dose-dependent inositol phosphate production was observed with both combinations (Fig. 8B), indicating that like NE, Labetalol acting on the α1AAR monomer/homomer is able to induce G protein-coupling but little or no β-arrestin recruitment. We then investigated the relative levels of agonism of Labetalol for β-arrestin recruitment and inositol phosphate production compared with NE. For β-arrestin, Labetalol was almost a full agonist compared with NE, with 10 μm Labetalol preincubation resulting in only a marginal reduction in NE response (Fig. 8C). In contrast, Labetalol was only a partial agonist for inositol phosphate signaling, a 10 μm dose substantially reducing the response to doses of NE ≤ 1 μm and shifting the NE EC50 >1300-fold to the right (Fig. 8D). Furthermore, as inositol phosphate assays cannot distinguish between coupling to the receptor heteromer complex versus the α1AAR monomer/homomer, it is unclear what proportions of these signals if any are due to the α1AAR-CXCR2 complex rather than the α1AAR monomer/homomer (Fig. 8B). Therefore, if anything, these data overestimate the amount of Labetalol agonism at the receptor complex for the Gq/11 signaling pathway.
      Extended BRET (eBRET) kinetic profiles were generated using submaximal concentrations of NE and Labetalol (Fig. 8E). The addition of 10 μm Labetalol had little effect on the BRET signal resulting from 100 nm NE, in contrast to the effect of 10 μm Terazosin on the NE-induced BRET (Fig. 8E). The agonism of 300 nm Labetalol with respect to β-arrestin recruitment to the receptor complex was clearly observed and was inhibited by 10 μm Terazosin, which exhibited no agonism on its own. Together, these data indicate that although Labetalol is a partial agonist for inositol phosphate signaling from α1AAR, it is effectively a full agonist for β-arrestin recruitment to the α1AAR-CXCR2 complex, thereby defining it as a β-arrestin-biased ligand (
      • Rajagopal S.
      • Rajagopal K.
      • Lefkowitz R.J.
      Teaching old receptors new tricks: biasing seven-transmembrane receptors.
      ).

      DISCUSSION

      We have provided evidence for CXCR2 expression in the prostate stroma, a region specifically associated with α1AAR expression in vivo (
      • Morishima S.
      • Suzuki F.
      • Nishimune A.
      • Yoshiki H.
      • Akino H.
      • Yokoyama O.
      • Muramatsu I.
      Visualization and tissue distribution of α1L-adrenoceptor in human prostate by the fluorescently labeled ligand Alexa-488-silodosin.
      ). Visualization of the α1AAR was not carried out due to the lack of reliable α1AAR antibodies (
      • Jensen B.C.
      • Swigart P.M.
      • Simpson P.C.
      Ten commercial antibodies for α-1-adrenergic receptor subtypes are nonspecific.
      ). We then proceeded to demonstrate a “biochemical fingerprint” of the α1AAR-CXCR2 heteromer by uncovering novel and specific pharmacology attributable to the heteromer complex in HEK293 cells. This has been done primarily using a BRET-based approach, which although not giving absolute proof of direct physical association between the receptors, does provide strong evidence of both functional interaction and close proximity in a “macromolecular complex.”
      Using GPCR-HIT, we have shown that α1AAR, which has been published to interact with β-arrestin very weakly if at all in transfected HEK293 cells (
      • Stanasila L.
      • Abuin L.
      • Dey J.
      • Cotecchia S.
      Different internalization properties of the α1a- and α1b-adrenergic receptor subtypes. The potential role of receptor interaction with β-arrestins and AP50.
      ), can recruit and form a stable complex with β-arrestin when it is co-expressed with CXCR2, but not CCR2, V2R, or OxR1. Indeed, it even appears that co-expression of V2R does not have this effect despite specific close proximity with α1AAR in these cells as suggested by BRET saturation assays. Our evidence for α1AAR-CXCR2 complex formation with β-arrestin provided from BRET-based assays has been supported by visualization of co-localization using confocal microscopy. Indeed, the observation of CXCL8 causing α1AAR internalization is particularly compelling, especially as NE was not seen to induce internalization of α1AAR in the absence of CXCR2. Indeed, this nicely illustrates a role CXCR2 may play in regulating α1AAR.
      Intriguingly, not only the α1AR antagonist Terazosin but also the CXCR2-specific inverse agonist SB265610 was observed to inhibit NE-induced β-arrestin recruitment to the receptor complex. This inhibition does not appear to be due to disruption of the heteromer as such, as treatment with Terazosin, SB265610, or both did not inhibit the BRET signal between α1AAR/Rluc8 and CXCR2/Venus. Indeed, agonist treatment also did not affect the BRET between receptors, implying that heteromerization in this particular case occurs constitutively. Therefore, the observed inhibition of the GPCR-HIT BRET signals is likely to be due to blockade of receptor activation, blockade of actual β-arrestin2/Venus binding to the complex, and/or blockade of an allosteric effect across the complex that facilitates β-arrestin recruitment to the heteromer.
      There has been debate in the literature for several years regarding whether or not GPCR heteromerization, and indeed homomerization, is constitutive or ligand-modulated. Not that long ago most homo- and heteromerization was believed to occur constitutively, with receptors forming complexes in the endoplasmic reticulum and trafficking to the plasma membrane together (
      • Bulenger S.
      • Marullo S.
      • Bouvier M.
      Emerging role of homo- and heterodimerization in G-protein-coupled receptor biosynthesis and maturation.
      ) as well as internalizing together (
      • Milligan G.
      A day in the life of a G protein-coupled receptor. The contribution to function of G protein-coupled receptor dimerization.
      ). More recently, evidence has been presented of examples where homo- and heteromerization appears to be more dynamic (
      • Lohse M.J.
      Dimerization in GPCR mobility and signaling.
      ,
      • Hern J.A.
      • Baig A.H.
      • Mashanov G.I.
      • Birdsall B.
      • Corrie J.E.
      • Lazareno S.
      • Molloy J.E.
      • Birdsall N.J.
      Formation and dissociation of M1 muscarinic receptor dimers seen by total internal reflection fluorescence imaging of single molecules.
      ). Indeed BRET, including in the GPCR-HIT configuration, has recently been used to investigate the heteromer between the gastric inhibitory polypeptide (GIP) receptor and glucagon-like peptide-1 (GLP-1) receptor, with the authors proposing a model of dynamic heteromer formation induced by GLP-1 and dissolved by GIP (
      • Schelshorn D.
      • Joly F.
      • Mutel S.
      • Hampe C.
      • Breton B.
      • Mutel V.
      • Lütjens R.
      Lateral allosterism in the glucagon receptor family. Glucagon-like peptide 1 induces G-protein-coupled receptor heteromer formation.
      ). Furthermore GPCR-HIT in this case, as it has in our current study, enabled conclusions to be drawn regarding functional implications of heteromerization with respect to β-arrestin recruitment. Interestingly, the difference is that α1AAR-CXCR2 appears to be a constitutively formed heteromer that recruits β-arrestin in contrast to the α1AAR monomer/homomer, whereas the GIP receptor-GLP-1 receptor heteromer appears to form dynamically, with the heteromer recruiting β-arrestin less well than the GLP-1 receptor monomer/homomer (
      • Schelshorn D.
      • Joly F.
      • Mutel S.
      • Hampe C.
      • Breton B.
      • Mutel V.
      • Lütjens R.
      Lateral allosterism in the glucagon receptor family. Glucagon-like peptide 1 induces G-protein-coupled receptor heteromer formation.
      ). These differences illustrate that the novel pharmacology resulting from GPCR heteromerization can take various forms even just in terms of β-arrestin recruitment and can differ for each individual combination of receptors.
      Our findings suggest that an allosteric effect occurs between α1AAR and CXCR2 that modulates the functioning of the constitutively formed heteromer complex with regard to β-arrestin recruitment. There are essentially three potential scenarios for explaining our observations upon stimulation with NE, depending upon whether the β-arrestin2 associates with the α1AAR-CXCR2 heteromer via interaction with CXCR2, α1AAR, or both. The concept of asymmetric activation of GPCR heteromers is being increasingly recognized and has been discussed recently (
      • Rovira X.
      • Pin J.P.
      • Giraldo J.
      The asymmetric/symmetric activation of GPCR dimers as a possible mechanistic rationale for multiple signaling pathways.
      ,
      • Maurice P.
      • Kamal M.
      • Jockers R.
      Asymmetry of GPCR oligomers supports their functional relevance.
      ), so multiple scenarios are plausible.
      The first scenario, which we will term the “trans hypothesis,” involves NE-bound α1AAR allosterically activating (or trans-activating) CXCR2 that in turn binds β-arrestin2 (Fig. 9B). In this case, agonist binding to one of the protomers results in the activation of both protomers in the heteromer. This would account for the substantially weaker BRET signals obtained when the α1AAR is labeled rather than CXCR2 (Fig. 2). As the BRET signal is inversely proportional to distance to the sixth power (
      • Pfleger K.D.
      • Eidne K.A.
      Illuminating insights into protein-protein interactions using bioluminescence resonance energy transfer (BRET).
      ), NE-induced recruitment of β-arrestin2/Venus to interact with CXCR2/Rluc8 (Fig. 9C) would be expected to result in a much larger signal than recruiting β-arrestin2/Venus to interact with untagged CXCR2 (Fig. 9D), with the energy transfer then occurring across the complex from α1AAR/Rluc8. Arguably “trans” β-arrestin recruitment to CXCR2 in the α1AAR-CXCR2 heteromer is also consistent with the ability of not only the α1AR antagonist Terazosin (Fig. 9F) but also the CXCR2-specific inverse agonist SB265610 (Fig. 9H) to block NE-induced β-arrestin recruitment to the receptor complex.
      Figure thumbnail gr9
      FIGURE 9Model of trans hypothesis of β-arrestin recruitment to the α1AAR-CXCR2 heteromer. CXCL8 activation of CXCR2 results in recruitment of β-arrestin2 (β-arr) to CXCR2, which may or may not heteromerize with α1AAR (A). One hypothesis to explain our observations after NE treatment is that NE activation of α1AAR results in β-arrestin2 recruitment to the α1AAR-CXCR2 heteromer via interaction with CXCR2 (B). Consistent with this hypothesis are the different strengths of energy transfer shown in depending upon whether the Rluc8 fusion (black star) is with CXCR2 (C) or α1AAR (D). In terms of inhibitor treatments, Terazosin did not affect CXCL8-induced recruitment of β-arrestin2 to CXCR2 (E); however, it did inhibit both NE-induced Gq/11 coupling and β-arrestin2 recruitment (F). In contrast, SB265610 inhibited both CXCL8-induced (G) and NE-induced (H) β-arrestin2 recruitment without apparently affecting Gq/11 coupling, although unlike GPCR-HIT, the measurement of inositol phosphate production does not discriminate between α1AAR monomers/homomers and α1AAR-CXCR2 heteromers.
      The second scenario, which we will term the “cis hypothesis,” involves CXCR2 allosterically modulating the α1AAR as a consequence of heteromerization, with the result that NE-activated α1AAR is able to bind β-arrestin2 with high affinity when it could not as a monomer/homomer. Despite α1AAR containing two putative high affinity GPCR kinase (GRK) phosphorylation sites as defined by Oakley et al. (
      • Oakley R.H.
      • Laporte S.A.
      • Holt J.A.
      • Barak L.S.
      • Caron M.G.
      Association of β-arrestin with G protein-coupled receptors during clathrin-mediated endocytosis dictates the profile of receptor resensitization.
      ,
      • Oakley R.H.
      • Laporte S.A.
      • Holt J.A.
      • Barak L.S.
      • Caron M.G.
      Molecular determinants underlying the formation of stable intracellular G protein-coupled receptor-β-arrestin complexes after receptor endocytosis.
      ), it is phosphorylated much more weakly than α1BAR, which contains none of these serine/threonine clusters in the C-terminal tail (
      • Vázquez-Prado J.
      • Medina L.C.
      • Romero-Avila M.T.
      • González-Espinosa C.
      • García-Sáinz J.A.
      Norepinephrine- and phorbol ester-induced phosphorylation of α(1a)-adrenergic receptors. Functional aspects.
      ). This difference in phosphorylation is consistent with the difference in β-arrestin recruitment observed between the two receptor subtypes (
      • Stanasila L.
      • Abuin L.
      • Dey J.
      • Cotecchia S.
      Different internalization properties of the α1a- and α1b-adrenergic receptor subtypes. The potential role of receptor interaction with β-arrestins and AP50.
      ). Both of these observations are, therefore, counterintuitive but can be explained by recent studies that have provided evidence for differential GPCR phosphorylation resulting in a signaling “bar code” (
      • Tobin A.B.
      • Butcher A.J.
      • Kong K.C.
      Location, location, location … site-specific GPCR phosphorylation offers a mechanism for cell-type-specific signalling.
      ,
      • Butcher A.J.
      • Prihandoko R.
      • Kong K.C.
      • McWilliams P.
      • Edwards J.M.
      • Bottrill A.
      • Mistry S.
      • Tobin A.B.
      Differential G-protein-coupled receptor phosphorylation provides evidence for a signaling bar code.
      ,
      • Nobles K.N.
      • Xiao K.
      • Ahn S.
      • Shukla A.K.
      • Lam C.M.
      • Rajagopal S.
      • Strachan R.T.
      • Huang T.Y.
      • Bressler E.A.
      • Hara M.R.
      • Shenoy S.K.
      • Gygi S.P.
      • Lefkowitz R.J.
      Distinct phosphorylation sites on the β2-adrenergic receptor establish a barcode that encodes differential functions of β-arrestin.
      ,
      • Liggett S.B.
      Phosphorylation barcoding as a mechanism of directing GPCR signaling.
      ). This is the concept that, depending on the cellular context or even the ligand used to activate the receptor, distinct subsets of potential sites are phosphorylated, thereby enabling an additional layer of signaling diversity to be achieved depending on the particular receptor conformation and set of conditions (
      • Tobin A.B.
      • Butcher A.J.
      • Kong K.C.
      Location, location, location … site-specific GPCR phosphorylation offers a mechanism for cell-type-specific signalling.
      ,
      • Butcher A.J.
      • Prihandoko R.
      • Kong K.C.
      • McWilliams P.
      • Edwards J.M.
      • Bottrill A.
      • Mistry S.
      • Tobin A.B.
      Differential G-protein-coupled receptor phosphorylation provides evidence for a signaling bar code.
      ,
      • Nobles K.N.
      • Xiao K.
      • Ahn S.
      • Shukla A.K.
      • Lam C.M.
      • Rajagopal S.
      • Strachan R.T.
      • Huang T.Y.
      • Bressler E.A.
      • Hara M.R.
      • Shenoy S.K.
      • Gygi S.P.
      • Lefkowitz R.J.
      Distinct phosphorylation sites on the β2-adrenergic receptor establish a barcode that encodes differential functions of β-arrestin.
      ,
      • Liggett S.B.
      Phosphorylation barcoding as a mechanism of directing GPCR signaling.
      ). Therefore, an allosteric effect of CXCR2 that changes the α1AAR conformation could conceivably alter the phosphorylation bar code, enabling the α1AAR to be differentially phosphorylated and thereby changing its affinity for β-arrestin2. If this is the reality, the explanation for the different strength of BRET signals in the two tag orientations (Fig. 9, C and D) would presumably be attributed to the orientation involving α1AAR/Rluc8 (Fig. 9D) simply being much less favorable for energy transfer. Furthermore, as a consequence of its binding to the C-terminal tail of CXCR2, SB265610 may be able to influence a putative allosteric effect of CXCR2 on α1AAR that alters the phosphorylation bar code on the α1AAR C-terminal tail, thereby indirectly inhibiting β-arrestin2 binding to α1AAR. The third possibility is that β-arrestin2 interacts with binding sites on both protomers in the heteromer, which would again imply an alteration of the phosphorylation bar code on α1AAR to enable this to occur.
      Our findings can also be considered in terms of biased signaling. Indeed, the observed heteromer-biased β-arrestin recruitment reflects the difference between α1AAR being a G protein-biased receptor and the α1AAR-CXCR2 heteromer being unbiased (
      • Mustafa S.
      • Ayoub M.A.
      • Pfleger K.D.
      Uncovering GPCR heteromer-biased ligands.
      ). In terms of ligand-bias in the heteromer context, we have identified Labetalol as exhibiting such properties.
      In a clinical setting, Labetalol is utilized for its antagonism of adrenoceptors, and in terms of inositol phosphate production, this in vivo effect is supported by the relatively weak partial agonism observed. However, although studies have indicated a lack of Labetalol-induced β-arrestin recruitment to the β2AR (
      • Wisler J.W.
      • DeWire S.M.
      • Whalen E.J.
      • Violin J.D.
      • Drake M.T.
      • Ahn S.
      • Shenoy S.K.
      • Lefkowitz R.J.
      A unique mechanism of β-blocker action. Carvedilol stimulates β-arrestin signaling.
      ), our findings indicate strong Labetalol-induced β-arrestin recruitment to the α1AAR-CXCR2 heteromer, highlighting that the concept of heteromerization needs to be taken into account when investigating ligand-biased signaling. Indeed, the allosterism between the receptors that is proposed to enable β-arrestin recruitment to the heteromer after α1AAR activation is likely to facilitate the ability of Labetalol to act as a biased full agonist for β-arrestin recruitment, thereby potentially explaining why biased agonism was not observed with the β2AR monomer/homomer.
      Whether the biased agonism of Labetalol is clinically relevant (unwanted or fortuitous) is far from clear and requires extensive investigation, as the precise mechanism of action of Labetalol is itself not fully elucidated. This was illustrated when an infant was accidentally given a massive overdose with amazingly limited clinical effect (
      • Thorsteinsson A.
      • Johannesdottir A.
      • Eiríksson H.
      • Helgason H.
      Severe labetalol overdose in an 8-month-old infant.
      ). The explanation was unclear, although altered distribution or structure of α-ARs was suggested as possibly playing a role. More generally, the potential clinical relevance of biased agonism is becoming increasingly apparent, with recent work from Lefkowitz and co-workers (
      • Wisler J.W.
      • DeWire S.M.
      • Whalen E.J.
      • Violin J.D.
      • Drake M.T.
      • Ahn S.
      • Shenoy S.K.
      • Lefkowitz R.J.
      A unique mechanism of β-blocker action. Carvedilol stimulates β-arrestin signaling.
      ) implicating a role for β-arrestin signaling via adrenoceptors in the regulation of apoptosis. Our demonstration of heteromer-biased β-arrestin recruitment takes these important concepts one step further.
      We postulate that heteromerization provides another mechanism by which α1AAR can exhibit distinct pharmacological profiles and consequently exert its diverse biological effects. Indeed, such heteromerization may account for the recent evidence of α1AAR-β-arrestin2 complex formation specifically in the prostate stroma (
      • Hennenberg M.
      • Schlenker B.
      • Roosen A.
      • Strittmatter F.
      • Walther S.
      • Stief C.
      • Gratzke C.
      β-Arrestin-2 is expressed in human prostate smooth muscle and a binding partner of α1A-adrenoceptors.
      ) despite the poor interaction observed in transfected HEK293 cells (
      • Stanasila L.
      • Abuin L.
      • Dey J.
      • Cotecchia S.
      Different internalization properties of the α1a- and α1b-adrenergic receptor subtypes. The potential role of receptor interaction with β-arrestins and AP50.
      ). More broadly, we suggest that heteromerization provides a mechanism by which certain GPCRs can either form a functional complex with β-arrestin without necessarily physically interacting with the protein themselves or alter their own ability to interact with β-arrestin. Therefore, these concepts and the approaches used to investigate them have substantial implications for the wider GPCR field and for drug discovery at these important pharmaceutical targets.

      Acknowledgments

      We acknowledge the facilities and scientific and technical assistance of the Australian Microscopy and Microanalysis Research Facility at the Centre for Microscopy, Characterization, and Analysis, The University of Western Australia, a facility funded by the University, State, and Commonwealth Governments.

      Supplementary Material

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