Elsevier

Methods in Enzymology

Volume 521, 2013, Pages 219-238
Methods in Enzymology

Chapter Twelve - G-Protein-Coupled Heteromers: Regulation in Disease

https://doi.org/10.1016/B978-0-12-391862-8.00012-0Get rights and content

Abstract

Over the past decade, an increasing number of studies have shown that G-protein-coupled receptors including opioid and cannabinoid receptors associate to form heteromers. Moreover, G-protein-coupled receptor heteromerization leads to the modulation of the binding, signaling, and trafficking properties of individual receptors. Although very little information is available about the physiological role of receptor heteromers, some studies have shown that the levels of some heteromers are upregulated in disease states such as preeclamptic pregnancy, schizophrenia, Parkinson's, ethanol-induced liver fibrosis, and development of tolerance to morphine. The recent generation of antibodies that selectively recognize distinct heteromers and, of peptides that selectively disrupt them, have started to elucidate the contribution of heteromers to the disease state. Here, we describe the methods for the generation of heteromer-selective antibodies and elucidation of their levels and localization under normal and pathological conditions.

Introduction

Drugs of abuse such as opioids and cannabinoids act through G-protein-coupled receptors (GPCRs), the opioid and cannabinoid receptors. Three opioid (μ, δ, and κ) and cannabinoid (CB1R, CB2R, and GPR55) receptor subtypes have been identified (Balenga et al., 2011, Dietis et al., 2011, Di Marzo et al., 2011). Both receptors signal via Gαi/o proteins to activate similar signal transduction cascades leading to decreases in intracellular cyclic AMP levels, inhibition of neurotransmitter release, and to increases in mitogen-activated protein kinase phosphorylation (Bushlin et al., 2010, Cichewicz, 2004, Howlett et al., 2002, Vigano et al., 2005). Moreover, activation of either receptor induces similar physiological responses such as antinociception, sedation, reward, and emotional responses (Maldonado et al., 2006, Manzanares et al., 1999). This similarity in mechanisms of action and physiological responses suggests the possibility of interactions between the opioid and cannabinoid systems.

Opioid receptor subtypes can associate to form higher-order structures, a process known as heteromerization. For example, μ (μOR) and δ (δOR) opioid receptors heteromerize and these modulate binding, signaling, and morphine-mediated analgesia (Gomes et al., 2004, Gomes et al., 2000, Gomes et al., 2011, Kabli et al., 2010, Levac et al., 2002, Rozenfeld and Devi, 2007). Heteromerization between δOR and κ opioid receptors (κOR) leads to novel pharmacology and alteration of individual receptor-trafficking properties (Berg et al., 2012, Bhushan et al., 2004, Jordan and Devi, 1999). Furthermore, opioid receptors can heteromerize with other family A GPCRs such as α2A adrenergic (Jordan et al., 2003, Rios et al., 2004), β2 adrenergic (Jordan, Trapaidze, Gomes, Nivarthi, & Devi, 2001), chemokine (Chen et al., 2004, Hereld and Jin, 2008, Pello et al., 2008), substance P (Pfeiffer et al., 2003), or somatostatin receptors (Pfeiffer et al., 2002). Interestingly, heteromerization between CB1R and μOR, δOR, or angiotensin AT1 receptors (AT1Rs) leads to alterations in signaling and localization of CB1R (Rios et al., 2006, Rozenfeld et al., 2012, Rozenfeld et al., 2011). However, little information is available about the physiological role of GPCR heteromers due to a lack of appropriate tools to study them in endogenous tissues and to distinguish from receptor homomers. Studies using mainly coimmunoprecipitation techniques suggest the involvement of some GPCR heteromers in disease. Heteromers between dopamine D1–D2 receptors have been implicated in major depression (Pei et al., 2010), between AT1R and adrenergic α1D or AT1R and bradykinin B2 receptors with preeclamptic pregnancy (AbdAlla et al., 2005, Gonzalez-Hernandez Mde et al., 2010) and between dopamine receptor subtypes as well as dopamine D2 and adenosine 2A receptors in schizophrenia (Dziedzicka-Wasylewska et al., 2008, Faron-Gorecka et al., 2008, Fuxe et al., 2005, Maggio and Millan, 2010, Perreault et al., 2011). However, direct demonstration of heteromers in vivo has not been possible due to a lack of appropriate reagents.

We recently generated monoclonal antibodies (mAbs) that selectively recognize heteromers over individual receptor homomers using a subtractive immunization strategy. This enabled studies to directly explore the physiological role of GPCR heteromers. For example, these antibodies can be used to detect the presence of a heteromer in a specific tissue/region. A case in point is the detection of δOR–κOR heteromers in peripheral sensory neurons using δOR–κOR selective antibodies (Berg et al., 2012). Alternatively, the antibodies could implicate the heteromer in a disease state. μOR–δOR heteromer-selective antibodies detect increased heteromer levels in brain regions involved in pain processing following chronic morphine administration under conditions leading to the development of tolerance (Gupta et al., 2010), suggesting that they may play a role in tolerance. This is supported by studies showing that μOR–δOR heteromer disruption leads to enhanced morphine analgesia with a concomitant decrease in tolerance (He et al., 2011). CB1R–AT1R heteromer-selective antibodies detect a significant heteromer upregulation in hepatic stellate cells of rats chronically treated with ethanol (Rozenfeld et al., 2011), suggesting its involvement in ethanol-induced liver fibrosis. Here, we describe the generation of heteromer-selective antibodies and their use with enzyme-linked immunosorbent assays (ELISAs), immunofluorescence, immunoprecipitation, and Western blotting to detect levels and localization of heteromers in native tissues under normal and pathological conditions.

Section snippets

Generation of Heteromer-Selective mAbs

The advantages of mAbs to probe for heteromer levels in normal and disease states are that they recognize a single epitope, are highly specific, and can be produced in large quantities. The challenge in the generation of heteromer-selective mAbs is that the “heteromer-specific” epitope may be present in a cell or tissue at relatively low levels thus limiting the chances of being detected by antibody-producing cells. This limitation can be overcome through the use of a subtractive immunization

ELISA for Detection of Receptor Heteromers

ELISA, a technique commonly used to screen for the presence on an antigenic epitope on membrane preparations, in whole cells or tissue sections, can also detect GPCRs by using antibodies that recognize endogenous receptors or epitope tags (Flag, myc, or HA) present in the N-terminal region of the receptors. We use ELISA to screen for and determine the selectivity of heteromer-selective antibodies (Gupta et al., 2010, Rozenfeld et al., 2011). In the case of μ-δ mAb, we used membranes from (i)

Immunofluorescence for Visualization of Receptor Heteromers

Immunofluorescence uses fluorescently labeled secondary antibodies to visualize proteins in cells and tissues and can provide information about the tissue distribution of a given protein as well as its subcellular distribution. We used immunofluorescence to show that coexpression of μOR with δOR leads to intracellular retention of μOR–δOR heteromers in the Golgi apparatus, and this is rescued by the expression of RTP4 (Decaillot, Rozenfeld, Gupta, & Devi, 2008). We used this technique to

Immunoprecipitation and Western Blotting

Immunoprecipitation is a useful technique to demonstrate heteromerization between differentially epitope-tagged GPCRs. After cell lysis, one of the receptors is immunoprecipitated using antibodies recognizing the epitope tag present on the receptor (myc-tagged receptors are immunoprecipitated using anti-myc antisera). The immunoprecipitates are then subjected to SDS-PAGE under nonreducing conditions, and Western blots are probed with antibodies to the epitope tag present to the other receptor.

Summary and Perspectives

Recent evidence indicates that some GPCR heteromers are upregulated in disease states. However, the physiological role of these heteromers in pathology is not clearly understood due to the lack of tools to distinguish between heteromer- and homomer-mediated effects. The development of heteromer-selective antibodies and of specific heteromer disrupting TAT peptides could help elucidate their role in pathological conditions. This is clearly shown in the case of μOR–δOR heteromers where selective

Acknowledgment

L. A. D. is supported by NIH grants DA008863 and DA019521.

References (48)

  • B.A. Levac et al.

    Oligomerization of opioid receptors: Generation of novel signaling units

    Current Opinion in Pharmacology

    (2002)
  • R. Maggio et al.

    Dopamine D2-D3 receptor heteromers: Pharmacological properties and therapeutic significance

    Current Opinion in Pharmacology

    (2010)
  • R. Maldonado et al.

    Involvement of the endocannabinoid system in drug addiction

    Trends in Neurosciences

    (2006)
  • J. Manzanares et al.

    Pharmacological and biochemical interactions between opioids and cannabinoids

    Trends in Pharmacological Sciences

    (1999)
  • M. Pfeiffer et al.

    Heterodimerization of substance P and mu-opioid receptors regulates receptor trafficking and resensitization

    Journal of Biological Chemistry

    (2003)
  • M. Pfeiffer et al.

    Heterodimerization of somatostatin and opioid receptors cross-modulates phosphorylation, internalization, and desensitization

    Journal of Biological Chemistry

    (2002)
  • R.A. Salata et al.

    Application of an immune-tolerizing procedure to generate monoclonal antibodies specific to an alternate protein isoform of bovine growth hormone

    Analytical Biochemistry

    (1992)
  • H.M. Sleister et al.

    Strategies to generate antibodies capable of distinguishing between proteins with > 90% amino acid identity

    Journal of Immunological Methods

    (2001)
  • H.M. Sleister et al.

    Subtractive immunization: A tool for the generation of discriminatory antibodies to proteins of similar sequence

    Journal of Immunological Methods

    (2002)
  • D. Vigano et al.

    Molecular and cellular basis of cannabinoid and opioid interactions

    Pharmacology, Biochemistry, and Behavior

    (2005)
  • S. AbdAlla et al.

    Mesangial AT1/B2 receptor heterodimers contribute to angiotensin II hyperresponsiveness in experimental hypertension

    Journal of Molecular Neuroscience

    (2005)
  • K.A. Berg et al.

    Allosteric interactions between delta and kappa opioid receptors in peripheral sensory neurons

    Molecular Pharmacology

    (2012)
  • R.G. Bhushan et al.

    A bivalent ligand (KDN-21) reveals spinal delta and kappa opioid receptors are organized as heterodimers that give rise to delta(1) and kappa(2) phenotypes. Selective targeting of delta-kappa heterodimers

    Journal of Medicinal Chemistry

    (2004)
  • F.M. Decaillot et al.

    Cell surface targeting of mu-delta opioid receptor heterodimers by RTP4

    Proceedings of the National Academy of Sciences of the United States of America

    (2008)
  • Cited by (11)

    • The heterotetrameric structure of the adenosine A<inf>1</inf>-dopamine D<inf>1</inf> receptor complex: Pharmacological implication for restless legs syndrome

      2019, Advances in Pharmacology
      Citation Excerpt :

      If these proteins are separated < 17 nm, heteromers will be observed as red dots in a confocal microscope (Gomes, Sierra, et al., 2016; Söderberg et al., 2008; Taura, Fernández-Dueñas, & Ciruela, 2015). Alternative strategies to target GPCR heteromers are the generation of selective compounds (bivalent, multifunctional or small molecule ligands), which exhibit higher efficacy in cells expressing both receptors and in tissues from wild type animals, as compared with cell expressing individual receptors or tissues from animals lacking the individual receptors (Akgün et al., 2013; Gomes, Gupta, & Devi, 2013; Molero et al., 2015; Nimczick & Decker, 2015; Peterson et al., 2017; Qian, Vasudevan, et al., 2018; Qian, Wouters, et al., 2018). Likewise, the use of membrane-permeable peptides that target the dimerization interface or heteromer-selective antibodies that can recognize an epitope in the heteromer but not in the individual protomers, have been emerged as useful tools to detect the presence of GPCR heteromers in vivo (Gupta et al., 2010; He et al., 2011; Jacobs et al., 2018; Moreno et al., 2017; Navarro et al., 2015; Rivera-Oliver et al., 2018; Viñals et al., 2015).

    • Molecular Mechanisms of Cannabis Signaling in the Brain

      2016, Progress in Molecular Biology and Translational Science
      Citation Excerpt :

      There is also evidence that CB1 forms heteromers with a number of other G-coupled receptors including D1 and D2, orexin, μ-opioid, and adenosine2A. Little is known about the physiologic role of these G-protein receptor heteromers but there is evidence that they play a role in disease states such as Parkinson's and addiction.33 CB1 activation also causes an interaction and transduction via receptor and nonreceptor tyrosine kinases (reviewed in Ref. [20]).

    • Resonance energy transfer-based approaches to study GPCRs

      2016, Methods in Cell Biology
      Citation Excerpt :

      Consequently, over the years interesting findings have been obtained regarding the role of such organization in the maturation, activation, G protein coupling, downstream signaling, and regulation of GPCRs (Angers, Salahpour, & Bouvier, 2002; Bai, 2004; Bouvier, 2001; Bulenger, Marullo, & Bouvier, 2005; Devi, 2001; Ferre et al., 2009; Gomes et al., 2016; Jordan & Devi, 1999; Milligan, 2004b; Prinster, Hague, & Hall, 2005; Szidonya, Cserzo, & Hunyady, 2008; Terrillon & Bouvier, 2004b; Vischer, Castro, & Pin, 2015). The major advances in the field concern the heterodimerization between the different subfamilies of GPCRs including the orphan receptors and its impact on the functioning of GPCRs, as well as its implication in diverse physiological and pathophysiological models (Devi, 2001; Ferre et al., 2009; Gomes et al., 2016; Gomes, Gupta, & Devi, 2013; Jordan & Devi, 1999; Levoye, Dam, Ayoub, Guillaume, & Jockers, 2006; Prinster et al., 2005; Vischer et al., 2015). Therefore, heterodimerization would constitute one of the mechanisms of diversifying and finely regulating GPCR signaling as multiple signaling pathways are possible through a limited number of receptors expressed within a given organism.

    • Opioid receptor function is regulated by post-endocytic peptide processing

      2014, Journal of Biological Chemistry
      Citation Excerpt :

      The relative decrease in cell surface receptors (% inhibition over control) after 60 min of recycling with different inhibitors was calculated by taking the corresponding values in the absence of the inhibitor as 100%. ELISA was carried out as described previously (18, 29, 30) to determine cell surface receptor levels following recycling experiments. Briefly, following a brief fixation with paraformaldehyde (as described above) cells were incubated for 1 h at 4 °C with PBS containing 3% BSA followed by a 16-h incubation at 4 °C with a 1:1000 dilution (in PBS containing 1% BSA) of anti-FLAG M1 mouse monoclonal antibody (Sigma-Aldrich) to detect FLAG epitope-tagged δOR, a 1:1000 dilution of anti-myc mouse monoclonal antibody (Santa Cruz Biotechnology) to detect myc-epitope-tagged δOR, or a 1:500 dilution of rat δOR antibody to detect endogenous δOR.

    View all citing articles on Scopus
    View full text