Chapter Twelve - G-Protein-Coupled Heteromers: Regulation in Disease
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.
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2019, Advances in PharmacologyCitation 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 ScienceCitation 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 BiologyCitation 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 ChemistryCitation 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.
Targeting Cannabinoid 1 and Delta Opioid Receptor Heteromers Alleviates Chemotherapy-Induced Neuropathic Pain
2019, ACS Pharmacology and Translational ScienceThe endocannabinoid system as a target in cancer diseases: Are we there yet?
2019, Frontiers in Pharmacology