cells Article Expression of Melatonin and Dopamine D3 Receptor Heteromers in Eye Ciliary Body Epithelial Cells and Negative Correlation with Ocular Hypertension Irene Reyes-Resina 1,2,3, *, Hanan Awad Alkozi 4 , Anna del Ser-Badia 3,5 , Juan Sánchez-Naves 6 , Jaume Lillo 1,3 , Jasmina Jiménez 3 , Jesús Pintor 4 , Gemma Navarro 3,7, * and Rafael Franco 3,8, * 1 Department of Biochemistry and Molecular Biomedicine, School of Biology, Universitat de Barcelona, 08028 Barcelona, Spain; lillojaume@gmail.com 2 Neuroplasticity Research Group, Leibniz Institute for Neurobiology, 39118 Magdeburg, Germany 3 Centro de Investigación en Red, Enfermedades Neurodegenerativas, CiberNed, Instituto de Salud Carlos III, 28029 Madrid, Spain; delserbadia@gmail.com (A.d.S.-B.); jasminajc@gmail.com (J.J.) 4 Department of Biochemistry and Molecular Biology, Faculty of Optics and Optometry, University Complutense of Madrid, 28037 Madrid, Spain; hanan-q1@live.com (H.A.A.); jpintor@ucm.es (J.P.) 5 Department de Bioquímica i Biologia Molecular, Institut de Neurociències, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, 08193 Barcelona, Spain 6 Department of Ophthalmology, Balearic Islands Institute of Ophthalmology, 07013 Palma de Mallorca, Mallorca, Spain; juansanchez.naves@gmail.com 7 Department of Biochemistry and Physiology, School of Pharmacy and Food Sciences, Universitat de Barcelona, 08027 Barcelona, Spain 8 School of Chemistry, Universitat de Barcelona, 08028 Barcelona, Spain * Correspondence: ire-reyes@hotmail.com (I.R.-R.); g.navarro@ub.edu (G.N.); rfranco123@gmail.com or rfranco@ub.edu (R.F.); Tel.: +34-934021208 (I.R.-R. & G.N.)  Received: 20 November 2019; Accepted: 2 January 2020; Published: 8 January 2020  Abstract: Background: Experiments in the late nineties showed an inverse relationship in the eye levels of melatonin and dopamine, thereby constituting an example of eye parameters that are prone to circadian variations. The underlying mechanisms are not known but these relevant molecules act via specific cell surface dopamine and melatonin receptors. This study investigated whether these receptors formed heteromers whose function impact on eye physiology. We performed biophysical assays to identify interactions in heterologous systems. Particular heteromer functionality was detected using Gi coupling, MAPK activation, and label-free assays. The expression of the heteroreceptor complexes was assessed using proximity ligation assays in cells producing the aqueous humor and human eye samples. Dopamine D3 receptors (D3 Rs) were identified in eye ciliary body epithelial cells. We discovered heteromers formed by D3 R and either MT1 (MT1 R) or MT2 (MT2 R) melatonin receptors. Heteromerization led to the blockade of D3 R-Gi coupling and regulation of signaling to the MAPK pathway. Heteromer expression was negatively correlated with intraocular hypertension. Conclusions: Heteromers likely mediate melatonin and dopamine actions in structures regulating intraocular pressure. Significant expression of D3 R–MT1 R and D3 R–MT1 R was associated with normotensive conditions, whereas expression diminished in a cell model of hypertension. A clear trend of expression reduction was observed in samples from glaucoma cases. The trend was marked but no statistical analysis was possible as the number of available eyes was 2. Keywords: circadian rhythm; glaucoma; G-protein-coupled receptor GPCR heteromer; melatonin MT1 and MT2 receptors; G protein coupling; mitogen-activated protein kinase pathway (MAPK); label-free dynamic mass redistribution (DMR); retina; human 59HCE cells Cells 2020, 9, 152; doi:10.3390/cells9010152 www.mdpi.com/journal/cells Cells 2020, 9, 152 2 of 20 1. Introduction The ciliary body, hidden behind the iris, is a complex, highly specialized tissue with a surface containing a series of radially arranged structures known as ciliary processes [1], which are densely vascularized and covered by highly active secretory cells to comply with the function of ciliary processes, i.e., aqueous humor production [2]. The aqueous humor is a clear transparent fluid which bathes the anterior and posterior chambers of the eye [3–5]. It participates in numerous functions such as providing nutrients to the cornea, lens, and trabecular meshwork. More importantly, it also contributes to keeping the force that supports intraocular tissue, thereby giving the eye a certain intraocular pressure (IOP) [6]. IOP is maintained within normal values through a dynamic balance between its constant production by the ciliary processes and its drainage. A lack of balance in the aqueous humor dynamics, usually leading to an increment of IOP, is a significant risk factor in specific ocular pathologies such as glaucoma, the second leading cause of blindness worldwide [7]. Several factors contribute to the homeostasis of IOP, among others, the episcleral vein pressure, the ratio between production and drainage of aqueous humor, the influence of hormones, the innervation by cranial nerves V and VII, and the circadian rhythm. Evidence showed that IOP follows variations throughout the day, being maximal in the early morning and reaching its minimal levels during the night [8]. These fluctuations are small, between 3–5 mm Hg, in healthy subjects. However, they are significantly higher in the glaucomatous eye [9]. The primary modulator of eye circadian rhythms seems to be melatonin. Melatonin, an indoleamine that participates in the control of circadian rhythms, is a neurohormone first identified in the pineal gland. However, a decade after its discovery by the dermatologist Aaron Lerner and colleagues [10], it gained particular interest because of its multifunctional role. Apart from being synthesized out of the pineal gland, melatonin is also considered an antioxidant and it contributes to minimize the circadian disruption resulting from jet lag or in shift workers [11,12]. Melatonin is synthesized by the retina [13] but, in subsequent studies, other sources were detected, namely, the iris, the ciliary body [14], the crystalline lens [15,16], the Harderian gland [17] and the lacrimal gland [18]. It seems that melatonin can function directly as a free radical scavenger, but it mainly acts via specific cell surface melatonin receptors. In mammals, two melatonin receptors, MT1 (MT1 R) and MT2 (MT2 R) have been cloned and characterized [19,20]. These receptors are present in numerous ocular structures; for instance, they are expressed in the cornea, and among other functions, they accelerate corneal wound healing [21,22]. They mediate regulation of circadian rhythms and neuroendocrine processes in the retina and in the non-pigmented ciliary body epithelium, which mediates IOP control (see References [23,24] and references therein). Melatonin receptors are members of the 7-transmembrane G protein-coupled receptor family (GPCRs). Both MT1 and MT2 receptors belong to the class A of rhodopsin-like GPCRs (https: //gpcrdb.org/) and contain seven hydrophobic transmembrane helices. Melatonin receptors have a well-established canonical signaling pathway, where the stimulation of MT1 R and MT2 R results in adenylate cyclase (AC) and protein kinase A inactivation by Gi-coupling [25–27]. However, it has been reported that, depending on the tissue or the species studied, melatonin receptors could couple to other G proteins to, for instance, activate phospholipase C-α (PLCα) [28] or inhibit guanylate cyclase (GC) [29]. Interestingly, studies show that the activation of melatonin receptors in the ciliary processes would lead to an increase in cAMP levels [30]. Other possibilities include the influence derived from interactions between these receptors and other receptors or other non-receptor proteins [31]. In this sense, direct interactions between melatonin receptors and GPR50 have been reported. This orphan GPCR may directly interact with the two melatonin receptors, although the interaction does not alter MT2 R but MT1 R coupling to Gi [32]. The MT2 R also interacts directly with 5-hydroxytryptamine 5HT2c receptors, the heteromer leading to biased agonism for the melatonin receptor ligand, agomelatine [33]. Although little is known about melatonin receptor heteromerization in the eye, a recent report has demonstrated Cells 2020, 9, 152 3 of 20 the heteromerization of melatonin and α1 -adrenergic receptors that alters canonical coupling as heteromerization impedes coupling to the cognate G proteins, Gi and Gq, respectively [26,34]. Dopamine is present in eye structures, and the extracellular level of this neurotransmitter regulates the synthesis of melatonin (in the eye) via dopamine receptors and cAMP as a second messenger [35–38]. The first evidence of an inverse correlation between dopamine and melatonin was found in the pigeon eye. Microdialysis assays found that dopamine concentration decreased when melatonin levels increased. In addition, melatonin injection suppressed the release of dopamine. Subsequent studies in the same laboratory confirmed that melatonin/dopamine interactions were complex but relevant for circadian rhythms in the eye [39,40]. However, the mechanisms underlying dopamine–melatonin relationships in the eye have not yet been elucidated. Dopamine receptors are grouped into two subfamilies: D1 -like, which includes D1 and D5 , and D2 -like, which includes D2 , D3 , and D4 [26]. Dopamine receptor expression has been addressed directly in the retina. Dopamine D4 receptors were detected in the vertebrate retina [41] and D2 and D3 receptors were identified in the human retina by targeted mass spectrometry and positron emission tomography [42]. Indirect evidence based on pharmacological assays and on responses in D3 receptor (D3 R) KO mice showed that D3 R activation leads to IOP reduction [43–45]. In preliminary experiments, we have detected the D3 receptor in human ciliary body epithelial cells. The present work aimed to address the potential interaction between dopamine D3 and melatonin receptors, along with the functional consequences of these interactions. The expressions of D3 R–MT1 R and D3 R–MT2 R heteroreceptor complexes were quantified in a ciliary body-based cell model of elevated IOP, and they were assessed in samples from human normotensive and hypertensive eyes. The results proved heteromerization and provided evidence of a negative correlation between ocular hypertension and heteromer expression. 2. Materials and Methods This work adhered to the guidelines detailed in Reference [46] and to the Helsinki declaration regarding the ethics of working with human samples. The studies were designed to generate groups of equal size, using randomization and blinded analysis. Immuno-based assays were conducted in line with guidelines detailed in Alexander et al., 2018 [47]. 2.1. Drugs (RS)-trans-7-Hydroxy-2-[N-propyl-N-(30 -iodo-20 -propenyl)amino]tetralin (7-OH-PIPAT) maleate (mixed D2 R/D3 R agonist), 3,5-Dichloro-N-[[(2S)-1-ethyl-2-pyrrolidinyl]methyl]-2-hydroxy-6- methoxybenzamide (raclopride: D3 R antagonist), N-Acetyl-5-methoxytryptamine (melatonin), N-qcetyl-2-benzyltryptamine (luzindole: non-selective MTR antagonist), N-[(1S)-1-[[4-[(2S)-2-[[(2,4- dichlorophenyl)sulfonyl]amino]-3-hydroxy-1-oxopropyl]-1-piperazinyl]carbonyl]-3-methylbutyl] benzo[b]thiophene-2-carboxamide (GSK1016790A: selective TRPV4 agonist), and cis-4-phenyl-2- propionamidotetralin (4PPDOT, a selective MT2 R antagonist) were purchased from Tocris Bioscience (Bristol, UK). N-[2-(2-methoxy-6H-isoindolo[2,1-a]indol-11-yl)ethyl]butanamide (IIK7, a selective MT2 receptor agonist [48]) was purchased from Sigma-Aldrich (St. Louis, MO, USA). 2.2. Cell Culture and Transient Transfection A human non-pigmented ciliary epithelial (59HCE) immortalized cell line was supplied by Dr. Coca-Prados (Yale University). Cells were grown in high glucose Dulbecco’s modified Eagle’s medium (Gibco/Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA) and 0.05 mg/mL penicillin/streptomycin (Gibco/Invitrogen) at 37 ◦ C in a humidified atmosphere of 5% CO2 . After the culture reached the confluence, the cells were detached with 0.25% trypsin and seeded into 6 well plates or glass coverslips. All the experiments were performed using cells under 10–15 passages to assure assay reproducibility. Then, 59HCE cells were treated with a selective Cells 2020, 9, 152 4 of 20 TRPV4 agonist, GSK1016790A (1–10,000 nM range), over 18 h, for the performance of the proximity ligation assay. HEK-293T cells grown in DMEM (Gibco/Invitrogen, Carlsbad, CA, USA) were processed as previously described in References [49,50]. Briefly, HEK-293T cells growing in 6-well dishes were transfected transiently with the corresponding cDNA using the PEI (PolyEthylenImine, Sigma-Aldrich, St. Louis, MO, USA) method. The cells were incubated (for 4h) with the corresponding cDNA together with PEI (5.47 mM in nitrogen residues) and 150 mM NaCl in a serum-starved medium. After 4 h, the medium was replaced by a fresh complete culture medium. 2.3. Fusion Proteins and Expression Vectors The human cDNAs for the D3 , MT1 , MT2 , and GHS-R1a receptors cloned in pcDNA3.1 were amplified without their stop codons using sense and antisense primers. The primers harbored either unique BamHI and HindIII sites for D3 ; EcoRI and KpnI sites for GHS-R1a or Hind III; and BamH1 sites for MT1 and MT2 . The fragments were subcloned to be in frame with an enhanced yellow fluorescent protein (pEYFP-N1; Clontech, Heidelberg, Germany) or an Rluc (pRluc-N1; PerkinElmer, Wellesley, MA) on the C-terminal end of the receptor to produce D3 -RLuc, MT1 –YFP, MT2 –YFP and GHS-R1a-YFP fusion proteins. 2.4. Bioluminescence Resonance Energy Transfer (BRET) Assays HEK-293T cells growing in 6-well plates were transiently co-transfected with a constant amount of cDNA encoding for D3 R fused to Renilla luciferase (D3 -Rluc) and with increasing amounts of cDNAs corresponding to MT1 R, MT2 R or ghrelin receptor GHS-R1a fused to the yellow fluorescent protein (MT1 -YFP, MT2 -YFP, GHS-R1a-YFP). 48 h post-transfection cells were washed twice in quick succession in HBSS (137 mM NaCl; 5 mM KCl; 0.34 mM Na2 HPO4; 0.44 mM KH2 PO4 ; 1.26 mM CaCl2 ; 0.4 mM MgSO4 ; 0.5 mM MgCl2 and 10 mM HEPES, pH 7.4) supplemented with 0.1% glucose (w/v), detached by gently pipetting and resuspended in the same buffer. To assess the number of cells per plate, we determined protein concentration using a Bradford assay kit (Bio-Rad, Munich, Germany) with bovine serum albumin dilutions as standards. To quantify YFP-fluorescence expression, we distributed the cells (20 µg protein) in 96-well microplates (black plates with a transparent bottom; Porvair, Leatherhead, UK). Fluorescence was read using a Mithras LB 940 (Berthold, Bad Wildbad, Germany) equipped with a high-energy xenon flash lamp, using a 10-nm bandwidth excitation and emission filters at 485 and 530 nm, respectively. YFP-fluorescence expression was determined as the fluorescence of the sample minus the fluorescence of cells expressing protein-Rluc alone. For the BRET measurements, the equivalent of 20 µg of cell suspension was distributed in 96-well microplates (white plates; Porvair), and we added 5 µM coelenterazine H (PJK GMBH, Kleinblittersdorf, Germany). Then, 1 min after coelenterazine H addition, the readings were collected using a Mithras LB 940 (Berthold, Bad Wildbad, Germany), which allowed the integration of the signals detected in the short-wavelength filter at 485 nm (440–500 nm) and the long-wavelength filter at 530 nm (510–590 nm). To quantify receptor-Rluc expression, we performed luminescence readings 10 min after addition of 5 µM coelenterazine H. The net BRET is defined as [(long-wavelength emission)/(short-wavelength emission)]-Cf where Cf corresponds to [(long-wavelength emission)/(short-wavelength emission)] for the Rluc construct expressed alone in the same experiment. The BRET curves were fitted assuming a single phase by a non-linear regression equation using the GraphPad Prism software (San Diego, CA, USA). BRET values are given as milli BRET units (mBU: 1000 × net BRET). 2.5. cAMP Level Determination Two hours before initiating the experiment, 59HCE cells or HEK-293T cell-culture medium were exchanged to serum-starved DMEM medium. Then, the cells were detached, re-suspended in growing medium containing 50 µM zardaverine, and plated in 384-well microplates (2500 cells/well), pretreated (15 min) with the corresponding antagonists—or vehicle—and stimulated with agonists (15 min) Cells 2020, 9, 152 5 of 20 before adding 0.5 µM forskolin or vehicle. Readings were performed after 1 h incubation at 25 ◦ C. Homogeneous time-resolved fluorescence energy transfer (HTRF) measures were obtained using the Lance Ultra cAMP kit (PerkinElmer, Waltham, MA, USA) (see Reference [51]). Fluorescence at 665 nm was analyzed on a PHERAstar Flagship microplate reader equipped with an HTRF optical module (BMG Lab technologies, Offenburg, Germany). 2.6. Extracellular Signal-Regulated Kinase Phosphorylation Assays To determine extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, 40,000 59HCE or HEK-293T cells/well were plated in transparent Deltalab 96-well plates and kept in the incubator for 48 h. Then, 2 to 4 h before the experiment the medium was replaced by serum-free medium. The cells were pre-treated at 25 ◦ C for 10 min with antagonists or vehicle and stimulated for an additional 7 min with selective agonists. The cells were then washed twice with cold PBS before the addition of lysis buffer (a 20 min treatment). Afterward, 10 µL of each supernatant were placed in white ProxiPlate 384-well plates and ERK 1/2 phosphorylation was determined using an AlphaScreen® SureFire® kit (Perkin Elmer), following the instructions of the supplier, and using an EnSpire® Multimode Plate Reader (PerkinElmer, Waltham, MA, USA). The value of reference (100%) was the value achieved in the absence of any treatment (basal). The effect of ligands was given in percentage with respect to the basal value. 2.7. Label-Free Dynamic Mass Redistribution (DMR) Assay Cell mass redistribution induced upon receptor activation was detected by illuminating the underside of a biosensor with polychromatic light. We then measured the changes in the wavelength of the reflected monochromatic light, which is a sensitive function of the index of refraction. The magnitude of the wavelength shift (in picometers) is directly proportional to the amount of mass redistribution. HEK-293T or 59HCE cells were seeded in 384-well sensor microplates to obtain 70–80% confluent monolayers constituted by approximately 10,000 cells per well. Previous to the assay, the cells were washed twice with assay buffer (HBSS with 20 mM HEPES, pH 7.15) and incubated for 2 h with assay-buffer containing 0.1% DMSO (24 ◦ C, 30 µL/well). Hereafter, the sensor plate was scanned, and a baseline optical signature was recorded for 10 min before adding 10 µL of the selective antagonists for 30 min, followed by the addition of 10 µL of the selective agonists. All test compounds were dissolved in assay buffer. Then, DMR responses elicited by the agonists were monitored for at least 5000 s in an EnSpire® Multimode Plate Reader (PerkinElmer, Waltham, MA, USA). The results were analyzed using EnSpire Workstation software v 4.10. 2.8. Human Eye Postmortem Samples Donor human eyes were obtained from the Fundación Banco de Sangre y Tejidos de las Islas Baleares (Balearic Islands Blood and Tissue Bank). This biobank is supervised by the regional Balearic Islands Government, which is in charge of enforcing the principles in Helsinki declaration. All donations are made after appropriate filling and signing of informed consent by patients or their legal representatives. Two eyes of healthy normal subjects and two of glaucoma patients were used for the assays. The eyes were enucleated and collected without the cornea in sterile tubes and maintained in 4% paraformaldehyde in 0.1 M phosphate buffer (PB) (pH 7.2–7.4) at 4 ◦ C until posterior processing. Eyes were dissected under a stereomicroscope (Zeiss), with a 0.8 mm curved tip forceps and sterile dissecting scissors, where we collected the iris and ciliary processes. Several washes in PBS were performed, and then, the specimens were cryoprotected in a sucrose gradient (from 11% to 33%) and embedded in tissue freezing medium (Tissue-Tek© OCT) until frozen with liquid N2 . Vertical sections of control and glaucomatous human samples (10 µm thick) were collected using a cryostat (Leica, Nussloch, Germany) and were mounted from the same region. The samples were maintained at −20 ◦ C until use. Cells 2020, 9, 152 6 of 20 2.9. Immunofluorescence Studies HEK-293T cells were fixed in 4% paraformaldehyde for 15 min and then washed twice with PBS containing 20 mM glycine before permeabilization with the same buffer containing 0.2% Triton X-100 (5 min incubation). The cells were treated for 1 h with PBS containing 1% bovine serum albumin and labeled with a mouse anti-Rluc (1/100; MAB4400, Millipore) antibody and subsequently treated with Cy3-conjugated anti-mouse (1/200; 715-166-150; Jackson ImmunoResearch (red)) IgG secondary antibody (1 h each). Alternatively, cells were labeled with rabbit anti-D3 R (#ab42114), anti-MT1 R (#ab203038), or anti-MT2 R (#ab115336) antibodies, all from Abcam (Cambridge, UK) and subsequently treated with Cy3-conjugated anti-rabbit (1/200; #711-166-152; Jackson ImmunoResearch (red)) IgG secondary antibody (1 h each). The samples were washed several times and mounted with 30% Mowiol (Calbiochem). Samples were observed under a Leica SP2 confocal microscope (Leica Microsystems). 2.10. In Situ and In Vitro Proximity Ligation Assay (PLA) The proximity ligation assay (PLA) allows the detection of molecular interactions between two endogenous proteins ex vivo. PLA requires both receptors to be sufficiently close (<16 nm) to allow double-strand formation of the complementary DNA probes conjugated to the antibodies. Using the PLA, the heteromerization of D3 R with MT1 and MT2 receptors was detected in non-pigmented epithelial ciliary body cells (59HCE) and, in situ, in the human ciliary body sections of healthy and glaucomatous donors. The presence/absence of receptor–receptor molecular interactions in the samples was detected using the Duolink II In Situ PLA Detection Kit (developed by Olink Bioscience, Uppsala, Sweden; and now distributed by Sigma-Aldrich as Duolink® using PLA® Technology). The PLA probes were obtained after conjugation of the primary anti-D3 R antibody (ab42114, Abcam, Cambridge, UK) to a PLUS oligonucleotide (DUO92009, Sigma-Aldrich, St. Louis, MO, USA), and the anti-MT1 R (ab203038, Abcam, Cambridge, UK) and -MT2 R (ab115336, Abcam, Cambridge, UK) antibodies to a MINUS oligonucleotide (DUO92010, Sigma-Aldrich, St. Louis, MO, USA). The specificity of antibodies was tested in non-transfected HEK-293T cells (see Appendix A Figure A3). Samples were fixed in 4% paraformaldehyde for 15 min and then washed twice with PBS containing 20 mM glycine before permeabilization with PBS-glycine containing 0.2% Triton X-100 (5 min incubation for the 59HCE cells and 15 min for the ciliary body sections). After permeabilization, the samples were washed in PBS at room temperature and incubated in a preheated humidity chamber for 1 h at 37 ◦ C with the blocking solution provided in the PLA kit. Then, the samples were incubated overnight with the PLA probe-linked antibodies (1:100 dilution for all antibodies) at 4 ◦ C. After washing, the samples were incubated with the ligation solution for 1 h, and then washed and subsequently incubated with the amplification solution for 100 min (both steps at 37 ◦ C in a humid chamber). Nuclei were stained with Hoechst (1/100; Sigma-Aldrich, St. Louis, MO, USA). Mounting was performed using 30% Mowiol (Calbiochem). Negative controls were performed by omitting the anti-D3 R-PLUS antibody. Samples were observed using a Leica SP2 confocal microscope (Leica Microsystems, Mannheim, Germany) equipped with an apochromatic 63× oil-immersion objective (N.A. 1.4), and with 405 nm and 561 nm laser lines. For each field of view, we acquired a stack of two channels (one per staining) and 5 Z planes with a step size of 1 µm. The ratio r (number of red spots/cell) was determined on the maximum projection of each image stack using the Duolink Image tool software. 2.11. Data Analysis Data, expressed as the mean ± SEM, were obtained from at least five independent experiments. Data comparisons were analyzed by one-way ANOVA or two-way ANOVA, followed by Bonferroni’s post-hoc test. The normality of populations and homogeneity of variances were tested before the ANOVA. Statistical analysis was undertaken only when each group size was at least n = 5, n being the number of independent variables (technical replicates were not treated as independent variables). Cells 2020, 9, 152 7 of 20 Cells 2020, 9, x FOR PEER REVIEW 7 of 20 Differences were considered significant when P ≤ 0.05. Statistical analyses were carried out with were carried GraphPad outsoftware Prism with GraphPad version 5Prism software (San Diego, CA,version 5 (San Diego, USA). Outliers CA,not tests were USA). used,Outliers tests and all data were not points used, (mean ofand all datawere replicates) points (mean used of replicates) for the analyses. were used for the analyses. 3. Results 3. Results 3.1. Dopamine D33 Receptors Receptors Interact Interact with with Melatonin Melatonin MT MT11 Receptors Receptors in in the the HEK-293T HEK-293T Cells Cells To determine whether To whether thethedopamine dopamineDD 3 receptor 3 receptor(D3(D R)3 R) couldcouldinteract withwith interact the melatonin MT1 the melatonin receptor, MT we first 1 receptor, performed we first performed immunocytochemical immunocytochemical assays assays in ina aheterologous heterologousexpression expression system. system. HEK-293T cells were transfected with cDNAs coding coding for for DD33R-Rluc or MT11R-YFP. Expression of D33R-Rluc was wasdetected detected using using an anti-Rluc an anti-Rluc primary primary antibody antibody followed followed by a secondary by a secondary Cy3- Cy3-conjugated conjugated antibody, andantibody, expressionand of MTexpression 1 R-YFP was of detected MT1R-YFP was using thedetected using the YFP’s YFP’s fluorescence. fluorescence. Receptor expression Receptor was foundexpression in differentwas found in different cell compartments, cell compartments, including the plasma membrane including the plasma (Appendix membrane A Figure A1A, (Appendix left and centerA Figure A1A, panels). Whenleft and center the cells panels). were When theDcells co-expressing 3 were R-Rluc co-expressing and MT 1 R-YFP,D a 3 R-Rluc and significant MT1R-YFP, degree a significant degree of co-localization of co-localization was observed (yellow in was observed Figure 1A). (yellow in Figure 1A). Figure 1. 1. Molecular interaction between D33 and MT11 receptors, receptors, and heteromer-mediated signaling. heteromer-mediated signaling. (A) Confocal microscopy microscopy images images of of HEK-293T HEK-293Tcellscellsco-expressing co-expressingDD 3 R-Rluc 3R-Rluc (2(2μg) and µg)and MTMT 1 R-YFP 1R-YFP (2 (2 μg).µg).D3Dreceptor 3 receptor (red)was (red) wasidentified identifiedby by immunocytochemistry immunocytochemistry using using anti-Rluc antibodies. antibodies. MT MT11 receptor receptor (green) (green)waswas identified fromfrom identified the fluorescence of YFP-containing the fluorescence fusion proteins. of YFP-containing fusion Co-localization proteins. Co- is shown in the panel on the right (yellow). Cell nuclei were stained with Hoechst localization is shown in the panel on the right (yellow). Cell nuclei were stained with Hoechst (blue(blue channel). Scale bar: 20 µm. (B) Scheme of the bioluminescence resonance energy transfer (BRET) channel). Scale bar: 20 μm. (B) Scheme of the bioluminescence resonance energy transfer (BRET) assay. (C,D) BRET saturation assay. (C,D) experiments performed BRET saturation using HEK-293T experiments performed cells co-transfected using HEK-293T with D3 R-Rluc cDNA cells co-transfected (0.7Dµg) with 3R- and Rlucincreasing cDNA (0.7amounts μg) and of MT1 R-YFP increasing cDNA of amounts (0–1.4 MT1µg cDNA) R-YFP cDNA (C) (0–1.4 or GHSR-1a-YFP μg cDNA) (C) cDNA (0–2.5 µg or GHSR-1a- cDNA) YFP cDNA as a negative (0–2.5 μgcontrol cDNA) (D). as BRET data are a negative expressed control as thedata (D). BRET meanare ± S.D. of 8 different expressed as the experiments mean ± S.D. performed in duplicates. mBU: milliBret units. HEK-293T cells transfected of 8 different experiments performed in duplicates. mBU: milliBret units. HEK-293T cells with cDNA encoding for transfected D with 3 R (1 µg) and MT cDNA encoding 1 R (1 µg) were pre-treated or not with receptor antagonists (1 µM for D3R (1 μg) and MT1R (1 μg) were pre-treated or not with receptor raclopride for D 3R or 1 µM luzindole antagonists (1 μMfor MT1 R) and raclopride for then D3R subsequently treatedfor or 1 μM luzindole with MTagonists (100 nM 7-OH-PIPAT for 1R) and then subsequently treated with agonists (100 nM 7-OH-PIPAT for D3R or 1 μM melatonin for MT1R), alone or in combination. (E) cAMP data were expressed as a % over 0.5 μM forskolin-induced levels. (F) ERK1/2 phosphorylation was analyzed using an AlphaScreen®SureFire® kit (Perkin Elmer). ERK1/2 Cells 2020, 9, 152 8 of 20 D3 R or 1 µM melatonin for MT1 R), alone or in combination. (E) cAMP data were expressed as a % over 0.5 µM forskolin-induced levels. (F) ERK1/2 phosphorylation was analyzed using an AlphaScreen® SureFire® kit (Perkin Elmer). ERK1/2 phosphorylation data are expressed as % with respect to basal levels. In cAMP accumulation and MAPK activation assays, values are the mean ± S.E.M. of 6 different experiments performed in triplicates. One-way ANOVA followed by Bonferroni’s multiple comparison post-hoc tests were used for statistical analysis. (*** p < 0.001; versus treatment with forskolin in cAMP or basal in pERK assays). (### p < 0.001; versus treatment with 7-OH-PIPAT alone). (&&& p < 0.001; versus treatment with melatonin alone). (G) label-free dynamic mass redistribution (DMR) tracings are representing the picometer-shifts of reflected light wavelengths over time upon ligand treatment. As co-localization is not direct evidence of interaction, we used an energy-transfer biophysical approach aimed at identifying direct physical interactions. Bioluminescence resonance energy transfer (BRET) assays were performed in HEK-293T cells expressing a constant amount of D3 R-Rluc and increasing amounts of MT1 R-YFP (Figure 1B). A saturation BRET curve was obtained (BRETmax 24.6 mBU, BRET50 14.9) (Figure 1C), indicating that D3 R and MT1 R physically interacted. As a negative control, D3 R was co-expressed with a non-interacting partner, the ghrelin receptor GHSR-1a. In this case, the cells were co-transfected with a constant amount of D3 R-Rluc and increasing amounts of GHSR-1aYFP. A linear signal was obtained, indicating the lack of interaction between these receptors (Figure 1D). These results indicated that D3 and MT1 may form heteroreceptor complexes in a heterologous expression system. 3.2. Functional Characterization of D3 -MT1 Heteroreceptor Complexes in HEK-293T Cells Before estimating whether D3 R and MT1 R signaling properties were altered by D3 R–MT1 R heteromerization, we performed assays in single-transfected cells. These preliminary assays consisted of dose-response curves, to select the single agonist concentration for use in further assays performed in cells expressing the two receptors. Cognate heterotrimeric G proteins are Gi/o for melatonin and D3 receptors [26]. Thus, activation of these receptors leads to a decrease in cytosolic cAMP levels. We have confirmed that this is the case in single transfected cells (Appendix A Figure A1B,C; see also our recent report [52]), thereby reproducing data reported in References [34,53]. HEK-293T cells expressing D3 R or MT1 and incubated with the selective agonists, 7-OH-PIPAT for D3 and melatonin for MT1 , led to significant decreases in the forskolin-induced cytosolic cAMP levels. The blockade was afforded by the respective antagonist, raclopride, or luzindole (Appendix A Figure A1B,C). Next, we analyzed the mitogen-activated protein kinase (MAPK) signaling pathway, which may be mediated by G-protein-dependent or independent mechanisms. Experiments performed in single-transfected cells HEK-293T cells expressing D3 R or MT1 R led to a dose-response increase in phosphorylated ERK1/2 levels, which was reversed by the respective antagonists (Appendix A Figure A2A,B). Finally, we used a label-free approach based on the cell’s dynamic mass redistribution (DMR). Increasing agonist concentrations were assayed in single-transfected HEK-293T cells expressing D3 R or MT1 R treated with 7-OH-PIPAT or with melatonin, respectively. In both cases, we could observe significant DMR outputs that were reverted by the respective antagonists (Appendix A Figure A2D,E). Next, HEK-293T cells were transfected with both D3 R and MT1 R and then treated with 100 nM 7-OH-PIPAT and 1 µM melatonin, alone or in combination. Melatonin induced a decrease in the forskolin-induced cAMP levels to a similar extent to that produced in cells only expressing MT1 R. In contrast, 7-OH-PIPAT did not produce any effect (Figure 1E). These results suggest that the co-expression of MT1 R blocks the 7-OH-PIPAT-induced D3 R activation and Gi-mediated signaling. The effect in simultaneous treatment with agonists was similar to that produced by melatonin alone. When the cells were pre-treated with antagonists, we could observe that the melatonin effect was not only abolished by the MT1 R antagonist, luzindole, but also by the D3 R antagonist, raclopride (Figure 1E). This cross-antagonism, consisting of the antagonist of one receptor blocking the signal of the partner receptor, is often found in GPCR heteroreceptor complexes. Cells 2020, 9, 152 9 of 20 When the MAPK signaling pathway was analyzed in the HEK-293T cells co-expressing D3 and MTCells 1 receptors, a significant 2020, 9, x FOR PEER REVIEW ERK1/2 phosphorylation was observed when the cells were treated 9 ofwith 20 7-OH-PIPAT or melatonin. These results showed that MT1 R did not block 7-OH-PIPAT-induced D3 R D3R signaling signaling in the MAPK in the MAPK pathway pathway (Figure(Figure 1F). Interestingly, 1F). Interestingly, the effect the effect waswas muchmuch lowerlower when whenthethe cells cells were were stimulated stimulated simultaneously simultaneously with both with both agonists. agonists. Simultaneous Simultaneous co-activation co-activation resultingresulting in in reduced reduced signaling is known as negative cross-talk and has been reported signaling is known as negative cross-talk and has been reported for other GPCR heteromers. When cells for other GPCR heteromers. were pre-treated Whenwithcells were pre-treated antagonists, with antagonists, a bi-directional a bi-directional cross-antagonism cross-antagonism was detected, was which consisted detected, which consisted of the antagonists of either receptor blocking the activation of the antagonists of either receptor blocking the activation of the partner receptor in the heteromer. of the partner receptor Indeed, the in D3the heteromer.raclopride R antagonist Indeed, the D3R antagonist inhibited not onlyraclopride the effect ofinhibited not only 7-OH-PIPAT butthe alsoeffect of 7- of the effect OH-PIPAT but also the effect of melatonin, and the MT1R antagonist luzindole inhibited the effect melatonin, and the MT1 R antagonist luzindole inhibited the effect of each agonist (Figure 1F). of each agonist (Figure 1F). DMR assays performed in the cells co-expressing D3 R and MT1 R led to results that were similar DMR assays performed in the cells co-expressing D3R and MT1R led to results that were to those observed in [cAMP] determination assays. While melatonin produced a significant effect similar to those observed in [cAMP] determination assays. While melatonin produced a significant on DMR, 7-OH-PIPAT did not produce any effect (Figure 1G). When the cells were pre-treated with effect on DMR, 7-OH-PIPAT did not produce any effect (Figure 1G). When the cells were pre- antagonists, we observed a cross-antagonism phenomenon, as the melatonin effect was not only treated with antagonists, we observed a cross-antagonism phenomenon, as the melatonin effect was inhibited not onlybyinhibited luzindole, but also bybut by luzindole, raclopride. also by raclopride. Together, Together, the results showed the the results showed that thatconsequences of D3 -MT the consequences of 1Dheteroreceptor complex formation 3-MT1 heteroreceptor complex included a blockade of D R-Gi coupling, a negative cross-talk in signaling formation included a blockade of D3R-Gi coupling, a negative cross-talk in signaling 3 towards the MAPK towardspathway, the and a cross-antagonism. MAPK pathway, and a cross-antagonism. 3.3.3.3. Dopamine DD Dopamine 3 Receptors Interact with Melatonin MT Receptors in HEK-293T Cells 3 Receptors Interact with Melatonin MT22Receptors in HEK-293T Cells WeWe considered the the considered possibility of the possibility of Dthe 3 R also D3R interacting with the also interacting MTthe with 2 R.MT Accordingly, we followed 2R. Accordingly, we thefollowed same strategy described earlier for MT the same strategy described earlier 1 R. Immunocytochemical for MT1R. Immunocytochemical assays showed of assays showed the presence thethe receptor in different presence cell compartments, of the receptor including in different cell the cell including compartments, membranethe (Appendix A Figure cell membrane A1A, right). (Appendix A In Figure double-transfected cells, A1A, right). In a high degree of double-transfected co-localization cells, a high degree between D3 R and MT of co-localization R between 2 was Dobserved 3R and MT2R2A). (Figure was Next, observed (Figureassay, the BRET 2A). schematized Next, the BRET assay, schematized in Figure 2B, undoubtedlyin Figure proved2B,that undoubtedly D3 R might proved form that D3R might heteroreceptor form with complexes heteroreceptor MT2 R in a complexes heterologous with MT2R in system. expression a heterologous expression The saturation curve wassystem. The saturation characterized curve by a BRET was max of characterized 30.7 mBU andby a a BRET BRET of max 50 of 30.7 38.2. mBU and a BRET 50 of 38.2. Figure2.2.Molecular Figure Molecularinteraction interactionbetween between D D33 and and MT receptors, and MT22 receptors, and heteromer-mediated heteromer-mediatedsignaling. signaling. (A) Confocal microscopy images of HEK-293T cells co-expressing D3R-Rluc (2 μg) and MT2R-YFP (2 μg). D3 receptor (red) was identified by immunocytochemistry using anti-Rluc antibodies. MT2 receptor (green) was identified from the fluorescence of YFP-containing fusion proteins. Co- Cells 2020, 9, 152 10 of 20 (A) Confocal microscopy images of HEK-293T cells co-expressing D3 R-Rluc (2 µg) and MT2 R-YFP (2 µg). D3 receptor (red) was identified by immunocytochemistry using anti-Rluc antibodies. MT2 receptor (green) was identified from the fluorescence of YFP-containing fusion proteins. Co-localization is shown in the panel on the right (yellow). Cell nuclei were stained with Hoechst (blue channel). Scale bar: 20 µm. (B) Scheme of the BRET assay. (C) BRET saturation experiments performed using HEK-293T cells co-transfected with D3 R-Rluc cDNA (0.7 µg) and increasing amounts of MT2 R-YFP cDNA (0–2.3 µg cDNA). BRET data are expressed as the mean ± S.D. of 8 different experiments performed in duplicates. mBU: milliBret units. Panels (D–F) Signaling in HEK-293T cells transfected with cDNA encoding for D3 R (1 µg) and MT2 R (1 µg) were pre-treated with antagonists (1 µM raclopride for D3 R or 1 µM 4PPDOT for MT2 R) or vehicle, and then subsequently treated with selective agonists (100 nM 7-OH-PIPAT for D3 R and 300 nM IIK7 for MT2 R), individually or in combination. (D) [cAMP] (expressed as % with respect to treatment with 0.5 µM forskolin) was detected by TR-FRET. (E) ERK1/2 phosphorylation was analyzed using an AlphaScreen® SureFire® kit (Perkin Elmer). ERK1/2 phosphorylation data are expressed as % with respect to basal levels. In cAMP accumulation and MAPK signaling assays, the values are the mean ± S.E.M. of 6 different experiments performed in triplicates, and one-way ANOVA followed by Bonferroni’s multiple comparison post-hoc tests were used for statistical analysis. (** p < 0.01, *** p < 0.001; versus treatment with forskolin in cAMP or basal in pERK assays). (# p < 0.05, ### p < 0.001; versus treatment with 7-OH-PIPAT alone). (& p < 0.05, &&& p < 0.001; versus treatment with IIK7 alone). (F) DMR tracings representing the picometer-shifts of reflected light wavelengths over time upon ligand treatment. 3.4. Functional Characterization of the D3 -MT2 Heteroreceptor Complexes in HEK-293T Cells Dose-response assays in HEK-293T cells expressing MT2 R were performed using a selective MT2 R agonist, IIK7. The results were consistent with Gi coupling, with MAPK activation, and with marked DMR readings. Pretreatment with the MT2 R antagonist 4P-PDOT blocked the MT2 R agonist-induced effect (Appendix A Figures A1D and A2C,F). In forskolin-pretreated HEK-293T cells co-expressing D3 R and MT2 R, treatment with 7-OH-PIPAT did not decrease cAMP levels, while IIK7 was able to produce a signal similar to that produced in single-transfected cells. This result indicated that co-expression of MT2 R blocks 7-OH-PIPAT-induced Gi-mediated signaling (Figure 2D), in full resemblance to what was observed for the D3 R–MT1 R heteromer. However, for the D3 R-MT2 R heteromer, no cross-antagonism was detected. When activation of the MAPK pathway was analyzed in cotransfected HEK-293T cells, we observed the same phenomena encountered for the D3 R–MT1 R heteromer. Both agonists were able to elicit a signal when used separately but a negative cross-talk was detected in simultaneous co-activation. In this experiment, the bi-directional cross-antagonism effect was observed, as both raclopride and 4P-PDOT were able to block the signal elicited by either D3 R or MT2 R activation (Figure 2E). Finally, when the DMR responses were analyzed, 7-OH-PIPAT did not produce any signal, while IIK7 produced a significant effect. Again, as in the cAMP assays, no cross-antagonism was detected. The IIK7-induced signal was only blocked by MT2 R antagonist 4P-PDOT and not by the D3 R antagonist raclopride. To sum up, the results showed that the consequences of D3 -MT2 heteroreceptor complex formation included a blockade of D3 R-Gi coupling and a negative cross-talk plus bi-directional cross-antagonism when signaling towards the MAPK pathway was analyzed. 3.5. Detection and Functional Characterization of D3 -MT1 and D3 -MT2 Heteroreceptor Complexes in Human Non-Pigmented Ciliary Body Epithelial Cells To determine whether D3 R–MT1 R and D3 R–MT2 R heteromers occur in natural sources, we took advantage of a technique that allows the detection of clusters of two receptors in fixed cells or tissue sections, i.e., the in situ proximity ligation assay (PLA). The PLA was performed in the non-pigmented human ciliary body epithelial 59HCE cell line, which expresses three receptors. Specific antibodies Cells 2020, 9, 152 11 of 20 against D3 , MT1 , and MT2 receptors were used (see Appendix A Figure A3) and punctuated red marks were visualized surrounding stained nuclei. This outcome demonstrated the existence of D3 -MT1 and D 3 -MT Cells 2 receptor 2020, complexes/clusters 9, x FOR PEER REVIEW (Figure 3A, top and bottom left—0 nM). 11 of 20 Figure3.3.DD33-MT Figure -MT11 and and D D33-MT2 heteroreceptor expression in human human 59HCE 59HCE cells. cells. Proximity Proximity ligation ligation assay assay(PLA) (PLA)was performed was performed as described in Methods as described to detecttoD3detect in Methods -MT1 and D3 -MT D3-MT 1 and 2 receptor D 3 -MTcomplexes 2 receptor in the 59HCE complexes innon-pigmented ciliary body epithelial the 59HCE non-pigmented cell line, ciliary body using cell epithelial specific line,antibodies against using specific D3 and antibodies either MT or MT against D13 and either 2 receptors. (A) Cells were treated with different concentrations of GSK1016790A, MT1 or MT2 receptors. (A) Cells were treated with different concentrations of aGSK1016790A, TRPV4 agonist. Representative a TRPV4 agonist.images corresponding Representative imagesto corresponding stacks of 5 sequential to stacksplanes of 5 are shown. sequential Cell nuclei planes are were shown.stained with Hoechst Cell nuclei (blue) and were stained withheteroreceptor Hoechst (blue)clusters appear as red clusters and heteroreceptor dots. (B)appear Ratio (r; number of red spots/cell-containing spots) is the mean ± S.E.M. of counts in 5 different as red dots. (B) Ratio (r; number of red spots/cell-containing spots) is the mean ± S.E.M. of counts fields fromin every sample (n = 5). One-way ANOVA followed by Bonferroni’s multiple comparison 5 different fields from every sample (n = 5). One-way ANOVA followed by Bonferroni’s multiple post-hoc test were used forpost-hoc comparison statisticaltest analysis. were (* p < 0.05, used *** p < 0.001; for statistical versus(*0 nM analysis. p < GSK1016790A). Scaleversus 0.05, *** p < 0.001; bar: 200µm.nM GSK1016790A). Scale bar: 20 μm. Next, we studied the signaling properties of D3 -MT1 and D3 -MT2 heteroreceptor complexes in the 59HCE Next, cells using the we studied theapproaches described in signaling properties ofprevious D3-MT1 andsubsections. When cytosolic complexes D3-MT2 heteroreceptor cAMP levels in were analyzed in the cells treated with 0.5 µM forskolin, the 59HCE cells using the approaches described in previous the D R agonist 7-OH-PIPAT, in 3 subsections. When cytosolicagreement cAMP with levelsthewere heteromer’s analyzedprint in theidentified in the with cells treated HEK-293T 0.5 μMcells, did not the forskolin, produce any effect D3R agonist (Figure 4A,D). 7-OH-PIPAT, in agreement with the heteromer’s print identified in the HEK-293T cells, did not produce any effect (Figure 4A,D). Cells 2020, 9, 152 12 of 20 Cells 2020, 9, x FOR PEER REVIEW 12 of 20 Figure 4. Effect of Figure 4. of dopamine dopamine and and melatonin melatonin receptor receptor agonists agonists inin human human 59HCE 59HCE cells. cells. Signaling was assayed in the 59HCE cells pre-treated with antagonists (1 µM assayed in the 59HCE cells pre-treated with antagonists (1 μM raclopride for D33R,raclopride for D R, 11 µM μM luzindole luzindole for for MT MT1R,R, or or 11 µM μM 4PPDOT 4PPDOT for for MT MT22R)R) or or vehicle, vehicle, and and then then subsequently subsequently treated treated with with agonists agonists (100 (100 nM 7-OH-PIPAT for nM 7-OH-PIPAT for DD33RRand andeither either1 1μM melatoninforfor µMmelatonin MT MT R (A–C) 1R1(A–C) or 300 or 300 nMnM IIK7IIK7 for 2MT for MT R (D–2R (D–F)), individually or in combination. (A,D) cAMP production is F)), individually or in combination. (A,D) cAMP production is expressed as % over 0.5 μM expressed as % over 0.5 µM forskolin-induced forskolin-induced [cAMP] [cAMP] increases. (B,E) ERK1/2 increases. (B,E) ERK1/2 phosphorylation phosphorylation data data are are expressed expressed as as %% with with respect respect to basal levels. In cAMP accumulation and MAPK signaling assays, the values are the mean to basal levels. In cAMP accumulation and MAPK signaling assays, the values are the mean ± S.E.M. of ± S.E.M. of 55 different different experiments experiments performed performed in in triplicates, and one-way triplicates, and one-way ANOVA ANOVA followed followed by by Bonferroni’s multiple comparison post-hoc tests were used for statistical analysis. Bonferroni’s multiple comparison post-hoc tests were used for statistical analysis. (*** p < 0.001; (*** p < 0.001; versus versus basal basal in in pERK pERK assays). (### pp < assays). (### 0.001; versus < 0.001; versus treatment treatment withwith 7-OH-PIPAT 7-OH-PIPAT alone). alone). (&(& pp < 0.05, < 0.05, &&& p < &&& p < 0.001; versus treatment with melatonin or IIK7 alone). (C,F) DMR tracings representing the 0.001; versus treatment with melatonin or IIK7 alone). (C,F) DMR tracings representing the picometer-shifts picometer-shiftsofofreflected reflected light wavelengths light wavelengths overover timetime uponupon ligandligand treatment. Note that treatment. Note data related that data to D3 R agonists related are the same to D3R agonists are thein the sameupper in theand lower upper panels; and lowerfor clarity,for panels; weclarity, decided wetodecided keep sixtopanels keep instead of including all the information in three panels. six panels instead of including all the information in three panels. Interestingly, and as previously described by Alkozi et al., [34], none of the melatonin receptor Interestingly, and as previously described by Alkozi et al., [34], none of the melatonin receptor agonists, melatonin or IIK7, were able to decrease cAMP levels in these cells. Next, we analyzed ERK1/2 agonists, melatonin or IIK7, were able to decrease cAMP levels in these cells. Next, we analyzed phosphorylation, which was significantly enhanced when 7-OH-PIPAT was added (Figure 4B,E). ERK1/2 phosphorylation, which was significantly enhanced when 7-OH-PIPAT was added (Figure When the cells were treated with 7-OH-PIPAT in combination with melatonin or with IIK7, a negative 4B,E). When the cells were treated with 7-OH-PIPAT in combination with melatonin or with IIK7, a cross-talk was observed, whereas a bi-directional cross antagonism occurred for both the D3 R–MT1 R negative cross-talk was observed, whereas a bi-directional cross antagonism occurred for both the and D3 R–MT2 R heteromers. Finally, interpretation of the DMR data resulting from activation of the D3R–MT1R and D3R–MT2R heteromers. Finally, interpretation of the DMR data resulting from receptors in the two heteromers was complex (Figure 4C,F). Perhaps the most relevant finding was the activation of the receptors in the two heteromers was complex (Figure 4C,F). Perhaps the most bi-directional cross-antagonism detected for both D3 R–MT1 R and D3 R–MT2 R heteromers expressed in relevant finding was the bi-directional cross-antagonism detected for both D3R–MT1R and D3R– the 59HCE cells. Apart from the evidence on heteromer formation, the results showed that neither D3 , MT2R heteromers expressed in the 59HCE cells. Apart from the evidence on heteromer formation, MT1 , or MT2 receptors were coupled functionally to Gi and that D3 R–MT1 R and D3 R–MT2 R signaling the results showed that neither D3, MT1, or MT2 receptors were coupled functionally to Gi and that was biased towards the MAPK pathway. D3R–MT1R and D3R–MT2R signaling was biased towards the MAPK pathway. 3.6. Differential Expression of D3 -MT1 and D3 -MT2 Heteroreceptor Complexes in the Glaucomatous Eye 3.6. Differential Expression of D3-MT1 and D3-MT2 Heteroreceptor Complexes in the Glaucomatous Eye We wondered whether intraocular hypertension correlated with the altered expression of dopamine We wondered whether intraocular hypertension correlated with the altered expression of and melatonin heteroreceptor complexes. First of all, we took advantage of an in vitro model of dopamine and melatonin heteroreceptor complexes. First of all, we took advantage of an in vitro model of hypertension, described in Reference [54], consisting of the activation of the transient Cells 2020, 9, 152 13 of 20 Cells 2020, 9, x FOR PEER REVIEW 13 of 20 hypertension, described in Reference [54], consisting of the activation of the transient receptor potential receptor potential vanilloid 4 (TRPV4) vanilloid channel. 4 (TRPV4) channel.inSuch Such activation 59HCEactivation in 59HCE cells using cells usingmimics GSK1016790A GSK1016790A the ion mimicsthat fluxes thedrive ion the fluxes thatindrive increase the increase hydrostatic pressure in thathydrostatic occurs in the pressure that occurs in the hypertensive/glaucomatous hypertensive/glaucomatous eye. eye. TRPV4 TRPV4 stimulation by application of stimulation by application was the agonist, GSK1016790A, of the agonist, followed byGSK1016790A, PLA to detect wasD3followed the R-containingby heteromers. PLA to detect the D3R-containing Remarkably, heteromers. concentrations increasing GSK1016790A Remarkably, translated increasing GSK1016790A into a significantconcentrations decrease in the translated PLA signal into aforsignificant D3 -MT1 and decrease D3 -MT in2 the PLA signal heteromers, andfortheD3effect -MT1 and dose-dependent. was D3-MT2 heteromers, The and the effectwas reduction wasupdose-dependent. to 45% for D3 R–MT The reduction 1 R heteromerwas up to 45% expression, for and D R– 3up MT95% to 1R heteromer in the case expression, of D3 R–MT and2 R up to 95% in(Figure heteromers the case of D3Therefore, 3A,B). R–MT2R heteromers we could (Figure conclude3A,B). that Therefore, we mimicking could conclude glaucomatous that mimicking conditions glaucomatous in a cell model reduces theconditions expression in aofcell D3model -MT1 and reduces D3 -MT the 2 expression of Dcomplexes. heteroreceptor 3-MT1 and D3-MT2 heteroreceptor complexes. Finally, exploratory Finally, exploratory experiments experiments on on heteromer heteromer expression expression were were performed performed in in the the postmortem postmortem samples obtained from glaucomaglaucoma patients patients and and age-matched age-matched controls. controls. Given the limitedlimited availability availability of these unique samples, which in part reflects ethical issues, we had access to two control (healthy) two glaucomatous and two glaucomatous eyes. Accordingly, statistical analysis could not be performed performed on the resultsresults described below. described below.PLAPLAwas was performed performed in ciliary in ciliary bodybody sections sections of glaucoma of glaucoma patientspatients and age- and age-matched matched In controls. controls. In thefrom the samples samples from normotensive normotensive eyes, marked eyes, marked of expression expression D3 R–MT1of D3R–MT R and 1R and D3 R–MT 2R D3R–MT2R heteromers heteromers was found in wasthefound basalinmembrane the basal of membrane of the non-pigmented the non-pigmented epithelium. epithelium. No heteromer No heteromer expression expression was found was in thefound stroma in of thethestroma ciliaryofprocesses the ciliary(Figure processes (Figure 5A,D). 5A,D). APLA A negligible negligible signal PLAfound was signalinwas thefound negativein the negative controls controlsby performed performed omitting byoneomitting one of the of the primary primary(Figure antibodies antibodies 5C). (Figure When 5C). When samples samples cases from glaucoma fromwere glaucoma analyzed, cases were analyzed, we observed we reduction a significant observed ina the significant number reduction dots in heteromers for both the number(Figure dots for5B,E), both with heteromers (Figure up to a 50% 5B,E), with reduction in D3upR–MTto a1 R50% reduction heteromer in D3R– expression MT1aRreduction and heteromer expression of up to 45% inand a reduction the case of D3 R–MT of 2up to 45% in (Figure R heteromers the case 5F).ofTogether D3R–MTwith 2R heteromers the results (Figure 5F). obtained Together in the 59HCEwith the cells, results these data obtained point toina the 59HCEofcells, reduction these data point toheteroreceptor dopamine–melatonin a reduction of dopamine–melatonin complexes heteroreceptor under conditions of elevated complexes intraocularunder conditions of elevated intraocular pressure. pressure. Figure Figure 5.5. D33-MT -MT11 and and D D33-MT -MT22 receptor receptor heteromer heteromer expression expression in in the human ciliary body. body. In situ situ proximity ligation assays were performed as described in Materials and Methods using proximity ligation assays were performed as described in Materials and Methods using human human ciliary body ciliarysections body from age-matched sections intraocular pressure from age-matched (IOP)pressure intraocular normotensive (IOP)individuals (control) normotensive (A,C,D) individuals (control) (A,C,D) and glaucoma patients (B,E). Confocal microscopy images (superimposed sections) are shown. D3-MT1 (A,B) and D3-MT2 (D,E) receptor heteromers appear as red clusters and Hoechst-stained nuclei appear in blue. (C) A negative control obtained by omitting one of the Cells 2020, 9, 152 14 of 20 and glaucoma patients (B,E). Confocal microscopy images (superimposed sections) are shown. D3 -MT1 (A,B) and D3 -MT2 (D,E) receptor heteromers appear as red clusters and Hoechst-stained nuclei appear in blue. (C) A negative control obtained by omitting one of the primary antibodies is shown. The ratio (number of red spots/cell) for the indicated samples is shown in (F). Data are the mean ± S.E.M. of counts in 6 different fields (each image covered an area of 238 µm2 ) from every sample from the controls (n = 2) or glaucoma patients (n = 2). Scale bar: 40 µm. 4. Discussion Dopamine, one of the primary neurotransmitters of the central nervous system, participates in almost any higher-order function, from motor control to emotion control, and from cognition to behavior. Dopamine is also a neurohormone acting in the periphery via the same receptors that are expressed in the brain. The difference is that the relevant actions of dopamine are mainly exerted by neuronal receptors, while in the periphery, these receptors are expressed in almost any kind of cell type. A prospection study, made in our laboratory, showed the expression of the D3 R in different cells of the eye structure, including those that controlled IOP. These expression results fit with pharmacological studies showing that different D3 R agonists may lower IOP and that the effect is lost in D3 R-/- mice [43–45]. From earlier studies by the laboratory of S. George [55–57] it is known that dopamine receptors may directly interact with themselves to form homodimers or with other GPCRs. In a previous study, we also discovered D1 -D3 heteroreceptor complexes with high relevance in Parkinson’s disease-associated dyskinesia [53]. For these reasons, we explored whether the D3 R expressed in ocular structures could form functional heteromers with other GPCR of significant relevance for eye physiology. Melatonin is an example of a neurohormone produced by the pineal gland that may also be produced (and released) elsewhere. Enzymes that participate in the synthesis of melatonin are expressed in different cell types and the indoleamine participates in IOP regulation. Although the retina was among the first identified structures able to synthesize melatonin [58,59], other eye cell types express melatonin synthesizing enzymes (see 1. Introduction). Our results decipher one of the mechanisms underlying the dopamine–melatonin interactions described to occur in the eye. Based on prototypic heteromer prints that occur similarly in heterologous expression systems and natural cell lines, and by further confirmation in natural sources by PLA, we discovered novel physiologically relevant receptor–receptor interactions. The D3 R, expressed in ciliary body epithelial cells, may interact with either MT1 R or MT2 R. Canonical coupling of melatonin receptors individually expressed in a heterologous system is to Gi (see References [25,52] and references therein). Dopamine receptors are either coupled to Gs (D1 and D5 ) or Gi (D2 , D3 and D4 ). Remarkably, in the D3 R–MT1 R and the D3 R–MT2 R heteromer, the dopamine receptor did not couple to Gi but was still functional, as dopamine receptor agonists could activate the MAPK pathway. Indeed, heteromerization produced a biased functionality, i.e., a given agonist of the D3 R, e.g., 7-OH-PIPAT may engage Gi-mediated signaling but only in the right context [60]. Our results prove antagonistic interactions, thus constituting a base to understand why the levels of dopamine and melatonin in the eye are oppositely varying according to the 24-h circadian rhythm [39,40]. Not only the presence of melatonin receptors and heteromer formation alters D3 R-mediated signaling, but coactivation of receptors—in heteromers—by moderate levels of the indoleamine and of dopamine would result in a decrease in the overall signal transduction output. Accordingly, these heteromers mediate dopamine/melatonin antagonistic actions in the eye. Considering IOP regulation by melatonin, circadian rhythms, and opposite melatonin/dopamine circadian levels in the eye; the results of heteromer expression in both a cell model of eye-related hypertension, namely 59HCE cells treated with a TRPV4 agonist, and the human eye (from IOP normotensive and hypertensive individuals) were relevant. They provided evidence of a correlation between heteromer D3 R–MT1 R and D3 R–MT2 R complex disruption and elevated IOP. Despite the few studies addressing GPCR heteromer expression in disease, often the results are quite relevant. Cells 2020, 9, 152 15 of 20 Cells On the2020, one9, xhand, FOR PEER theyREVIEW provide the basis for confirming whether the heteromer is expressed or not 15 and, of 20 accordingly, whether the heteromer may be a therapeutic target. On the other hand, the differential Based onmay functionality ourlead results, we think that of to understanding future directions should the consequences, at theinclude interventions signaling level, of theassaying IOP maintenance after infusions in the eye of a combination of melatonin and either D 3R agonists or antagonists at or the disassembly of heteroreceptor complexes. This will provide reliable information on the different times ofand pathophysiology the factors day in affecting wild typethe animals courseand in disease. of the a glaucoma animal model, such as the aged DBA/2J mice Based on [61,62]. From our results, wethethinkresults of such that future analysis,should directions a potential includetherapy would assaying interventions arise for IOP the assessment of the hypertensive eye. after infusions in the eye of a combination of melatonin and either D3 R agonists or antagonists at different times of the day in wild type animals and in a glaucoma animal model, such as the aged DBA/2J Author Contributions: Conceptualization, H.A.A., J.P., G.N., and R.F.; data curation, I.R.-R. and G.N.; Formal mice [61,62]. From the results of such analysis, a potential therapy would arise for the assessment of analysis, I.R.-R. and A.d.S.-B.; Funding acquisition, J.P. and R.F; Investigation, I.R.-R., J.L., G.N., and A.d.S.-B.; the hypertensive Methodology, eye. I.R.-R., H.A.A., J.L., J.J., J.P., G.N., and R.F.; Project administration, J.J., J.P., and R.F.; Resources, J.S.-N., J.P. and R.F. Supervision, J.P. G.N., and R.F.; Validation, J.P. G.N. and R.F.; Visualization, J.P. G.N., and Author Contributions: Conceptualization, H.A.A., J.P., G.N., and R.F.; data curation, I.R.-R. and G.N.; Formal R.F.; Writing—original analysis, draft, I.R.-R. I.R.-R. and A.d.S.-B.; FundingH.A.A., and R.F.; acquisition, Writing—review J.P. and & editing, R.F; Investigation, I.R.-R., I.R.-R., H.A.A., J.L., G.N., andG.N., and A.d.S.-B.; R.F. All authors (except J.P., see Acknowledgements) have read and validated the submitted Methodology, I.R.-R., H.A.A., J.L., J.J., J.P., G.N., and R.F.; Project administration, J.J., J.P., and R.F.; Resources, version. All J.S.-N., J.P. and R.F. Supervision, J.P. G.N., and R.F.; Validation, J.P. G.N. authors (except J.P.) have read and agreed to the published version of the manuscript. and R.F.; Visualization, J.P. G.N., and R.F.; Writing—original draft, I.R.-R. H.A.A., and R.F.; Writing—review & editing, I.R.-R., H.A.A., G.N., and R.F. Funding: All authors This research (except wasAcknowledgements) J.P., see funded by grants from havethe Spanish read Ministerio and validated thede Economía submitted y Competitividad version. All authors (MINECO) (except Ref. read J.P.) have [SAF2013-44416-R], and agreed to the[SAF2016-77084-R] published version ofand [BFU2015-64405-R], and from the Spanish the manuscript. Ministerio Funding: This de Sanidad researchRef. was RETICS funded[RD12/0034/0003] by grants from the andSpanish [RD16/0008/0017]. Ministerio Hanan A. Alkozi de Economía is a fellowship y Competitividad holder of the Saudi Arabian government. MINECO grants may include (MINECO) Ref. [SAF2013-44416-R], [SAF2016-77084-R] and [BFU2015-64405-R], and from the Spanish EU FEDER funds. The “NBM” Ministerio Molecular de SanidadNeurobiology laboratory of theand Ref. RETICS [RD12/0034/0003] University of Barcelona [RD16/0008/0017]. is considered Hanan A. Alkozi isofa fellowship excellence holder (“grup of the Saudiby consolidat”) Arabian government. the regional MINECO Catalonian grants may Government, include which EU FEDER neither provides funds. fundsTheto“NBM” Molecular the consolidated Neurobiology laboratory nor laboratory to perform of thethe University research of Barcelona is considered of excellence (“grup consolidat”) by the here reported. regional Catalonian Government, which neither provides funds to the consolidated laboratory nor to perform the research here reported. Acknowledgments: In memoriam of our co-author Jesús Pintor, astounding as a scientist and yet a good person. Acknowledgments: In memoriam of our co-author Jesús Pintor, astounding as a scientist and yet a good person. Conflicts of Interest: Conflicts of Interest: The authors declare declare no no conflict conflict of of interest. interest. Appendix A Appendix A A1. Membrane Figure A1. Figure Membranereceptor receptorexpression and expression cAMP and responses cAMP in single-transfected responses HEK-293T in single-transfected cells. HEK-293T (A) Confocal microscopy images of HEK-293T cells transfected with cDNAs for D3 R-Rluc (2 µg), cells. (A) Confocal microscopy images of HEK-293T cells transfected with cDNAs for D3R-Rluc (2 MT R-YFP (2 µg), or MT2 R-YFP (2 µg). D3 receptors (red) were identified by immunocytochemistry μg),1 MT1R-YFP (2 μg), or MT2R-YFP (2 μg). D3 receptors (red) were identified by using anti-Rluc antibodies. MT1 and MT2 receptors (green) were identified from the fluorescence of immunocytochemistry using anti-Rluc antibodies. MT1 and MT2 receptors (green) were identified YFP-containing fusion proteins. Cell nuclei were stained with Hoechst (blue channel). Scale bar: from the fluorescence of YFP-containing fusion proteins. Cell nuclei were stained with Hoechst (blue channel). Scale bar: 20 μm. Panels (B–D) HEK-293T cells transfected with cDNAs encoding for D3R (1.5 μg) (B), MT1R (1.5 μg) (C) or MT2R (1.5 μg) (D) cells were pre-treated with vehicle or antagonists (1 μM raclopride for D3R, 1 μM luzindole for MT1R or 1 μM 4PPDOT for MT2R), and Cells 2020, 9, 152 16 of 20 20 µm. Panels (B–D) HEK-293T cells transfected with cDNAs encoding for D3 R (1.5 µg) (B), MT1 R (1.5 µg) (C) or MT2 R (1.5 µg) (D) cells were pre-treated with vehicle or antagonists (1 µM raclopride Cells 2020, 9, x FOR PEER REVIEW 16 of 20 for D3 R, 1 µM luzindole for MT1 R or 1 µM 4PPDOT for MT2 R), and then subsequently treated with different concentrations of selective agonists (10–1000 nM 7-OH-PIPAT for D3 R, 0.5–10 µM melatonin then subsequently treated with different concentrations of selective agonists (10–1000 nM 7-OH- for MT1 R or 5–100 nM IIK7 for MT2 R). cAMP accumulation was detected by TR-FRET in the presence PIPAT for D3R, 0.5–10 μM melatonin for MT1R or 5–100 nM IIK7 for MT2R). cAMP accumulation ofwas 0.5 µM forskolin. detected cAMP production by TR-FRET is expressed in the presence of 0.5 μMasforskolin. % of levels obtained cAMP by 0.5 µM production forskolin as is expressed (0 nM % agonist). Values are the mean ± S.E.M. of 6 different experiments performed in triplicates, of levels obtained by 0.5 μM forskolin (0 nM agonist). Values are the mean ± S.E.M. of 6 different and one-way ANOVA followed experiments by Bonferroni’s performed multiple in triplicates, andcomparison post-hoc tests one-way ANOVA were used followed for statistical by Bonferroni’s analysis. multiple p < 0.05, ***post-hoc (*comparison p < 0.001; versus treatment with forskolin). (### p < 0.001; versus treatment with 1 µM tests were used for statistical analysis. (* p < 0.05, *** p < 0.001; versus treatment melatonin or 100 (### with forskolin). nM IIK7 alone). p < 0.001; versus treatment with 1 μM melatonin or 100 nM IIK7 alone). Figure A2. ERK phosphorylation and DMR responses in single-transfected HEK-293T cells. HEK-293T Figure A2. ERK phosphorylation and DMR responses in single-transfected HEK-293T cells. HEK- cells transfected with cDNA encoding for D3 R (1.5 293T cells transfected with cDNA encoding for µg) (A,D), MT R (1.5 µg) (B,E), or MT2 R 2(1.5 D3R (1.5 μg) (A,D), 1MT1R (1.5 μg) (B,E), or MT µg) R (1.5 (C,F) were pre-treated with vehicle or antagonists (1 µM raclopride for D R, 1 µM luzindole μg) (C,F) were pre-treated with vehicle or antagonists (1 μM raclopride 3for D3R, 1 μM luzindole for for MT 1 R, orMT 1 µM 4PPDOT for MT2 R), 1R, or 1 μM 4PPDOT for and MT2then subsequently R), and treated with then subsequently treateddifferent concentrations with different of agonists concentrations of (30–1000 agonists (30–1000 nM 7-OH-PIPAT for D3R, 0.3–10 μM melatonin for MT1R, or 3–1000 nMR). nM 7-OH-PIPAT for D 3 R, 0.3–10 µM melatonin for MT 1 R, or 3–1000 nM IIK7 for MT 2 ERK1/2 IIK7 for phosphorylation (A–C) was analyzed using ® SureFire® kit (Perkin Elmer). ERK1/2 MT2R). ERK1/2 phosphorylation (A–C) wasan AlphaScreen analyzed using an AlphaScreen®SureFire® kit (Perkin phosphorylation Elmer). ERK1/2 phosphorylation data are expressed as % with respect (0 data are expressed as % with respect to basal levels to nM basalagonist). levels (0 Values are the nM agonist). mean ± S.E.M. of seven different experiments performed in triplicates, and one-way Values are the mean ± S.E.M. of seven different experiments performed in triplicates, and one-way ANOVA followed byANOVA Bonferroni’s multiple followed comparisonmultiple by Bonferroni’s post-hoccomparison tests were post-hoc used for tests statistical were analysis. (* p < 0.05, used for statistical p < 0.01, (* **analysis. ***p p<<0.05, 0.001; (## p < ** p < 0.01, *** p < 0.001; versus basal). (## p < 0.001; versus treatment withalone). versus basal). 0.001; versus treatment with 100 nM 7-OH-PIPAT 100 (D–F) DMR tracings representing nM 7-OH-PIPAT alone). (D–F) DMR the picometer-shifts of reflected tracings representing light wavelengths the picometer-shifts of over timelight reflected upon agonist ligand over wavelengths treatment. time upon agonist ligand treatment. Cells 2020, 9, 152 17 of 20 Cells 2020, 9, x FOR PEER REVIEW 17 of 20 Figure A3. The specificity of antibodies against D3 , MT1 , and MT2 receptors. Confocal microscopy Figure A3. The specificity of antibodies against D3, MT1, and MT2 receptors. Confocal microscopy images of the HEK-293T cells untransfected (bottom) or transfected with cDNAs coding for D3 , MT1 , images or MT2ofreceptors the HEK-293T cellsThe (0.5 µg). untransfected (bottom)with cells were treated or transfected with cDNAs primary antibodies coding against for D either D33,, MT MT1,1 , or MT 2 receptors (0.5 μg). The cells were treated with primary antibodies against either D3, MT1, or or MT2 receptors (as indicated), and subsequently treated with a Cy3 secondary antibody (red). 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