A fundamental question in G protein coupled receptor biology is how a single ligand acting at a specific receptor is able to induce a range of signaling that results in a variety of physiological responses. We focused on Type 1 cannabinoid receptor (CB1R) as a model GPCR involved in a variety of processes spanning from analgesia and euphoria to neuronal development, survival and differentiation. We examined receptor dimerization as a possible mechanism underlying expanded signaling responses by a single ligand and focused on interactions between CB1R and delta opioid receptor (DOR). Using co-immunoprecipitation assays as well as analysis of changes in receptor subcellular localization upon co-expression, we show that CB1R and DOR form receptor heteromers. We find that heteromerization affects receptor signaling since the potency of the CB1R ligand to stimulate G-protein activity is increased in the absence of DOR, suggesting that the decrease in CB1R activity in the presence of DOR could, at least in part, be due to heteromerization. We also find that the decrease in activity is associated with enhanced PLC-dependent recruitment of arrestin3 to the CB1R-DOR complex, suggesting that interaction with DOR enhances arrestin-mediated CB1R desensitization. Additionally, presence of DOR facilitates signaling via a new CB1R-mediated anti-apoptotic pathway leading to enhanced neuronal survival. Taken together, these results support a role for CB1R-DOR heteromerization in diversification of endocannabinoid signaling and highlight the importance of heteromer-directed signal trafficking in enhancing the repertoire of GPCR signaling.
References
[1]
Berghuis P, Rajnicek AM, Morozov YM, Ross RA, Mulder J, et al. (2007) Hardwiring the brain: endocannabinoids shape neuronal connectivity. Science 316: 1212–1216.
Guzman M, Sanchez C, Galve-Roperh I (2002) Cannabinoids and cell fate. Pharmacol Ther 95: 175–184.
[4]
Bromberg KD, Ma'ayan A, Neves SR, Iyengar R (2008) Design logic of a cannabinoid receptor signaling network that triggers neurite outgrowth. Science 320: 903–909.
[5]
Harkany T, Guzman M, Galve-Roperh I, Berghuis P, Devi LA, et al. (2007) The emerging functions of endocannabinoid signaling during CNS development. Trends Pharmacol Sci 28: 83–92.
[6]
Gomes I, Grushko JS, Golebiewska U, Hoogendoorn S, Gupta A, et al. (2009) Novel endogenous peptide agonists of cannabinoid receptors. FASEB J 23: 3020–3029.
[7]
Mackie K, Hille B (1992) Cannabinoids inhibit N-type calcium channels in neuroblastoma-glioma cells. Proc Natl Acad Sci U S A 89: 3825–3829.
[8]
Mackie K, Lai Y, Westenbroek R, Mitchell R (1995) Cannabinoids activate an inwardly rectifying potassium conductance and inhibit Q-type calcium currents in AtT20 cells transfected with rat brain cannabinoid receptor. J Neurosci 15: 6552–6561.
[9]
Ma'ayan A, Jenkins SL, Barash A, Iyengar R (2009) Neuro2A differentiation by Galphai/o pathway. Sci Signal 2: cm1.
[10]
Jin W, Brown S, Roche JP, Hsieh C, Celver JP, et al. (1999) Distinct domains of the CB1 cannabinoid receptor mediate desensitization and internalization. J Neurosci 19: 3773–3780.
[11]
Cichewicz DL, Cox ML, Welch SP, Selley DE, Sim-Selley LJ (2004) Mu and delta opioid-stimulated [35S]GTP gamma S binding in brain and spinal cord of polyarthritic rats. Eur J Pharmacol 504: 33–38.
[12]
Manzanares J, Corchero J, Fuentes JA (1999) Opioid and cannabinoid receptor-mediated regulation of the increase in adrenocorticotropin hormone and corticosterone plasma concentrations induced by central administration of delta(9)-tetrahydrocannabinol in rats. Brain Res 839: 173–179.
[13]
Ghozland S, Matthes HW, Simonin F, Filliol D, Kieffer BL, et al. (2002) Motivational effects of cannabinoids are mediated by mu-opioid and kappa-opioid receptors. J Neurosci 22: 1146–1154.
[14]
Ledent C, Valverde O, Cossu G, Petitet F, Aubert JF, et al. (1999) Unresponsiveness to cannabinoids and reduced addictive effects of opiates in CB1 receptor knockout mice. Science 283: 401–404.
[15]
Rios C, Gomes I, Devi LA (2006) mu opioid and CB1 cannabinoid receptor interactions: reciprocal inhibition of receptor signaling and neuritogenesis. Br J Pharmacol 148: 387–395.
[16]
Bushlin I, Rozenfeld R, Devi LACannabinoid-opioid interactions during neuropathic pain and analgesia. Curr Opin Pharmacol 10: 80–86.
[17]
Dill JA, Howlett AC (1988) Regulation of adenylate cyclase by chronic exposure to cannabimimetic drugs. J Pharmacol Exp Ther 244: 1157–1163.
[18]
Law PY, Koehler JE, Loh HH (1982) Comparison of opiate inhibition of adenylate cyclase activity in neuroblastoma N18tG2 and neuroblastoma x glioma NG108-15 hybrid cell lines. Mol Pharmacol 21: 483–491.
[19]
Shapira M, Gafni M, Sarne Y (1998) Independence of, and interactions between, cannabinoid and opioid signal transduction pathways in N18TG2 cells. Brain Res 806: 26–35.
[20]
Rubovitch V, Gafni M, Sarne Y (2004) The involvement of VEGF receptors and MAPK in the cannabinoid potentiation of Ca2+ flux into N18TG2 neuroblastoma cells. Brain Res Mol Brain Res 120: 138–144.
[21]
Korzh A, Keren O, Gafni M, Bar-Josef H, Sarne Y (2008) Modulation of extracellular signal-regulated kinase (ERK) by opioid and cannabinoid receptors that are expressed in the same cell. Brain Res 1189: 23–32.
[22]
Berrendero F, Maldonado R (2002) Involvement of the opioid system in the anxiolytic-like effects induced by Delta(9)-tetrahydrocannabinol. Psychopharmacology (Berl) 163: 111–117.
[23]
Berrendero F, Mendizabal V, Murtra P, Kieffer BL, Maldonado R (2003) Cannabinoid receptor and WIN 55 212-2-stimulated [35S]-GTPgammaS binding in the brain of mu-, delta- and kappa-opioid receptor knockout mice. Eur J Neurosci 18: 2197–2202.
[24]
Uriguen L, Berrendero F, Ledent C, Maldonado R, Manzanares J (2005) Kappa- and delta-opioid receptor functional activities are increased in the caudate putamen of cannabinoid CB1 receptor knockout mice. Eur J Neurosci 22: 2106–2110.
[25]
Gupta A, Decaillot FM, Gomes I, Tkalych O, Heimann AS, et al. (2007) Conformation-state sensitive antibodies to G-protein-coupled receptors. J Biol Chem 282: 5116–5124.
[26]
Cvejic S, Trapaidze N, Cyr C, Devi LA (1996) Thr353, located within the COOH-terminal tail of the delta opiate receptor, is involved in receptor down-regulation. J Biol Chem 271: 4073–4076.
[27]
Decaillot FM, Befort K, Filliol D, Yue S, Walker P, et al. (2003) Opioid receptor random mutagenesis reveals a mechanism for G protein-coupled receptor activation. Nat Struct Biol 10: 629–636.
[28]
Rozenfeld R, Devi LA (2008) Regulation of CB1 cannabinoid receptor trafficking by the adaptor protein AP-3. Faseb J 22: 2311–2322.
[29]
Fedorova E, Battini L, Prakash-Cheng A, Marras D, Gusella GL (2006) Lentiviral gene delivery to CNS by spinal intrathecal administration to neonatal mice. J Gene Med 8: 414–424.
[30]
Zhu Y, King MA, Schuller AG, Nitsche JF, Reidl M, et al. (1999) Retention of supraspinal delta-like analgesia and loss of morphine tolerance in delta opioid receptor knockout mice. Neuron 24: 243–252.
[31]
Gomes I, Filipovska J, Devi LA (2003) Opioid receptor oligomerization. Detection and functional characterization of interacting receptors. Methods Mol Med 84: 157–183.
[32]
Gomes I, Jordan BA, Gupta A, Trapaidze N, Nagy V, et al. (2000) Heterodimerization of mu and delta opioid receptors: A role in opiate synergy. J Neurosci 20: RC110.
[33]
Gupta A, Mulder J, Gomes I, Rozenfeld R, Bushlin I, et al. Increased abundance of opioid receptor heteromers after chronic morphine administration. Sci Signal 3: ra54.
[34]
Smith ER, Smedberg JL, Rula ME, Xu XX (2004) Regulation of Ras-MAPK pathway mitogenic activity by restricting nuclear entry of activated MAPK in endoderm differentiation of embryonic carcinoma and stem cells. J Cell Biol 164: 689–699.
[35]
Gupta A, Devi LA (2006) The use of receptor-specific antibodies to study G-protein-coupled receptors. Mt Sinai J Med 73: 673–681.
[36]
Rozenfeld R, Devi LA (2007) Receptor heterodimerization leads to a switch in signaling: beta-arrestin2-mediated ERK activation by mu-delta opioid receptor heterodimers. Faseb J 21: 2455–2465.
[37]
Hirst J, Robinson MS (1998) Clathrin and adaptors. Biochim Biophys Acta 1404: 173–193.
[38]
Rozenfeld RType I cannabinoid receptor trafficking: all roads lead to lysosome. Traffic 12: 12–18.
[39]
Fan SF, Shen KF, Scheideler MA, Crain SM (1992) F11 neuroblastoma x DRG neuron hybrid cells express inhibitory mu- and delta-opioid receptors which increase voltage-dependent K+ currents upon activation. Brain Res 590: 329–333.
[40]
Coutts AA, Anavi-Goffer S, Ross RA, MacEwan DJ, Mackie K, et al. (2001) Agonist-induced internalization and trafficking of cannabinoid CB1 receptors in hippocampal neurons. J Neurosci 21: 2425–2433.
[41]
Hong MH, Xu C, Wang YJ, Ji JL, Tao YM, et al. (2009) Role of Src in ligand-specific regulation of delta-opioid receptor desensitization and internalization. J Neurochem 108: 102–114.
[42]
Luttrell LM, Lefkowitz RJ (2002) The role of beta-arrestins in the termination and transduction of G-protein-coupled receptor signals. J Cell Sci 115: 455–465.
[43]
Shankar H, Michal A, Kern RC, Kang DS, Gurevich VV, et al. (2010) Non-visual arrestins are constitutively associated with the centrosome and regulate centrosome function. J Biol Chem 285: 8316–8329.
[44]
Molla-Herman A, Boularan C, Ghossoub R, Scott MGH, Burtey A, et al. (2008) Targeting of beta-arrestin 2 to the centrosome and primary cilium: role in cell proliferation control. PlosOne 3: e3728. doi:10.1371/journal.pone.0003728.
[45]
Ahn S, Kim J, Hara MR, Ren XR, Lefkowitz RJ (2009) beta -arrestin2 mediates anti-apoptotic signaling through regulation of bad phosphorylation. J Biol Chem.
[46]
Bergmann A (2002) Survival signaling goes BAD. Dev Cell 3: 607–608.
[47]
Urban JD, Clarke WP, von Zastrow M, Nichols DE, Kobilka B, et al. (2007) Functional selectivity and classical concepts of quantitative pharmacology. J Pharmacol Exp Ther 320: 1–13.
[48]
Rozenfeld R, Devi LAExploring a role for heteromerization in GPCR signalling specificity. Biochem J 433: 11–18.
[49]
Drake MT, Violin JD, Whalen EJ, Wisler JW, Shenoy SK, et al. (2008) beta-arrestin-biased agonism at the beta2-adrenergic receptor. J Biol Chem 283: 5669–5676.
