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Somatostatin Receptor 5 Is a Prominent Regulator of Signaling Pathways in Cells with Coexpression of Cannabinoid Receptors 1

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Somatostatin Receptor 5 Is a Prominent Regulator of Signaling Pathways in Cells with Coexpression of Cannabinoid Receptors 1 NSC 17416 No. of Pages 14 4 November 2016 Please cite this article in press as: Zou S et al. Somatostatin receptor 5 is a prominent regulator of signaling pathways in cells with coexpression of Cannabinoid receptors 1. Neuroscience (2016), http://dx.doi.org/10.1016/j.neuroscience.2016.10.056 1 Neuroscience xxx (2016) xxx–xxx 2 SOMATOSTATIN RECEPTOR 5 IS A PROMINENT REGULATOR OF 3 SIGNALING PATHWAYS IN CELLS WITH COEXPRESSION OF 4 CANNABINOID RECEPTORS 1 5 SHENGLONG ZOU, RISHI K. SOMVANSHI AND in the regulation of receptor trafficking and signal transduc- 6 UJENDRA KUMAR * tion pathways open new therapeutic avenue in cancer biol- Ó 7 Faculty of Pharmaceutical Sciences, The University of ogy and excitotoxicity. 2016 IBRO. Published by Elsevier 8 British Columbia, Vancouver, BC, Canada Ltd. All rights reserved. 9 Abstract—Endocannabinoids and somatostatin (SST) play critical roles in several pathophysiological conditions via Key words: somatostatin receptor 5, cannabinoid receptor 1, binding to different receptor subtypes. Cannabinoid recep- G protein-coupled receptor, heterodimerization, signaling. tor 1 (CB1R) and somatostatin receptors (SSTRs) are 10 expressed in several brain regions and share overlapping functions. Whether these two prominent members of G-pro- tein-coupled receptor (GPCR) family interact with each other INTRODUCTION 11 and constitute a functional receptor complex is not known. In the present study, we investigated the colocalization of Cannabinoid receptor 1 (CB1R) belongs to G-protein- 12 CB1R and SSTR5 in rat brain, and studied receptor internal- coupled receptor (GPCR) family and is the major 13 ization, interaction and signal transduction pathways in cannabinoid receptor (CBR) that is well-expressed in 14 HEK-293 cells cotransfected with human cannabinoid recep- both central and peripheral nervous systems. In central 15 tor 1 (hCB1R) and hSSTR5. Our results showed that CB1R nervous system (CNS), most of the physiological effects 16 9 and SSTR5 colocalized in rat brain cortex, striatum, and hip- of D -tetrahydrocannabinol, the main psychoactive 17 pocampus. CB1R was expressed in SSTR5 immunoprecipi- component in cannabis, are mediated by CB1R 18 tate prepared from the brain tissue lysate, indicating their (Matsuda et al., 1990). CB1R is widely expressed in the 19 association in a system where these receptors are endoge- CNS, and plays a crucial role in neurotransmission, neu- 20 nously expressed. In cotransfected HEK-293 cells, SSTR5 romodulation, and synaptic plasticity upon activation by 21 and CB1R existed in a constitutive heteromeric complex under basal condition, which was disrupted upon agonist its endogenous ligand, endocannabinoids (Howlett et al., 22 treatments. Furthermore, concurrent receptor activation 2002). Endocannabinoids bind to CB1R and inhibit the 23 led to preferential formation of SSTR5 homodimer and dis- release of several neurotransmitters from presynaptic 24 sociation of CB1R homodimer. We also discovered that sec- terminals and exert neuroprotective effects against 25 ond messenger cyclic adenosine monophosphate and excitotoxicity (Marsicano et al., 2003). Given that endo- 26 downstream signaling pathways were modulated in a cannabinoids exert such predominant role in synaptic 27 SSTR5-dominant and concentration-dependent manner in communication, any interference in this system could 28 the presence of receptor-specific agonist. In conclusion, initiate severe pathophysiological conditions. Previous 29 with predominant role of SSTR5, the functional conse- studies have reported decreased expression of CB1R in 30 quences of crosstalk between SSTR5 and CB1R resulting Alzheimer’s disease (AD), Parkinson’s disease and Hunt- 31 ington’s disease (HD) Bisogno and Di Marzo, 2010. CB1R 32 *Corresponding author. Address: Faculty of Pharmaceutical knockout mice display an increased susceptibility to exci- 33 Sciences, The University of British Columbia, Vancouver, BC V6T totoxin along with sustained neurodegeneration, indicat- 34 1Z3, Canada. Fax: +1-604-822-3035. ing the pivotal role of CB1R in neuroprotection (Mievis 35 E-mail address: [email protected] (U. Kumar). Abbreviations: AD, Alzheimer’s disease; cAMP, cyclic adenosine et al., 2011). The neuroprotective role of CB1R has been 36 monophosphate; CB1R, Cannabinoid receptor 1; CNS, central reported both in vivo and in vitro (Ramirez et al., 2005; Liu 37 nervous system; co-IP, co-immunoprecipitation; Cy3, cyanine 3; et al., 2009). In peripheral tissues, CB1R is associated 38 D2R, dopamine receptor 2; ERK1/2, extracellular signal-regulated kinase 1/2; FITC, fluorescein isothiocyanate; FSK, forskolin; GPCR, G with several pathophysiological functions, including car- 39 protein-coupled receptor; HA, hemagglutinin; hCB1R, human diac functions, energy metabolism and bone formation 40 cannabinoid receptor 1; HD, Huntington’s disease; HEPES, 4-(2-hydr (Pacher et al., 2006). Despite these promising therapeutic 41 oxyethyl)-1-piperazineethanesulfonic acid; hSSTR5, human 42 somatostatin receptor 5; IBMX, 3-isobutyl-1-methylxanthine; NGS, benefits, the use of cannabinoids in clinical practice is lim- normal goat serum; PAGE, polyacrylamide gel electrophoresis; ited, due to undesired psychoactive effects and increased 43 pbFRET, photobleaching fluorescence resonance energy transfer; incidence of schizophrenia (Mackie, 2006). 44 PBS, phosphate-buffered saline; PI3K, phosphoinositide-3 kinase; Like cannabinoids, somatostatin (SST) plays a critical 45 PKA, protein kinase A; RIPA, radioimmune precipitation assay; RT, room temperature; SP, stratum pyramidal layer; SST, somatostatin; role in many pathological conditions through binding to 46 SSTRs, somatostatin receptors; TBS, Tris-buffered saline. five different receptor subtypes, namely somatostatin 47 http://dx.doi.org/10.1016/j.neuroscience.2016.10.056 0306-4522/Ó 2016 IBRO. Published by Elsevier Ltd. All rights reserved. 1 NSC 17416 No. of Pages 14 4 November 2016 2 S. Zou et al. / Neuroscience xxx (2016) xxx–xxx 48 receptor 1–5 (SSTR1–5). SSTR subtypes are well- CB1R interacts with any SSTR subtypes and if such inter- 109 49 expressed in the CNS in a region- and receptor-specific action exists, what the functional consequences are. We 110 50 manner (Schindler et al., 1996; Patel, 1999; Ramirez recently have reviewed that SSTR5 is one of the most 111 51 et al., 2004; Kumar, 2007). Although the expression of dynamic receptors that displays significant diversity upon 112 52 SSTR5 in the CNS is relatively low in comparison to other heterodimerization with other members of GPCR family 113 53 SSTR subtypes, its pathophysiological significance in (Somvanshi and Kumar, 2012). CB1R heterodimerizes 114 54 neurological disorders has been supported by several with D2R and tends to switch its coupling from Gi to Gs 115 55 studies (Craft et al., 1999; Stroh et al., 1999; Kumar, (Glass and Felder, 1997). Our previous study demon- 116 56 2005; Watson et al., 2009). In AD patients, decreased strated D2R heterodimerization with SSTR5 (Rocheville 117 57 expression of SSTR5-like immunoreactivity has been et al., 2000a). It is tempting to determine whether this 118 58 observed in cortical brain regions (Kumar, 2005). SSTR5 phenomenon is unique for CB1R in the combination with 119 59 is suggested to play a role in SST analog-mediated mem- D2R or a common feature of CB1R. Accordingly, in the 120 60 ory facilitation in both AD patients and healthy adults present study, we employed multiple techniques to deter- 121 61 (Craft et al., 1999; Watson et al., 2009). Furthermore, mine the possible interaction between SSTR5 and CB1R 122 62 our recent study has demonstrated comparable neuro- in rat brain with endogenous expression and HEK-293 123 63 chemical changes in the striatum of HD transgenic R6/2 cells stably transfected with hSSTR5 and/or human 124 64 mice and mice deficient in SSTR1/5 (Rajput et al., cannabinoid receptor 1 (hCB1R). Our results show that 125 65 2011). In addition, SSTR5 is involved in protection of rat SSTR5 and CB1R are coexpressed in rat brain cortex, 126 66 retina against a-amino-3-hydroxy-5-methyl-4-isoxazole striatum and hippocampus and CB1R is expressed in 127 67 propionic acid-induced excitotoxicity (Kiagiadaki et al., SSTR5 immunoprecipitate. In cotransfected HEK-293 128 68 2010; Chen et al., 2014). Taken together, these observa- cells, SSTR5 and CB1R constituted a functional complex 129 69 tions suggest a potential beneficial role of SSTR5 in neu- and displayed novel properties in modulation of down- 130 70 rological disorders, including excitotoxicity. stream signaling. At present, no molecular mechanism 131 71 At present, it is well established that GPCRs function is on place that could help to minimize undesired side 132 72 as dimers or even higher-order oligomers in a manner effect of cannabinoids while enhancing its potential med- 133 73 distinct from the native receptor (Kumar and Grant, 2010; ical use. Results described here showing complex forma- 134 74 Ferre et al., 2014; Gomes et al., 2016). Numerous studies tion between CB1R and SSTR5 provide possibility of 135 75 have shown that SSTRs, as well as CBRs, form homo- developing a new therapeutic in drugs of abuse. 136 76 and/or heterodimers either within the family or with other 77 GPCRs including dopamine, opioid, orexin and adenosine EXPERIMENTAL PROCEDURES 137 78 receptors and members of receptor tyrosine kinase family 79 (Kearn et al., 2005; Ellis et al., 2006; Rios et al., 2006; Animals 138 80 Carriba et al., 2007; Hudson et al., 2010; Somvanshi
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