Proc

Proc. signal-regulated kinases (ERK1/2) activation. GPR55 and CB1 receptors can form heteromers, but the internalization of both receptors is not affected. In addition, we observe that the presence of GPR55 enhances CB1R-mediated ERK1/2 and nuclear element of triggered T-cell activation. Our data provide the 1st evidence that GPR55 can form heteromers with another 7TM/GPCR and that this interaction with the CB1 receptor offers functional effects cerebral cortex, hippocampus, and striatum) (1, 4). In contrast, CB2Rs are primarily found on immune cells (2, 5). The lipid receptor GPR55 is definitely highly indicated in the CNS, putamen, striatum, and hippocampus, as well as with intestine, bone marrow, spleen, immune cells, and endothelial cells (6C11). GPR55 was also recognized in cancer cells and malignancy cell lines (12C15). CB1Rs mainly couple to Gi/o proteins and therefore inhibit adenylyl cyclase, activate mitogen-activated protein kinases (MAPKs), and further activate several transcription factors. In addition, CB1Rs have been explained to mediate the activation of several potassium channels (16, 17). Multiple GPR55-mediated signaling pathways have been explained (6, 7, 18C20), whereby probably the most consistent reports suggest that GPR55 couples to G13 in recombinant HEK cells transiently or stably expressing GPR55 (9, 18, 19, 21C23). GPR55 signaling can be mediated via small GTPases (6, 21, 22) and the mobilization of intracellular calcium stores (21, 22, 24, 25). This prospects to the activation of several transcription factors, such as nuclear element of triggered T-cells (NFAT), nuclear element -light chain-enhancer of triggered B cells (NF-B), cyclic AMP response-element binding protein, and activating transcription element 2 (21, 22, 26). In addition, the activation of MAP kinases, such as p38 and ERK1/2 MAPKs were described to be induced after GPR55 activation (22, 26). Furthermore, the formation of filamentous actin (F-actin) upon GPR55 activation was reported in HEK293 cells and human being neutrophils and this process is definitely mediated by G13 and RhoA (6). The formation of F-actin is related to the induction of serum response elements (SRE) UNC3866 and is under control of the G13-mediated RhoA signaling (27, 28). The CB1R is definitely activated by several endogenous cannabinoid ligands, such as anandamide (AEA) and 2-arachidonoylglycerol (2-AG), as well as the psychoactive phytocannabinoid (9)-tetrahydrocannabinol, and synthetic compounds such as WIN55,212-2 or CP55940 ((2-[(1(38) and (39C41). The living of CB1R homomers was recognized by using antibodies specifically realizing CB1R dimers (42). In addition, it was reported that CB1Rs can form heteromers (3). The G protein coupling is definitely modified in CB1R-dopamine D2R heteromers (43), CB1R-orexin-1 receptor heteromers show different trafficking and signaling properties (44) and the signaling pathways are modulated in CB1R-adenosine A2A receptor heteromers (45). To time, it is unidentified whether GPR55 can develop useful heteromers with various other 7TM/GPCRs. Right here we present that GPR55 and CB1Rs may interact and modulate each others signaling properties physically. Our data present that GPR55 signaling is inhibited in the current presence of the CB1 receptor specifically. EXPERIMENTAL Techniques Cell Lifestyle, Transfections, and Steady Cell Lines HEK293 cells had been cultured in Dulbecco’s improved Eagle’s moderate (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Invitrogen) at 37 C within a 5% CO2, humidified atmosphere. HEK293 cells stably expressing the individual 3HA-GPR55 (HEK-GPR55) had been previously defined (21) and preserved in G418 (PAA) formulated with moderate (0.4 mg/ml). To create HEK293 cells stably expressing individual FLAG-CB1 by itself (HEK-CB1) or 3HA-GPR55 and FLAG-CB1 receptor (HEK-GPR55+CB1), HEK293 or HEK-GPR55 cells had been transfected with pcDNA3.1 encoding the FLAG-CB1 receptor using Lipofectamine 2000 (Invitrogen). Cells had been generated in selection mass media (0.5 mg/ml of Zeocin for HEK-CB1 or 0.8 mg/ml of G418 and 0.5 mg/ml of Zeocin for HEK-GPR55+CB1) and single colonies had been propagated. HEK-CB1 cells had been cultured in DMEM formulated with 0.2 mg/ml of Zeocin, and HEK-GPR55+CB1 cells had been preserved in DMEM containing 0.4 mg/ml of G418 and 0.2 mg/ml of Zeocin. All cells had been serum starved in Opti-MEM (Invitrogen) ahead of all tests. Transient transfections had been performed using Lipofectamine 2000 following manufacturer’s instructions. Particular GPR55 Agonist GSK319197A GSK319197A is certainly [4-(3,4-dichloro-phenyl)-piperazin-1-yl]-(4-fluoro-4-methanesulfonyl-biphenyl-2-yl)-methanone (example 13 from Ref. 46), a structural analog of GSK494581A (24) and CID2440433 (35). It really is one of some benzoylpiperazines originally defined as inhibitors from the glycine transporter subtype 1 (GlyT1) and proven also to become selective ligands of GPR55 (24, 35). Needlessly to say for illustrations in.Milan-Lobo L., Whistler J. activation, such as for example nuclear aspect of turned on T-cells and serum response component, aswell as extracellular signal-regulated kinases (ERK1/2) activation. GPR55 and CB1 receptors can develop heteromers, however the internalization of both receptors isn’t affected. Furthermore, we discover that the current presence of GPR55 enhances CB1R-mediated ERK1/2 and nuclear aspect of turned on T-cell activation. Our data supply the initial proof that GPR55 can develop heteromers with another 7TM/GPCR and that interaction using the CB1 receptor provides functional implications cerebral cortex, hippocampus, and striatum) (1, 4). On the other hand, CB2Rs are generally found on immune system cells (2, 5). The lipid receptor GPR55 is certainly highly portrayed in the CNS, putamen, striatum, and hippocampus, aswell such as intestine, bone tissue marrow, spleen, immune system cells, and endothelial cells (6C11). GPR55 was also discovered in cancer tissue and cancers cell lines (12C15). CB1Rs mostly few to Gi/o protein and thus inhibit adenylyl cyclase, activate mitogen-activated proteins kinases (MAPKs), and additional activate many transcription factors. Furthermore, CB1Rs have already been defined to mediate the activation of many potassium stations (16, 17). Multiple GPR55-mediated signaling pathways have already been defined (6, 7, 18C20), whereby one of the most constant reports claim that GPR55 lovers to G13 in recombinant HEK cells transiently or stably expressing GPR55 (9, 18, 19, 21C23). GPR55 signaling could be mediated via little GTPases (6, 21, 22) as well as the mobilization of intracellular calcium mineral shops (21, 22, 24, 25). This network marketing leads to the activation of many transcription factors, such as for example nuclear aspect of turned on T-cells (NFAT), nuclear aspect Rabbit Polyclonal to SGK (phospho-Ser422) -light chain-enhancer of turned on B cells (NF-B), cyclic AMP response-element binding proteins, and activating transcription aspect 2 (21, 22, 26). Furthermore, the activation of MAP kinases, such as for example p38 and ERK1/2 MAPKs had been described to become induced after GPR55 arousal (22, 26). Furthermore, the forming of filamentous actin (F-actin) upon GPR55 activation was reported in HEK293 cells and individual neutrophils which process is certainly mediated by G13 and RhoA (6). The forming of F-actin relates to the induction of serum response components (SRE) and it is in order from the G13-mediated RhoA signaling (27, 28). The CB1R is certainly activated by many endogenous cannabinoid ligands, such as for example anandamide (AEA) and 2-arachidonoylglycerol (2-AG), aswell as the psychoactive phytocannabinoid (9)-tetrahydrocannabinol, and artificial compounds such as for example WIN55,212-2 or CP55940 ((2-[(1(38) and (39C41). The lifetime of CB1R homomers was discovered through the use of antibodies specifically spotting CB1R dimers (42). Furthermore, it had been reported that CB1Rs can develop heteromers (3). The G proteins coupling is certainly changed in CB1R-dopamine D2R heteromers (43), CB1R-orexin-1 receptor heteromers display different trafficking and signaling properties (44) as well as the signaling pathways are modulated in CB1R-adenosine A2A receptor heteromers (45). To time, it is unidentified whether GPR55 can develop useful heteromers with various other 7TM/GPCRs. Right here we present that GPR55 and CB1Rs can in physical form interact and modulate each others signaling properties. Our data present that GPR55 signaling is certainly particularly inhibited in the current presence of the CB1 receptor. EXPERIMENTAL Techniques Cell Lifestyle, Transfections, and Steady Cell Lines HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Invitrogen) at 37 C in a 5% CO2, humidified atmosphere. HEK293 cells stably expressing the human 3HA-GPR55 (HEK-GPR55) were previously described (21) and maintained in G418 (PAA) made up of medium (0.4 mg/ml). To generate HEK293 cells stably expressing human FLAG-CB1 alone (HEK-CB1) or 3HA-GPR55 and FLAG-CB1 receptor (HEK-GPR55+CB1), HEK293 or HEK-GPR55 cells were transfected with pcDNA3.1 encoding the FLAG-CB1 receptor using Lipofectamine 2000 (Invitrogen). Cells were generated in selection media (0.5 mg/ml of Zeocin for HEK-CB1 or 0.8 mg/ml of G418 and 0.5 mg/ml of Zeocin for HEK-GPR55+CB1) and single colonies were propagated. HEK-CB1 cells were cultured in DMEM made up of 0.2 mg/ml of Zeocin, and HEK-GPR55+CB1 cells were maintained in DMEM containing 0.4 mg/ml of G418 and 0.2 mg/ml of Zeocin. All cells were serum starved in Opti-MEM (Invitrogen) prior to all experiments. Transient transfections were performed using Lipofectamine 2000 following the manufacturer’s instructions. Specific GPR55 Agonist GSK319197A GSK319197A is usually [4-(3,4-dichloro-phenyl)-piperazin-1-yl]-(4-fluoro-4-methanesulfonyl-biphenyl-2-yl)-methanone (example 13 from Ref. 46), a structural analog of GSK494581A (24) and CID2440433 (35). It is one of a series of benzoylpiperazines originally identified as inhibitors of the glycine transporter subtype. The membranes were then resuspended and protein concentration was measured using a Bradford assay. Homologous and Heterologous Competition Binding Assay Radioligand binding on HEK293, HEK-GPR55, HEK-CB1, and HEK-GPR55+CB1 cell membranes was performed essentially as reported in Ref. that this co-expression of FLAG-CB1 receptors in cells stably expressing HA-GPR55 specifically inhibits GPR55-mediated transcription factor activation, such as nuclear factor of activated T-cells and serum response element, as well as extracellular signal-regulated kinases (ERK1/2) activation. GPR55 and CB1 receptors can form heteromers, but the internalization of both receptors is not affected. In addition, we observe that the presence of GPR55 enhances CB1R-mediated ERK1/2 and nuclear factor of activated T-cell activation. Our data provide the first evidence that GPR55 can form heteromers with another 7TM/GPCR and that this interaction with the CB1 receptor has functional consequences cerebral cortex, hippocampus, and striatum) (1, 4). In contrast, CB2Rs are mainly found on immune cells (2, 5). The lipid receptor GPR55 is usually highly expressed in the CNS, putamen, striatum, and hippocampus, as well as in intestine, bone marrow, spleen, immune cells, and endothelial cells (6C11). GPR55 was also detected in cancer tissues and cancer cell lines (12C15). CB1Rs predominantly couple to Gi/o proteins and thereby inhibit adenylyl cyclase, activate mitogen-activated protein kinases (MAPKs), and further activate numerous transcription factors. In addition, CB1Rs have been described to mediate the activation of several potassium channels (16, 17). Multiple GPR55-mediated signaling pathways have been described (6, 7, 18C20), whereby the most consistent reports suggest that GPR55 couples to G13 in recombinant HEK cells transiently or stably expressing GPR55 (9, 18, 19, 21C23). GPR55 signaling can be mediated via small GTPases (6, 21, 22) and the mobilization of intracellular calcium stores (21, 22, 24, 25). This leads to the activation of several transcription factors, such as nuclear factor of activated T-cells (NFAT), nuclear factor -light chain-enhancer of activated B cells (NF-B), cyclic AMP response-element binding protein, and activating transcription factor 2 (21, 22, 26). In addition, the activation of MAP kinases, such as p38 and ERK1/2 MAPKs were described to be induced after GPR55 stimulation (22, 26). Furthermore, the formation of filamentous actin (F-actin) upon GPR55 activation was reported in HEK293 cells and human neutrophils and this process is usually mediated by G13 and RhoA (6). The formation of F-actin is related to the induction of serum response elements (SRE) and is under control of the G13-mediated RhoA signaling (27, 28). The CB1R is usually activated by numerous endogenous cannabinoid ligands, such as anandamide (AEA) and 2-arachidonoylglycerol (2-AG), as well as the psychoactive phytocannabinoid (9)-tetrahydrocannabinol, and synthetic compounds such as WIN55,212-2 or CP55940 ((2-[(1(38) and (39C41). The presence of CB1R homomers was detected by using antibodies specifically recognizing CB1R dimers (42). In addition, it was reported that CB1Rs can form heteromers (3). The G protein coupling is usually altered in CB1R-dopamine D2R heteromers (43), CB1R-orexin-1 receptor heteromers show different trafficking and signaling properties (44) and the signaling pathways are modulated in CB1R-adenosine A2A receptor heteromers (45). To date, it is unknown whether GPR55 can form functional heteromers with other 7TM/GPCRs. Here we show that GPR55 and CB1Rs can physically interact and modulate each others signaling properties. Our data show that GPR55 signaling is specifically inhibited in the presence of the CB1 receptor. EXPERIMENTAL PROCEDURES Cell Culture, Transfections, and Stable Cell Lines HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Invitrogen) at 37 C in a 5% CO2, humidified atmosphere. HEK293 cells stably expressing the human 3HA-GPR55 (HEK-GPR55) were previously described (21) and maintained in G418 (PAA) containing medium (0.4 mg/ml). To generate HEK293 cells stably expressing human FLAG-CB1 alone (HEK-CB1) or 3HA-GPR55 and FLAG-CB1 receptor (HEK-GPR55+CB1), HEK293 or HEK-GPR55 cells were transfected with pcDNA3.1 encoding the FLAG-CB1 receptor using Lipofectamine 2000 (Invitrogen). Cells UNC3866 were generated in selection media (0.5 mg/ml of Zeocin for HEK-CB1 or 0.8 mg/ml of G418 and 0.5 mg/ml of Zeocin for HEK-GPR55+CB1) and single colonies were propagated. HEK-CB1 cells were cultured in DMEM containing 0.2 mg/ml of Zeocin, and HEK-GPR55+CB1 cells were maintained in DMEM containing 0.4 mg/ml of G418 and 0.2 mg/ml of Zeocin. All cells were serum.Our data provide the first evidence that GPR55 can form heteromers with another 7TM/GPCR and that this interaction with the CB1 receptor has functional consequences cerebral cortex, hippocampus, and striatum) (1, 4). CB1 receptors alter each others signaling properties in human embryonic kidney (HEK293) cells. We demonstrate that the co-expression of FLAG-CB1 receptors in cells stably expressing HA-GPR55 specifically inhibits GPR55-mediated transcription factor activation, such as nuclear factor of activated T-cells and serum response element, as well as extracellular signal-regulated kinases (ERK1/2) activation. GPR55 and CB1 receptors can form heteromers, but the internalization of both receptors is not affected. In addition, we observe that the presence of GPR55 enhances CB1R-mediated ERK1/2 and nuclear factor of activated T-cell activation. Our data provide the first evidence that GPR55 can form heteromers with another 7TM/GPCR and that this interaction with the CB1 receptor has functional consequences cerebral cortex, hippocampus, and striatum) (1, 4). In contrast, CB2Rs are mainly found on immune cells (2, 5). The lipid receptor GPR55 is highly expressed in the CNS, putamen, striatum, and hippocampus, as well as in intestine, bone marrow, spleen, immune cells, and endothelial cells (6C11). GPR55 was also detected in cancer tissues and cancer cell lines (12C15). CB1Rs predominantly couple to Gi/o proteins and thereby inhibit adenylyl cyclase, activate mitogen-activated protein kinases (MAPKs), and further activate numerous transcription factors. In addition, CB1Rs have been described to mediate the activation of several potassium channels (16, 17). Multiple GPR55-mediated signaling pathways have been described (6, 7, 18C20), whereby the most consistent reports suggest that GPR55 couples to G13 in recombinant HEK cells transiently or stably expressing GPR55 (9, 18, 19, 21C23). GPR55 signaling can be mediated via small GTPases (6, 21, 22) and the mobilization of intracellular calcium stores (21, 22, 24, 25). This leads to the activation of several transcription factors, such as nuclear factor of activated T-cells (NFAT), nuclear factor -light chain-enhancer of activated B cells (NF-B), cyclic AMP response-element binding protein, and activating transcription factor 2 (21, 22, 26). In addition, the activation of MAP kinases, such as p38 and ERK1/2 MAPKs were described to be induced after GPR55 stimulation (22, 26). Furthermore, UNC3866 the formation of filamentous actin (F-actin) upon GPR55 activation was reported in HEK293 cells and human neutrophils and this process is mediated by G13 and RhoA (6). The formation of F-actin is related to the induction of serum response elements (SRE) and is under control of the G13-mediated RhoA signaling (27, 28). The CB1R is activated by numerous endogenous cannabinoid ligands, such as anandamide (AEA) and 2-arachidonoylglycerol (2-AG), as well as the psychoactive phytocannabinoid (9)-tetrahydrocannabinol, and synthetic compounds such as WIN55,212-2 or CP55940 ((2-[(1(38) and (39C41). The existence of CB1R homomers was detected by using antibodies specifically recognizing CB1R dimers (42). In addition, it was reported that CB1Rs can form heteromers (3). The G protein coupling is modified in CB1R-dopamine D2R heteromers (43), CB1R-orexin-1 receptor heteromers show different trafficking and signaling properties (44) and the signaling pathways are modulated in CB1R-adenosine A2A receptor heteromers (45). To day, it is unfamiliar whether GPR55 can form practical heteromers with additional 7TM/GPCRs. Here we display that GPR55 and CB1Rs can actually interact UNC3866 and modulate each others signaling properties. Our data display that GPR55 signaling is definitely specifically inhibited in the presence of the CB1 receptor. EXPERIMENTAL Methods Cell Tradition, Transfections, and Stable Cell Lines HEK293 cells were cultured in Dulbecco’s altered Eagle’s medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Invitrogen) at 37 C inside a 5% CO2, humidified atmosphere. HEK293 cells stably expressing the human being 3HA-GPR55 (HEK-GPR55) were previously explained (21) and managed in G418 (PAA) comprising medium (0.4 mg/ml). To generate HEK293 cells stably expressing human being FLAG-CB1 only (HEK-CB1) or 3HA-GPR55 and FLAG-CB1 receptor (HEK-GPR55+CB1), HEK293 or HEK-GPR55 cells were transfected with pcDNA3.1 encoding the FLAG-CB1 receptor using Lipofectamine 2000 (Invitrogen). Cells were generated in selection press (0.5 mg/ml of Zeocin for HEK-CB1 or 0.8 mg/ml of G418 and 0.5 mg/ml of Zeocin for HEK-GPR55+CB1) and single colonies were propagated. HEK-CB1 cells were cultured in DMEM comprising 0.2 mg/ml of Zeocin, and HEK-GPR55+CB1 cells were taken care of in DMEM containing 0.4 mg/ml of G418 and 0.2 mg/ml of Zeocin. All cells were serum starved in Opti-MEM (Invitrogen) prior to all experiments. Transient transfections were performed using Lipofectamine 2000 following a manufacturer’s instructions. Specific GPR55 Agonist GSK319197A GSK319197A is definitely [4-(3,4-dichloro-phenyl)-piperazin-1-yl]-(4-fluoro-4-methanesulfonyl-biphenyl-2-yl)-methanone (example 13 from Ref. 46), a structural analog.(2012) The GPCR-associated sorting protein 1 regulates ligand-induced down-regulation of GPR55. each others signaling properties in human being embryonic kidney (HEK293) cells. We demonstrate the co-expression of FLAG-CB1 receptors in cells stably expressing HA-GPR55 specifically inhibits GPR55-mediated transcription element activation, such as nuclear element of triggered T-cells and serum response element, as well as extracellular signal-regulated kinases (ERK1/2) activation. GPR55 and CB1 receptors can UNC3866 form heteromers, but the internalization of both receptors is not affected. In addition, we observe that the presence of GPR55 enhances CB1R-mediated ERK1/2 and nuclear element of triggered T-cell activation. Our data provide the 1st evidence that GPR55 can form heteromers with another 7TM/GPCR and that this interaction with the CB1 receptor offers functional effects cerebral cortex, hippocampus, and striatum) (1, 4). In contrast, CB2Rs are primarily found on immune cells (2, 5). The lipid receptor GPR55 is definitely highly indicated in the CNS, putamen, striatum, and hippocampus, as well as with intestine, bone marrow, spleen, immune cells, and endothelial cells (6C11). GPR55 was also recognized in cancer cells and malignancy cell lines (12C15). CB1Rs mainly couple to Gi/o proteins and therefore inhibit adenylyl cyclase, activate mitogen-activated protein kinases (MAPKs), and further activate several transcription factors. In addition, CB1Rs have been explained to mediate the activation of several potassium channels (16, 17). Multiple GPR55-mediated signaling pathways have been explained (6, 7, 18C20), whereby probably the most consistent reports suggest that GPR55 couples to G13 in recombinant HEK cells transiently or stably expressing GPR55 (9, 18, 19, 21C23). GPR55 signaling can be mediated via small GTPases (6, 21, 22) and the mobilization of intracellular calcium stores (21, 22, 24, 25). This prospects to the activation of several transcription factors, such as nuclear element of triggered T-cells (NFAT), nuclear element -light chain-enhancer of triggered B cells (NF-B), cyclic AMP response-element binding protein, and activating transcription element 2 (21, 22, 26). In addition, the activation of MAP kinases, such as p38 and ERK1/2 MAPKs were described to be induced after GPR55 activation (22, 26). Furthermore, the formation of filamentous actin (F-actin) upon GPR55 activation was reported in HEK293 cells and human being neutrophils and this process is definitely mediated by G13 and RhoA (6). The formation of F-actin is related to the induction of serum response elements (SRE) and is under control of the G13-mediated RhoA signaling (27, 28). The CB1R is usually activated by numerous endogenous cannabinoid ligands, such as anandamide (AEA) and 2-arachidonoylglycerol (2-AG), as well as the psychoactive phytocannabinoid (9)-tetrahydrocannabinol, and synthetic compounds such as WIN55,212-2 or CP55940 ((2-[(1(38) and (39C41). The presence of CB1R homomers was detected by using antibodies specifically recognizing CB1R dimers (42). In addition, it was reported that CB1Rs can form heteromers (3). The G protein coupling is usually altered in CB1R-dopamine D2R heteromers (43), CB1R-orexin-1 receptor heteromers show different trafficking and signaling properties (44) and the signaling pathways are modulated in CB1R-adenosine A2A receptor heteromers (45). To date, it is unknown whether GPR55 can form functional heteromers with other 7TM/GPCRs. Here we show that GPR55 and CB1Rs can actually interact and modulate each others signaling properties. Our data show that GPR55 signaling is usually specifically inhibited in the presence of the CB1 receptor. EXPERIMENTAL PROCEDURES Cell Culture, Transfections, and Stable Cell Lines HEK293 cells were cultured in Dulbecco’s altered Eagle’s medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Invitrogen) at 37 C in a 5% CO2, humidified atmosphere. HEK293 cells stably expressing the human 3HA-GPR55 (HEK-GPR55) were previously described (21) and maintained in G418 (PAA) made up of medium (0.4 mg/ml). To generate HEK293 cells stably expressing human FLAG-CB1 alone (HEK-CB1) or 3HA-GPR55 and FLAG-CB1 receptor (HEK-GPR55+CB1), HEK293 or HEK-GPR55 cells were transfected with pcDNA3.1 encoding the FLAG-CB1 receptor using Lipofectamine 2000 (Invitrogen). Cells were generated in selection media (0.5 mg/ml of Zeocin for HEK-CB1 or 0.8 mg/ml of G418 and 0.5 mg/ml of Zeocin for HEK-GPR55+CB1) and single colonies were propagated. HEK-CB1.