Co-expression of the human cannabinoid receptor coding region splice variants (hCB1) affects the function of hCB1 receptor complexes

Biased Type 1 Cannabinoid Receptor Signaling Influences Neuronal Viability in a Cell Culture Model of Huntington Disease

“…Human arrestin2 (b-arrestin1)-Renilla luciferase II (Rluc) C-terminal fusion protein was generated using the pcDNA3.1 plasmid and provided by Dr. Denis J Dupré (Dalhousie University, NS). The GFP 2 -Rluc fusion construct, and Rluc plasmids have also been described (Bagher et al, 2013). The Ga q dominant negative mutant [Glu 209 D Leu, Asp 277 D Asn (Q209L,D277N)] pcDNA3.1 plasmid was obtained from the cDNA Resource Center (Missouri S&T, Rolla, MO) (Lauckner et al, 2005).…”

“…Plasmids and Transfection. Human CB 1 -green fluorescent protein 2 (GFP 2 ) C-terminal fusion protein was generated using the pGFP 2 -N3 plasmid (PerkinElmer, Waltham, MA), as described previously (Bagher et al, 2013). Human arrestin2 (b-arrestin1)-Renilla luciferase II (Rluc) C-terminal fusion protein was generated using the pcDNA3.1 plasmid and provided by Dr. Denis J Dupré (Dalhousie University, NS).…”

Type 1 Cannabinoid Receptor Ligands Display Functional Selectivity in a Cell Culture Model of Striatal Medium Spiny Projection Neurons

“…Cells were grown at 33°C, 5% CO 2 in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 10 4 units/ml penicillin-streptomycin, and 400 g/ml Geneticin. Twenty-four hours of serum deprivation promotes the differentiation of STHdh Q7/Q7 cells into an adult neuron-like phenotype characterized by increased neurite outgrowth, GABA release, and increased expression of CB 1 , dopamine D 2 receptors, preproenkephalin, and dopamine and cAMP-related phosphoprotein 32 kDa (DARPP-32), typical of mature medium spiny projection neurons of the indirect motor pathway of the striatum (36,37,40). These cells are ideally suited for the characterization of cannabinoid ligand bias in vitro because they model a neuronal cell type that expresses CB 1 at high levels compared with other cell types in the central nervous system.…”

Antagonism of Dopamine Receptor 2 Long Affects Cannabinoid Receptor 1 Signaling in a Cell Culture Model of Striatal Medium Spiny Projection Neurons

“…The colocalization of endogenous CB 1 and D 2L in STHdh Q7/Q7 cells was observed using confocal microscopy as described (Bagher et al, 2013). Cells were incubated with primary monoclonal rabbit N-terminal CB 1 antibody (1:500; Cayman Chemical Company, Ann Arbor, MI) and primary monoclonal mouse N-terminal-D 2 antibody (1:200; Santa Cruz Biotechnology, Dallas, TX) overnight at 4°C.…”

“…Interactions between CB 1 and D 2L , Ga i , Ga s , and b-arrestin1 were assessed via BRET 2 as described previously (Ramsay et al, 2002;James et al, 2006;Bagher et al, 2013). For BRET 2 experiments using Ga i -Rluc or Ga s -Rluc fusion proteins, cells were cotransfected with untagged Gb 1 and Gg 2 in pcDNA3.1 (1) /Rluc concentration.…”