ABSTRACT
The extensive physiological influence of transmission through the CB2 cannabinoid receptor makes this G protein-coupled receptor (GPCR) a promising therapeutic target for treating neuropathic pain, inflammation, and immune disorders. However, there is little direct structural information pertaining to either GPCR or CB2-receptor ligand recognition and activation. The present work helps characterize experimentally the ligand-binding interactions of the human CB2 (hCB2) receptor. This study illustrates how our overall experimental approach, "ligand-assisted protein structure" (LAPS), affords direct determination of the requirements for ligand binding to the hCB2 receptor and discrimination among the binding motifs for ligands that activate therapeutically relevant GPCRs.
Subject(s)
Models, Molecular , Receptor, Cannabinoid, CB2/metabolism , Affinity Labels/metabolism , Amino Acid Sequence , Cell Line , Drug Discovery , Gene Expression Regulation , Humans , Ligands , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutant Proteins/metabolism , Mutation , Protein Binding , Receptor, Cannabinoid, CB1/metabolism , Receptor, Cannabinoid, CB2/chemistry , Receptor, Cannabinoid, CB2/genetics , Substrate SpecificityABSTRACT
Genome sequencing projects, and their available resources, have revealed two distinct genes encoding cannabinoid receptors, CB(1) and CB(2). Biochemical evidence in support of a third cannabinoid receptor includes signal transduction events and vasodilation in the vasculature of cannabinoid receptor knockout mice after exposure to the endogenous cannabinoid, anandamide. In addition, a nonpsychoactive ingredient in marijuana, abnormal cannabidiol, which does not activate the two characterized cannabinoid receptor homologues, has been shown to induce vasodilation in the endothelium. Our work distinguishes the biochemical differences by way of a phylogenetic analysis of cannabinoid receptors. Recently a putative orthologue to CB(1) and CB(2) has been identified in the urochordate, Ciona intestinalis, indicating the presence of cannabinoid receptors previous to the evolution of vertebrates. Moreover, the Ciona sequence shares equal identity to both cannabinoid paralogous sequences and no other GPCR sequence identified in an exhaustive database search is as similar. We propose that, although an alternate cannabinergic-activating pathway may be present, it does not include a GPCR (or other receptor type) phylogenetically related to the CB(1)/CB(2)Ciona lineage.
Subject(s)
Receptors, Cannabinoid/genetics , Animals , Humans , Phylogeny , Receptors, Cannabinoid/chemistry , Receptors, Cannabinoid/classification , Sequence Alignment , Signal TransductionABSTRACT
Vascular endothelial cells (VEC) provide an essential protective barrier between the vascular system and underlying tissues. Using VEC barrier models of human coronary artery cells and human and rat brain microvascular endothelial cells, we investigated the mechanism by which morphine affects lipopolysaccharide (LPS)-induced VEC permeability. We demonstrated that co-administration of morphine and LPS induced greater VEC apoptosis and permeability than morphine or LPS alone. The extent of induced apoptosis appeared to be cell-type dependent. Furthermore, RT-PCR analysis revealed that morphine and LPS up-regulated Fas expression. These data suggest potential crosstalk between the signaling pathways that mediate morphine- and LPS-triggered apoptosis in brain VEC.