title: Examining the critical roles of human CB2 receptor residues Valine 3.32 (113) and Leucine 5.41 (192) in ligand recognition and downstream signaling activities.
abstract: We performed molecular modeling and docking to predict a putative binding pocket and associated ligand-receptor interactions for human cannabinoid receptor 2 (CB2). Our data showed that two hydrophobic residues came in close contact with three structurally distinct CB2 ligands: CP-55,940, SR144528 and XIE95-26. Site-directed mutagenesis experiments and subsequent functional assays implicated the roles of Valine residue at position 3.32 (V113) and Leucine residue at position 5.41 (L192) in the ligand binding function and downstream signaling activities of the CB2 receptor. Four different point mutations were introduced to the wild type CB2 receptor: V113E, V113L, L192S and L192A. Our results showed that mutation of Val113 with a Glutamic acid and Leu192 with a Serine led to the complete loss of CB2 ligand binding as well as downstream signaling activities. Substitution of these residues with those that have similar hydrophobic side chains such as Leucine (V113L) and Alanine (L192A), however, allowed CB2 to retain both its ligand binding and signaling functions. Our modeling results validated by competition binding and site-directed mutagenesis experiments suggest that residues V113 and L192 play important roles in ligand binding and downstream signaling transduction of the CB2 receptor.
authors.affiliation: Department of Pharmaceutical Sciences and Computational Chemical Genomics Screening Center, School of Pharmacy, Pittsburgh, PA 15260, USA.
authors.forename: Mohammed
authors.initials: M
authors.lastname: Alqarni
authors.affiliation: Department of Pharmaceutical Sciences and Computational Chemical Genomics Screening Center, School of Pharmacy, Pittsburgh, PA 15260, USA; Joint Carnegie Mellon University-University of Pittsburgh Ph.D. Program, Department of Computational Biology and Structural Biology, School of Medicine, Pittsburgh, PA 15260, USA.
authors.forename: Kyaw Zeyar
authors.initials: KZ
authors.lastname: Myint
authors.affiliation: Department of Pharmaceutical Sciences and Computational Chemical Genomics Screening Center, School of Pharmacy, Pittsburgh, PA 15260, USA.
authors.forename: Qin
authors.initials: Q
authors.lastname: Tong
authors.affiliation: Department of Pharmaceutical Sciences and Computational Chemical Genomics Screening Center, School of Pharmacy, Pittsburgh, PA 15260, USA.
authors.forename: Peng
authors.initials: P
authors.lastname: Yang
authors.affiliation: Department of Pharmaceutical Sciences and Computational Chemical Genomics Screening Center, School of Pharmacy, Pittsburgh, PA 15260, USA.
authors.forename: Patrick
authors.initials: P
authors.lastname: Bartlow
authors.affiliation: Department of Pharmaceutical Sciences and Computational Chemical Genomics Screening Center, School of Pharmacy, Pittsburgh, PA 15260, USA.
authors.forename: Lirong
authors.initials: L
authors.lastname: Wang
authors.affiliation: Department of Pharmaceutical Sciences and Computational Chemical Genomics Screening Center, School of Pharmacy, Pittsburgh, PA 15260, USA.
authors.forename: Rentian
authors.initials: R
authors.lastname: Feng
authors.affiliation: Department of Pharmaceutical Sciences and Computational Chemical Genomics Screening Center, School of Pharmacy, Pittsburgh, PA 15260, USA; Joint Carnegie Mellon University-University of Pittsburgh Ph.D. Program, Department of Computational Biology and Structural Biology, School of Medicine, Pittsburgh, PA 15260, USA; Drug Discovery Institute, University of Pittsburgh, Pittsburgh, PA 15260, USA. Electronic address: xix15@pitt.edu.
authors.forename: Xiang-Qun
authors.initials: XQ
authors.lastname: Xie
mesh_terms: D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D001892:Camphanes / Q000737:chemistry* / Q000378:metabolism; D063386:Cannabinoid Receptor Agonists / Q000737:chemistry* / Q000378:metabolism; D063387:Cannabinoid Receptor Antagonists / Q000737:chemistry* / Q000378:metabolism; D003412:Cricetulus; D000242:Cyclic AMP / Q000737:chemistry / Q000378:metabolism; D003511:Cyclohexanols / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007930:Leucine / Q000737:chemistry* / Q000235:genetics; D008024:Ligands; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011720:Pyrazoles / Q000737:chemistry* / Q000378:metabolism; D043885:Receptor, Cannabinoid, CB2 / Q000819:agonists / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014633:Valine / Q000737:chemistry* / Q000235:genetics
chemical_list: D001892:Camphanes; D063386:Cannabinoid Receptor Agonists; D063387:Cannabinoid Receptor Antagonists; D003511:Cyclohexanols; D008024:Ligands; D011720:Pyrazoles; D043885:Receptor, Cannabinoid, CB2; D011994:Recombinant Proteins; C110630:SR 144528; C054649:3-(2-hydroxy-4-(1,1-dimethylheptyl)phenyl)-4-(3-hydroxypropyl)cyclohexanol; D000242:Cyclic AMP; D007930:Leucine; D014633:Valine
keywords: Adenylyl cyclase (AC activity); Cannabinoid receptor subtype 2 (CB2); Cyclic adenosine monophosphate (cAMP); Molecular modeling; Site-directed mutagenesis; Time-resolved fluorescence resonance energy (TR-FRET) transfer