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. In summary, 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.

Multi-Functional Diarylurea Small Molecule Inhibitors of TRPV1 with Therapeutic Potential for Neuroinflammation

“…Importantly, compound 15 also formed strong hydrophobic interactions with two reported key residues, Trp194 and Phe197. All these results were consistent with the previous studies [26, 53]: Trp194 in TM5 was reported to have an important role in CB2 receptor ligand binding and adenylyl cyclase (AC) activity. Moreover, replacing Phe197 with the corresponding Val282 of CB1 resulted in a 14-fold decrease of WIN55,212-2 affinity to CB2 but had no effect on CP55940, HU-210, or AEA binding [54].…”

Targeting cannabinoid receptor‐2 pathway by phenylacetylamide suppresses the proliferation of human myeloma cells through mitotic dysregulation and cytoskeleton disruption

“…As shown in Figure 2A, PAM possesses a docking pose similar to the SR144528 surrounded by the hydrophobic residues in a range of 3.1 to 4.2 Å , including the reported Trp258, V113, Leu196, Val261 as well as newly predicted residues Phe281 and Thr114, and also possess H-bonding interactions (in a range of 2.8 to 3.5 Å ) with reported polar residue Ser285 of the CB2 receptor as defined previously [28] ( Figure 2B). These data suggested that PAM is an important lead compound as a cannabinoid CB 2 receptor-selective ligand and behaves like an inverse agonist.…”

Hydrophobic Ligand Entry and Exit Pathways of the CB1 Cannabinoid Receptor

“…The interactions with W356 6.48 were observed only for ligands containing a long alkyl chain, such as anandamide, HU-210, and CP55940 (results not shown), penetrating deep inside the receptor. We also compared our results with those of Alqarni et al 5 done on CB2, concluding that residue V 3.32 is also present in the CB1 receptor (V196 3.32 ) and, similarly to CB2, participates in CB1 ligand binding by formation of hydrophobic interactions with the ligand's alkyl chain, stabilizing both anandamide and THC in their binding pockets.…”

“…Their results also indicated that W 6.48 in TM6 and residues in TM4 (V 4.56 −L 4.61 ) contribute greatly to the binding of the agonist. In another study by Alqarni et al, 5 molecular modeling and ligand docking combined with site-directed mutagenesis experiments revealed residues V 3.32 and L 5.41 to be important for ligand binding and downstream signaling in the CB2 receptor.…”