Computational analysis of the CB1 carboxyl-terminus in the receptor-G protein complex

Abstract

Despite the important role of the carboxyl-terminus (Ct) of the activated brain cannabinoid receptor one (CB1) in the regulation of G protein signaling, a structural understanding of interactions with G proteins is lacking. This is largely due to the highly flexible nature of the CB1 Ct that dynamically adapts its conformation to the presence of G proteins. In the present study, we explored how the CB1 Ct can interact with the G protein by building on our prior modeling of the CB1-Gi complex (Shim J-Y, Ahn KH, Kendall DA. The Journal of Biological Chemistry 2013;288:32449-32465) to incorporate a complete CB1 Ct (Glu416Ct–Leu472Ct). Based upon the structural constraints from NMR studies, we employed ROSETTA to predict tertiary folds, ZDOCK to predict docking orientation, and molecular dynamics (MD) simulations to obtain two distinct plausible models of CB1 Ct in the CB1-Gi complex. The resulting models were consistent with the NMR-determined helical structure (H9) in the middle region of the CB1 Ct. The CB1 Ct directly interacted with both Gα and Gβ and stabilized the receptor at the Gi interface. The results of site-directed mutagenesis studies of Glu416Ct, Asp423Ct, Asp428Ct, and Arg444Ct of CB1 Ct suggested that the CB1 Ct can influence receptor-G protein coupling by stabilizing the receptor at the Gi interface. This research provided, for the first time, models of the CB1 Ct in contact with the G protein.

Assessment of quality and consistency of monoclonal antibodies for CB1 and CB2 in head and neck squamous cell carcinoma

“…Both standard (Figure ) and confocal (Figure ) microscopy demonstrated diffuse expression of CB1 within SCC and NHK. Confocal imaging provided clearer resolution, and in two patient samples, numbered HN65 and HN123, respectively, CB1 was found to be expressed in higher density within the cellular membrane, which is consistent with the receptor's prior characterizations …”

“…Confocal imaging provided clearer resolution, and in two patient samples, numbered HN65 and HN123, respectively, CB1 was found to be expressed in higher density within the cellular membrane, which is consistent with the receptor's prior characterizations. 13 IHC was utilized to support the evidence of CB1 expression from WB and IF microscopy ( Figure 4). IHC staining was performed on five SCC samples.…”