1. The NPXXY Motif Regulates β-Arrestin Recruitment by the CB1 Cannabinoid Receptor.
- Author
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Leo LM, Al-Zoubi R, Hurst DP, Stephan AP, Zhao P, Tilley DG, Miess E, Schulz S, Abood ME, and Reggio PH
- Subjects
- Humans, beta-Arrestins metabolism, Cannabinoids, GTP-Binding Proteins metabolism, HEK293 Cells, Receptors, G-Protein-Coupled genetics, Receptors, G-Protein-Coupled metabolism, beta-Arrestin 1 genetics, beta-Arrestin 1 metabolism, Receptor, Cannabinoid, CB1 genetics, Receptor, Cannabinoid, CB1 metabolism
- Abstract
Background: Activation of signaling effectors by G-protein coupled receptors (GPCRs) depends on different molecular mechanisms triggered by conserved amino acid residues. Although studies have focused on the G-protein signaling state, the mechanism for β-arrestin signaling by CB1 is not yet well defined. Studies have indicated that transmembrane helix 7 (TMH7) and the highly conserved NPXXY motif can be subject to different conformational changes in response to biased ligands and could therefore participate in a molecular mechanism to trigger β-arrestin recruitment. Objective: To investigate the effect of mutations in the NPXXY motif on different signaling pathways activated by the CB1 receptor. Materials and Methods: Point mutations of the NPXXY motif and associated residues were generated in the CB1 receptor using site-directed mutagenesis and transfection into HEK-293 cells. Signaling by wild-type and mutant receptors was analyzed by quantifying inhibition of cAMP, and by β-arrestin recruitment assays. Results: We found that N7.49 and Y7.53 are essential for β-arrestin recruitment by CB1. N7.49A and Y7.53F impair β-arrestin signaling, with no effect on G-protein signaling. We found a regulatory role for residue I2.43; I2.43 interacts with Y7.53, affecting its positioning. Reducing steric bulk at I2.43 (I2.43A) enhances β-arrestin1 recruitment, while introducing a polar residue (I2.43T) reduces β-arrestin recruitment. Conclusions: These findings point to a novel mechanism for β-arrestin recruitment, implicating amino acids in the NPXXY motif as critical for the putative β-arrestin biased conformational state of Class A GPCRs.
- Published
- 2023
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