Functional Residues in Cannabinoid Receptor Type 1 β‐arrestin Biased Signaling
Authors: Leo Luciana M. , Zhao Pingwei , Zoubi Rufaida Al , Reggio Patricia H. , Abood Mary E. 
Published: June 2021

The study of Cannabinoid Receptor Type 1 (CB1) biased signaling is focused on the activation of G‐protein or β‐arrestin mediated signaling transduction. Coupling to either type of effector protein depends on stabilization of different receptor conformations that favor binding to G proteins or β‐arrestins. Coupling to G‐protein involves outward movement of the intracellular domain of transmembrane helix 6 (TM6), while coupling to β‐arrestins involves outward movement of TM7 and helix 8. The rotameric state of Y7.53 also varies in these structural arrangements. Molecular dynamics simulations show that during β‐arrestin biased signaling, the Y7.53 Chi1 dihedral is in the trans conformation. In order to investigate the importance of the position of this residue during CB1 biased signaling, we proposed three amino acid mutations that are predicted to stabilize Y7.53 in trans to different extents and therefore skew signaling bias toward β‐arrestin mediated signaling transduction. The mutations studied were: I2.43A, I2.43T and S7.57E. The ERK1/2 phosphorylation response to treatment with CB1 agonist 2‐arachidonoylglycerol (2‐AG) was analyzed in stably transfected HEK293 cells. We found no difference in the EC50 or Emax of cells expressing I2.43A, I2.43T or S7.57E when compared to wild‐type (WT) CB1, indicating that these mutations do not affect agonist potency or efficacy in the ERK1/2 phosphorylation response. Next, we evaluated ERK1/2 phosphorylation at various time points and in the presence of pertussis toxin (PTx), which inhibits Gαi/o coupling to the receptor. No difference was found between WT and I2.43A in the presence or absence of PTx. However, although the time course of ERK1/2 phosphorylation in response to 2‐AG is similar between WT and I2.43T, we found that the I2.43T response is insensitive to PTx treatment at all time points analyzed. This indicates that signaling through I2.43T is biased toward Gαi/o independent mechanisms. For the S7.57E mutation, there was an early PTx‐insensitive peak of ERK1/2 phosphorylation at 10 min. Yet, in the later time points, no difference was observed between WT and S7.57E in the presence of PTx. This result suggests that the S7.57E response to 2‐AG is skewed toward Gαi/o independent mechanisms only at earlier time points. Knockdown of β‐arrestins will reveal if the PTx‐insensitive responses observed in I2.43T and S7.57E are mediated by activation of β‐arrestins. Altogether, our data suggests that stabilization of Y7.53 in the trans conformation is important for β‐arrestin coupling to CB1. Understanding the role of these residues in receptor activation will assist in an understanding of how protein structure determines signaling bias. Further, study of these mutations can also help demonstrate the functional role of each effector protein in diverse biological systems.

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Support: RO1 DA003934

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.