CB1 Cannabinoid Receptor Juxtamembrane C‐terminal Phosphorylation in NT18TG2 Cells

Abstract

CB1 receptors primarily stimulate the G protein family of Gi/o. The aim of the present study is to investigate the effect of phosphorylation of the juxtamembrane C‐terminal domain of the CB1 receptor on individual Gi proteins activation in N18TG2 neuroblastoma cell membranes using an antibody‐capture[35S]‐GTPγS scintillation proximity assay (SPA). C‐terminal peptides were incubated with N18TG2 membranes in a reaction mix including [35S]‐GTPγS, NaCl, dithiothreitol and GDP for 1 h at 30 °C then terminated by solubilization of the membrane proteins, followed by incubation with specific antibodies. In N18TG2 membranes, the unphosphorylated peptide (Arg401 to Glt420) was able to stimulate [35S]‐GTPγS binding in a dose‐dependent fashion (30–300μM). When applied at 100μM the unphosphorylated peptide was able to stimulate [35S]‐GTPγS binding mediated by Gi1: Emax = 18%, Gi2: Emax = 34%, Gi3: Emax = 25%, Go: Emax =18% over basal, respectively. We investigated the effect of phosphorylation of the three Ser residues (Ser402, Ser411, and Ser415) (100μM) and found that phosphorylation of Ser402 was able to stimulate [35S]‐GTPγS binding mediated by Gi3: Emax = 33%, Go: Emax = 27% over basal. Phosphorylation of Ser415, was able to stimulate [35S]‐GTPγS binding mediated by Gi3: Emax = 38%, Go: Emax =29% over basal. Phosphorylation of Ser411 was able to stimulate [35S]‐GTPγS binding mediated by Gi3: Emax = 31%, Go: Emax =33.6% over basal. The order of potency for Go activation was pSer411>;pSer415>;pSer402>;unphosphorylated and for Gi3 activation was pSer415>;pSer411>;pSer402>;unphosphorylated. In conclusion, these data show that the 8th helix C‐terminal peptides were able to stimulate G protein activation in N18TG2 cell membranes, and the activation could be augmented by phosphorylation of Ser residues.