The membrane proximal region of the cannabinoid receptor CB₁ N-terminus can allosterically modulate ligand affinity

The human cannabinoid receptor, CB₁, a G protein-coupled receptor (GPCR), contains a relatively long (∼110 a.a.) amino terminus, whose function is still not defined. Here we explore a potential role for the CB₁ N-terminus in modulating ligand binding to the receptor. Although most of the CB₁ N-terminus is not necessary for ligand binding, previous studies have found that mutations introduced into its conserved membrane proximal region (MPR) do impair the receptors ability to bind ligand. Moreover, within the highly conserved MPR (∼ residues 90-110) lie two cysteine residues that are invariant in all CB₁ receptors. We find these two cysteines (C98 and C107) form a disulfide in heterologously expressed human CB₁, and this C₉₈-C₁₀₇ disulfide is much more accessible to reducing agents than the previously known disulfide in extracellular loop 2 (EL2). Interestingly, the presence of the C ₉₈-C₁₀₇ disulfide modulates ligand binding to the receptor in a way that can be quantitatively analyzed by an allosteric model. The C ₉₈-C₁₀₇ disulfide also alters the effects of allosteric ligands for CB₁, Org 27569 and PSNCBAM-1. Together, these results provide new insights into how the N-terminal MPR and EL2 act together to influence the high-affinity orthosteric ligand binding site in CB₁ and suggest that the CB₁ N-terminal MPR may be an area through which allosteric modulators can act.