Figure 8

The CB1R carries the HINT1-RGSZ signaling module. Upper panel: In solubilized PAG synaptosomal membranes, MOR and CB1R co-precipitated PKCI/HINT1 and RGSZ proteins. In contrast, the δ-opioid receptor (DOR) showed little association with these proteins. Middle panel: Zinc increases the association of PKCγ with PAG MOR and CB1R. PAG synaptosomes were solubilized and incubated with zinc (30 nM) for 4 h at 4°C. MORs and CB1Rs were immunoprecipitated, and co-precipitation of PKCγ was evaluated. Lower panel: WIN55,212-2 promotes the recruitment of PKCγ to the CB1 receptor. Mice were injected icv with a desensitizing dose of 20 nmol WIN55,212-2. The CB1 receptors were immunoprecipitated (IP) from PAG synaptosomes (P2) obtained at the indicated intervals following agonist administration. For each interval, the PAG from six to eight mice were pooled. Since this dose of WIN55,212-2 promotes internalization of PAG CB1Rs, equal loading was verified by examining the signals obtained by immunodetection of the heavy chain of the anti-CB1R IgGs. IgGs were detached from the immunoprecipitated CB1 receptors, processed in parallel gel/blots, and detected using the appropriate secondary antibody. Co-precipitation studies were performed under non-denaturing conditions, and the MOR- and CB1R-associated proteins were immunodetected with antibodies directed against PKCI/HINT1, PKCγ, RGSZ1, and RGSZ2.