Agonist-induced internalization of brain CB1Rs. A. The recombinant CB1 receptor (rCB1R) was labeled by both Nt and 1EL antibodies. The CB1R in solubilized PAG synaptosomal membranes was precipitated with 1EL or Nt antibodies and then immunodetected using the other antibody, yielding a ladder pattern. This banding of CB1Rs was also observed in the purified glycosylated fraction of PAG membranes, indicating the presence of multiple glycosylated species. B. Cannabinoid agonists used at doses that desensitize analgesia caused the internalization of surface CB1Rs (P2) into the cytosol (S3). ACEA and WIN55,212-2 caused internalization, but methanandamide caused almost none. C. Upper panel: Presence of CB1Rs in the cytosolic fraction of brain tissue from mice that did not receive WIN55,212-2. The mice were killed and the PAG S3 fraction was subjected to subcellular fractionation in order to determine the presence of CB1Rs. Lower panel: Mice were injected icv with WIN55,212-2, then sacrificed 30 min later, and the PAG S3 cytosolic fraction was analyzed for the presence and distribution of internalized CB1Rs. Subcellular markers: Rab5 (BD 610281), EEA1 (early endosome antigen I; BD 610456), Rab4 (BD 610888), Rab11 (BD 610656), Lamp-1 (lysosomal-associated membrane protein I; BD 61043). Right panel: The PAG cytosolic fraction was analyzed at various time intervals following administration of WIN55,212-2 in order to test for the presence of the CB1R.