FIGURE 3.

Both CB₁ receptor agonists and orexin A produce internalization of CB₁ receptor: effect of orexin A requires OX₁ receptor. Cells of clone B6 (A and B) were uninduced (− doxycycline) or induced to express VSV-G-SNAP-CB₁ (+ doxycycline). These were challenged with the identified ligands (CP55940 and WIN55212-2 are CB₁ receptor agonists, whereas SR141716A is an antagonist at this receptor. Orexin A (OxA) is an agonist of the OX₁ receptor) for 40 min. Cell surface VSV-G-SNAP-CB₁ was detected via anti-VSV-G ELISA (A) or the binding of SNAP-Lumi4Tb (B). C, similar studies were performed on cells able to express inducible VSV-G-SNAP-CB₁ but that lack expression of HA-CLIP-OX₁, and cell surface VSV-G-SNAP-CB₁ was detected by the binding of SNAP-Lumi4Tb. ***, p < 0.001 (different from doxycycline). Combined data from n = 3 experiments (means ± S.E.) are shown. VS, VSV-G + SNAP tag; HC, HA + CLIP tag.