FIG. 2.

Truncation of the last 14 amino acid residues of the CB₁ receptor does not prevent agonist-induced internalization in HEK293 cells. A, Immunostaining of HEK293 cells stably expressing either wild-type or V460Z CB₁ receptors. Cells were treated with vehicle (a, d) or stimulated with 100 nM CP 55,940 alone (b, e) or 100 nM CP 55,940 in the presence of 1 µM SR141716A (c, f) for the indicated times. Both wild-type and V460Z CB₁ receptors were internalized following agonist treatment. Endocytosis was prevented by the addition of SR141716A. Scale bar, 20 µm. Images are representative of a minimum of three independent experiments. B, Quantitative detection of CB₁ receptor removal from the cell surface. HEK293 cells stably expressing either wild-type, A470Z, V464Z or V460Z CB₁ receptors were treated with 100 nM CP 55,940 for times indicated and the time course of receptor internalization was determined (see Experimental Procedures). All of the mutant receptors internalized at a similar rate and to the same extent as wild-type CB₁ receptors. Data are mean ± SEM with n = 15–20 from three to four experiments. C, Time course of wild-type and V460Z CB₁ receptor internalization following 1 µM WIN 55,212-2 (WIN, solid lines) or 100 nM HU-210 (HU, dashed lines) stimulation. Both receptors internalized at a similar rate and extent relative to each other in the presence of either agonist. However, the rate of WIN 55,212-2-induced internalization for both receptors was more rapid than for HU-210-induced endocytosis (see Results). Data are mean ± SEM; n = 6 from three experiments performed in duplicate.