FIGURE 9.
Acceleration of endogenously expressed α2AAR endocytosis by βAR co-activation requires spinophilin. A, neurons with (SpWT) and without (Sp−/−) spinophilin expression were stimulated with clonidine (1 μm) as indicated. Neurons were subjected to α2AAR prelabeling method as in Fig. 1 (B and C), with endocytosis indicated by the appearance of intracellular punctae containing internalized receptors (arrows). ISO pretreatment to co-activate βARs was done as in Fig. 4. B, quantitation showing an acceleration of clonidine-mediated α2AAR endocytosis basally in Sp−/− neurons, with significantly enhanced internalization at both the 10- and 15-min time points. C, quantitation showing that no enhancement of clonidine-mediated α2AAR endocytosis by βAR co-activation with ISO is observed in Sp−/− neurons. Confocal images are representative of at least three independent samples, and quantitation was performed over at least 12–14 neurons. *, p < 0.01; **, p < 0.0001 versus SpWT.