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. 2011 Feb 22;286(15):13561–13573. doi: 10.1074/jbc.M110.215723

FIGURE 6.

FIGURE 6.

Inhibition of receptor internalization selectively blocks receptor dephosphorylation at Ser-341/343. CHO-sst2A cells were equilibrated in the absence (●) or presence of either 0.45 m sucrose (○) or 50 μm dynasore (▿) for 15 min at 37 °C. Panel A, cells were then stimulated with 10 nm SS14 for the times shown in the continued absence or presence of either sucrose or dynasore. After washing with cold PBS, cells were fixed, and cell surface receptors were measured by ELISA as described under “Experimental Procedures.” Surface receptors at each time point were expressed as the percentage of the nonstimulated control group (t = 0). Panel A shows that the rate of receptor internalization in untreated cells followed first order kinetics. In three independent experiments, the half-time for sst2A receptor internalization was 4.9 ± 0.8 min for untreated cells, and sucrose and dynasore completely blocked sst2A receptor internalization. Panels B–E, rate of receptor dephosphorylation was measured in untreated CHO-sst2A cells (●) or in the presence of either 0.45 m sucrose (○) or 50 μm dynasore (▿). Cells were preincubated with inhibitors as above and then stimulated with 10 nm SS14 for an additional 15 min to allow phosphorylation to reach a steady state. Following a rapid wash, cells were incubated at 37 °C for the times shown in fresh medium without SS14 in the continued absence (●) or presence of either sucrose (○) (panels B and D) or dynasore (▿) (panels C and E). Following fixation, receptor phosphorylation was measured on Ser-341/343 (panels B and C) and Thr-353/354 (panels D and E) with an in-cell ELISA. Nonlinear regression curve fitting to a single exponential decay in nine independent experiments gave a half-time of 7.9 ± 0.9 min for Ser-341/343 dephosphorylation and 7.6 ± 0.4 min for Thr-353/354 dephosphorylation in untreated cells. In the presence of sucrose or dynasore, Ser-341/343 dephosphorylation was largely blocked. In contrast, dephosphorylation of Thr-353/354 was unaffected by the endocytosis inhibitors (t½ was 9.5 ± 1.4 min (n = 3) with sucrose and 10.9 ± 1.4 (n = 2) with dynasore).