Figure 2.
GH specifically induces temporal changes in GHR-GHR complementation. A, GH signaling in γ2A-JAK2-GHR-Nluc cells, in which GHR-Nluc is stably expressed in the γ2A-JAK2 background. Cells were serum starved for 6 hours and stimulated ±GH (500 ng/mL) for 10 minutes. Detergent cell extracts were resolved by SDS-PAGE and sequentially immunoblotted with anti-pY, anti-GHRcyt-AL47, anti-p-JAK2, anti-JAK2AL33, anti-p-STAT5, and anti-STAT5. The mature form of GHR is indicated by a bracket and the precursor form is indicated by an arrowhead. Note that the position of the pY GHR-Nluc (detected by anti-pY) and the mature GHR-Nluc (both bracketed) are identical. The blot shown is representative of three such experiments. B, GH concentration-dependent changes in complementation. γ2A-JAK2-GHR-Nluc cells were transiently transfected with an expression plasmid encoding GHR-Cluc. After basal bioluminescence was determined, GH at indicated concentrations was added and bioluminescence was serially determined at 5-minute intervals over 30 minutes. Data are expressed as mean ± SE of GH-induced signal as a percentage above baseline signal (n = 4 per condition). For 250 ng/mL GH vs 50 ng/mL GH, the value as P < .05 at 5 minutes. For 500 ng/mL GH vs 50 ng/ml GH, the value was P < .05 at 5 minutes. For 100 ng/mL, 250 ng/mL, and 500 ng/mL GH, the value was P < .05 for 20 minutes, 25 minutes, and 30 minutes each vs 5 minutes. For 250 ng/mL and 500 ng/mL GH, the value was P < .05 for 30 minutes each vs 10 minutes. For 500 ng/mL, the value was P < .05 for 15 minutes vs 5 minutes. The figure shown is representative of three such experiments. C, GH induces sustained JAK2 phosphorylation. γ2A-JAK2-GHR-Nluc cells were transiently transfected with GHR-Cluc, serum starved for 6 hours, and then treated with 500 ng/mL GH for 0–30 minutes, as indicated. Detergent cell extracts were resolved by SDS-PAGE and sequentially immunoblotted to detect phosphorylated and total JAK2. The blot shown is representative of three such experiments. D, GHR-specific antagonist, B2036, inhibits GH-induced JAK2 activation. γ2A-JAK2-GHR-Nluc cells were transiently transfected with GHR-Cluc, serum starved for 6 hours, and then treated ±GH (500 ng/mL, 10 min), with B2036 (20 μg/mL, 10 min) alone, or cotreated with GH plus B2036. Detergent cell extracts were resolved by SDS-PAGE and sequentially immunoblotted to detect phosphorylated and total JAK2. The blot shown is representative of three such experiments. E, GHR-specific antagonist, B2036, inhibits GH-induced changes in GHR-GHR complementation. γ2A-JAK2-GHR-Nluc cells transiently expressing GHR-Cluc were treated with GH (500 ng/mL), B2036 (20 μg/mL), or GH plus B2036, as indicated, after basal bioluminescence was determined. Bioluminescence was measured serially thereafter over 40 minutes. The data are expressed as mean ± SE of GH-induced signal as a percentage above baseline signal (n = 3 per condition). For GH vs GH+B2036, the value was P < .05 at each time point, except at 30 minutes, 35 minutes, and 40 minutes. The figure shown is representative of three such experiments.