Figure 7.
Basal and GH-augmented trGHR-trGHR luciferase complementation. A, Specific luciferase complementation of trGHR-Nluc with trGHR-Cluc. γ2A-JAK2 cells were transiently transfected with expression plasmids encoding the indicated chimeras. Bioluminescence was determined in triplicate (inset shows actual color coded signals) and is displayed graphically as mean ± SE total flux (photons per second × 1000). See Materials and Methods for details. The figure shown is representative of two such experiments. B, GH signaling in γ2A-JAK2-trGHR-Nluc/trGHR-Cluc cells, in which trGHR-Nluc and trGHR-Cluc are stably coexpressed in the γ2A-JAK2 background. Cells were serum starved for 6 hours and stimulated ±GH (500 ng/mL) for 10 minutes. Detergent cells extracts were resolved by SDS-PAGE and sequentially immunoblotted with anti-Nluc, anti-Cluc, anti-p-JAK2, and anti-JAK2AL33. The blot shown is representative of two such experiments. C, GH concentration-dependent changes in complementation. γ2A-JAK2-trGHR-Nluc/trGHR-Cluc cells were treated with GH at the indicated concentrations after basal bioluminescence was determined. Bioluminescence was thereafter serially determined at 5 minutes intervals over 40 minutes. Data are expressed as mean ± SE of GH-induced signal as a percentage above baseline signal (n = 3 per condition). For each time point, a value of P < .05 was attained for signals produced by each GH concentration with the exception of the following: 20 minutes, 25 minutes, 30 minutes, 35 minutes, and 40 minutes, 250 ng/mL GH vs 500 ng/mL GH; and 40 minutes, 100 ng/mL GH vs 250 ng/mL. The figure shown is representative of two such experiments. D, GHR-specific antagonist, B2036, inhibits GH-induced JAK2 activation via ICD-truncated GHR chimeras. γ2A-JAK2-trGHR-Nluc/trGHR-Cluc cells were 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 two such experiments. E, GHR-specific antagonist, B2036, inhibits GH-induced changes in trGHR-trGHR complementation. γ2A-JAK2-trGHR-Nluc/trGHR-Cluc cells 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 25 minutes. Data are expressed as mean ± SE of GH-induced signal as a percentage above baseline signal (n = 3 per condition). GH vs GH+B2036: P < .05 at each time point. The figure shown is representative of two such experiments.