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. 1997 Oct 14;94(21):11381-6.
doi: 10.1073/pnas.94.21.11381.

Insulin and insulin-like growth factor-I acutely inhibit surface translocation of growth hormone receptors in osteoblasts: a novel mechanism of growth hormone receptor regulation

K C Leung  1 , M J Waters, I Markus, W R Baumbach, K K Ho
Affiliations
Free PMC article

Insulin and insulin-like growth factor-I acutely inhibit surface translocation of growth hormone receptors in osteoblasts: a novel mechanism of growth hormone receptor regulation

K C Leung et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

We previously have demonstrated that insulin and insulin-like growth factor-I (IGF-I) down-regulate growth hormone (GH) binding in osteoblasts by reducing the number of surface GH receptors (GHRs). The present study was undertaken to investigate the mechanism of GHR down-regulation. Treatment with 5 nM insulin or IGF-I for 18 hr significantly decreased surface GH binding to 26.4 +/- 2.9% and 23.0 +/- 2.7% of control (mean +/- SE; P < 0.05), respectively. No corresponding reductions in the mRNA level and total cellular content of GHR were found, nor was the rate of receptor internalization affected. The effects on GHR translocation were assessed by measuring the reappearance of GH binding of whole cells after trypsinization to remove the surface receptors. GH binding of control cultures significantly increased (P < 0.05) over 2 hr after trypsinization, whereas no recovery of binding activity was detected in insulin and IGF-I-treated cultures, indicating that GHR translocation was impaired. Studies on the time course of GHR down-regulation revealed that surface GH binding was reduced significantly by 3-hr treatment (P </= 0.0005), whereas GHR translocation was completely abolished by 75-90 min with insulin and IGF-I. The inhibition of receptor translocation by insulin, but not IGF-I, was attenuated by wortmannin. In conclusion, insulin and IGF-I down-regulated GH binding in osteoblasts by acutely impairing GHR translocation, with their effects exerted through distinct postreceptor signaling pathways.

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Figures

Figure 1
Figure 1
Northern and Western studies of GHR. (a) Northern analysis. Total RNA was isolated from the cultures after treatment with insulin or IGF-I, and abundance of mRNA for GHR, GH binding protein (GHBP), and β-actin was quantified by Northern analysis. Positions of the 18S and 28S rRNA were shown. (b) Western analysis. Total protein content was harvested from control and treated cultures and was studied using Western blotting technique with an anti-GHR antiserum. Position of the 137-kDa molecular mass marker was shown. Similar results were obtained in a repeated experiment.
Figure 2
Figure 2
GHR distribution. After treatment with insulin or IGF-I, total (□), surface (░⃞), and intracellular GH binding (▪) were determined as described in Materials and Methods. The control values for total, surface, and intracellular GH binding were 2,459 ± 209 cpm/mg, 112 ± 20 cpm/106 cells, and 1,723 ± 103 cpm/mg, respectively. Vs. control, ∗, P < 0.05; ‡, P < 0.005; and †, P = 0.0001.
Figure 3
Figure 3
GHR internalization. GH binding assay was undertaken at 22°C for 2 hr. At the end of the binding assay, the surface-bound radioligand was removed by washing with 50 mM HCl at 4°C. The remaining cell incorporated radioactivity accounted for the amount of radioligand internalized with GHRs.
Figure 4
Figure 4
GHR translocation. GHR translocation was measured as the recovery of GH binding on the cell surface after removal of the surface GHR by trypsinization. The trypsinized cells were allowed to recover at 22°C for the time periods indicated, followed by GH binding assay. Results from a representative experiment were shown, and similar findings were obtained in four other experiments. □, Control; •, insulin; ○, IGF-I. Vs. 0 hr, ∗, P < 0.05.
Figure 5
Figure 5
Time course of inhibition. (a) Surface GH binding. Cultures were incubated in serum-free conditions for 18 hr, treated with insulin or IGF-I for the indicated time periods, and assayed for surface GH binding of cell monolayers. •, insulin; ○, IGF-I. GH binding of the control at time 0 was 1,065 ± 92 cpm/106 cells. Vs. control, ‡, P = 0.001; ∗, P = 0.0005, and †, P < 0.0001. (b) GHR translocation. Cultures were incubated in serum-free conditions for 18 hr, followed by treatment with insulin or IGF-I for the durations as indicated. Cells were then briefly trypsinized, and recovery of surface GH binding was measured 2 hr after trypsinization. □, control; •, insulin; ○, IGF-I. Vs. control at the same time point, ‡, P = 0.02; †, P = 0.007; ∗, P = 0.0006; ¶, P = 0.0002; and §, P = 0.0001.
Figure 6
Figure 6
Effect of wortmannin. Cultures were treated with dimethyl sulfoxide vehicle (□) or 100 nM wortmannin (░⃞) at 37°C for 0.5 hr, followed by treatment with insulin or IGF-I for 1.5 hr. Then, cells were briefly trypsinized, and recovery of surface GH binding was measured 2 hr after trypsinization. Vs. dimethyl sulfoxide control, ∗, P < 0.05 and ‡, P < 0.01. vs. dimethyl sulfoxide insulin; †, P < 0.05. vs. wortmannin control; ¶, P = 0.01.
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