Studies with a Growth Hormone Antagonist and Dual-fluorescent Confocal Microscopy Demonstrate that the Full-length Human Growth Hormone Receptor, but Not the Truncated Isoform, Is Very Rapidly Internalized Independent of Jak2-Stat5 Signaling

Abstract

We have investigated trafficking of two negative regulators of growth hormone receptor (GHR) signaling: a human, truncated receptor, GHR1-279, and a GH antagonist, B2036. Fluorescent-labeled growth hormone (GH) was rapidly internalized by the full-length GHR, with >80% of the hormone internalized within 5 min of exposure to GH. In contrast, <5% of labeled GH was internalized by cells expressing truncated GHR1-279. Using another truncated receptor, GHR1-317 fused to enhanced green fluorescent protein (EGFP), we have exploited fluorescence energy transfer to monitor the trafficking of ligand-receptor complexes. The data confirmed that internalization of this truncated receptor is very inefficient. It was possible to visualize the truncated GHR1-317-EGFP packaged in the endoplasmic reticulum, its rapid movement in membrane bound vesicles to the Golgi apparatus, and subsequent transport to the cell membrane. The GH antagonist, B2036, blocked Jak2-Stat5-mediated GHR signaling but was internalized with a similar time course to native GH. The results: 1) demonstrate the rapid internalization of GH when studied under physiological conditions; 2) confirm the hypothesis that internalization of cytoplasmic domain truncated human GHRs is very inefficient, which explains their dominant negative action; and 3) show that the antagonist action of B2036 is independent of receptor internalization.

A46R and A52R from vaccinia virus are antagonists of host IL-1 and toll-like receptor signaling

“…Fig. 4C shows that the induction of this promoter by ectopic expression of either IL-1RI and IL-1RAcP or MyD88 was potently inhibited by coexpression of A52R, whereas the relative induction of a reporter gene linked to the STAT5-dependent lactogenesis hormone response element (33) by the recombinant human GH genotropin, which is not TIR-dependent, was the same in the presence and absence of A52R.…”

“…The IL-8 promoter reporter plasmid was constructed by subcloning the 5Ј noncoding region of the human IL-8 gene, including part of the first exon into pGL3-basic vector (Promega). The lactogenesis hormone response element (LHRE)-luc reporter gene containing the STAT5-binding element LHRE fused to luciferase has been described (33).…”

Analytical characterization of NOTA-modified somatropins

“…The potential involvement of the GH system in tumor promotion and progression, which has been critically reviewed in references [32] and [33], as well as the internalization of the receptor-ligand complex [34], makes hGHR a potential tumor target for the development of somatropin-based radiopharmaceuticals.…”

Pegvisomant: structure and function

“…This strongly suggests that the antagonist, B2036, binds to a receptor dimer. It is clear that the antagonist, B2036, prevents JAK2 phosphorylation and STAT5 signalling (Maamra et al 1999). However, the antagonist receptor complex is still rapidly internalised into cells expressing the human GH receptor (Maamra et al 1999).…”

“…It is clear that the antagonist, B2036, prevents JAK2 phosphorylation and STAT5 signalling (Maamra et al 1999). However, the antagonist receptor complex is still rapidly internalised into cells expressing the human GH receptor (Maamra et al 1999). This internalisation is associated with a down-regulation of GH binding and signalling with a refractory period of around 2-3 h (Ross et al 2001).…”