A Short Isoform of the Human Growth Hormone Receptor Functions as a Dominant Negative Inhibitor of the Full-Length Receptor and Generates Large Amounts of Binding Protein

Abstract

The GH receptor (GHR) is a member of the cytokine receptor family. Short isoforms resulting from alternative splicing have been reported for a number of proteins in this family. RT-PCR experiments, in human liver and cultured IM-9 cells, using primers in exon 7 and 10 of the GHR, revealed three bands reflecting alternative splicing of GHR mRNA: the predicted product at 453 bp and two other products at 427 and 383 bp. The 427-bp product (GHR1-279) utilized an alternative 3'-acceptor splice site 26 bp downstream in exon 9; the predicted C-terminal residues are six frameshifted exon 9 codons ending in an inframe stop codon. The 383-bp product (GHR1-277) resulted from skipping of exon 9; the predicted C-terminal residues are three frame-shifted exon 10 codons ending in an in-frame stop codon. RNase protection experiments confirmed the presence of the GHR1-279 variant in IM-9 cells and human liver. The proportion of alternative splice to full length was 1-10% for GHR1-279 and less than 1% for GHR1-277. The function of GHR1-279 was examined after subcloning in an expression vector and transient transfection in 293 cells. Scatchard analysis of competition curves for [125l]-hGH bound to cells transfected either with GHR full length (GHRfl) or GHR1-279 revealed a 2-fold reduced affinity and 6-fold increased number of binding sites for GHR1-279. The increased expression of GHR1-279 was confirmed by cross-linking studies. The media of cells transfected with GHR1-279 contained 20-fold more GH-binding protein (GHBP) than that found in the media of cells transfected with the full-length receptor. Immunoprecipitation and Western blotting experiments, using a combination of antibodies directed against extracellular and intracellular GHR epitopes, demonstrated that GHRfl and GHR1-279 can form heterodimers and that the two forms also generate a 60-kDa GHBP similar in size to the GHBP in human serum. Functional tests using a reporter gene, containing Stat5-binding elements, confirmed that while the variant form was inactive by itself, it could inhibit the function of the full-length receptor. We have demonstrated the presence of a splice variant of the GHR in human liver encoding a short form of the receptor similar in size to a protein previously identified in human liver and choroid plexus. Expression studies in 293 cells support the hypothesis that while the expression of the splice variant accounts for only a small proportion of the total GHR transcript, it produces a short isoform that modulates the function of the full-length receptor, inhibits signaling, and generates large amounts of GHBP. The differential expression of GHR receptor short forms may regulate the production of GHBP, and truncated receptors may act as transport proteins or negative regulators of GHR signaling.

Ligand-independent growth hormone receptor dimerization occurs in the endoplasmic reticulum and is required for ubiquitin system-dependent endocytosis

“…2), which implies that either the TMD or the cytosolic domain or both contain the dimerizing domain. A role for the latter seems unlikely because cotransfection of a full-length GHR and a signaling-deficient GHR truncation mutant, lacking most of the cytosolic domain, decreases the signaling capacity of the cells probably by formation of heterodimers (36). Therefore, we speculate that the dimerization of the GHR is mediated by way of the TMD, which is in accordance with recent findings that the TMD of the EpoR (21), the EGF receptor (37), the MHC class II ␣ and ␤ subunits (32), and glycophorin A (38) are sufficient for (ligand-independent) dimerization.…”

Metalloprotease-mediated GH Receptor Proteolysis and GHBP Shedding

“…That is, despite infectious overexpression: 1) rbGHR del 297-406 and rbGHR 1-274-Myc-His underwent basal and PMA-induced cleavage and GHBP shedding with kinetics similar to what we previously showed; 2) PMA-induced proteolysis and GHBP shedding was inhibited by IC3; and 3) GH antagonized PMA's ability to cause receptor cleavage. In addition, rbGHR 1-274-Myc-His is analogous to a naturally occurring alternatively spliced GHR form that is known to effectively yield proteolytically shed GHBP in other systems (31,32).…”

“…It is known that a naturally occurring GHR splice variant lacking nearly the entire cytoplasmic domain generates substantial amounts of proteolytically shed GHBP (31,32). For remnant purification, we designed and adenovirally expressed a rbGHR mutant, rbGHR 1-274-Myc-His , that encodes the first 274 residues of the receptor followed by C-terminal Myc and His tags.…”

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

“…Activation of the Jak-Stat pathway is dependent on an intact cytoplasmic domain of the receptor forming multisubunit complexes with associated tyrosine kinases. Recently, we (2) and others (3) identified a truncated form of the receptor, GHR1-279, in normal human liver and certain human cell lines. This truncated receptor lacks 97% of the cytoplasmic domain of the receptor and has a dominant negative action on Jak-Stat signaling (2).…”