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

The GH receptor (GHR) is a member of the cytokine receptor family. Sho rt isoforms resulting from alternative splicing have been reported for a number of proteins in this family. RT-PCR experiments, in human liv er 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 spl ice site 26 bp downstream in exon 9; the predicted C-terminal residues are six frame-shifted exon 9 codons ending in an inframe stop codon. The 383-bp product (GHR1-277) resulted from skipping of exon 9; the pr edicted C-terminal residues are three frame-shifted exon 10 codons end ing in an in-frame stop codon. RNase protection experiments confirmed the presence of the GHR1-279 variant in IM-9 cells and human liver. Th e proportion of alternative splice to full length was 1-10% for GHR1-2 79 and less than 1% for GHR1-277. The function of GHR1-279 was examine d after subcloning in an expression vector and transient transfection in 293 cells. Scatchard analysis of competition curves for [I-125]hGH bound to cells transfected either with GHR full length (GHRf) 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 c onfirmed by cross-linking studies. The media of cells transfected with GHR1-279 contained 20-fold more GH-binding protein (GHBP) than that f ound in the media of cells transfected with the full-length receptor. Immunoprecipitation and Western blotting experiments, using a combinat ion 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, conta ining Stat5-binding elements, confirmed that while the variant form wa s inactive by itself, it could inhibit the function of the full-length receptor. We have demonstrated the presence of a splice variant of th e GHR in human liver encoding a short form of the receptor similar in size to a protein previously identified in human liver and choroid ple xus. Expression studies in 293 cells support the hypothesis that while the expression of the splice variant accounts for only a small propor tion of the total GHR transcript, it produces a short isoform that mod ulates the function of the full-length receptor, inhibits signaling, a nd generates large amounts of GHBP. The differential expression of GHR receptor short forms may regulate the production of GHBP, and truncat ed receptors may act as transport proteins or negative regulators of G HR signaling.