BES2003 Poster Presentations Cytokines and Growth Factors (9 abstracts)
A growth hormone receptor (GHR)- glycophosphatidylinositol (GPI) chimera acts as a receptor antagonist
NG Gavalas 1 , SL Pradhananga 1 , C Beghadi 2 , M Maamra 1 , H Zarkesh-Esfahani 1 , J Sayers 3 , P Artymiuk 4 , C Reymond 2 & RJM Ross 1
1Division of Clinical Sciences North, Sheffield University, Sheffield, UK; 2University of Lausanne, Switzerland; 3Division of Genomic Medicine, Sheffield University, Sheffield, UK; 4Department of Molecular Biology and Biotechnology, Sheffield University, Sheffield, UK.
We have previously demonstrated that a truncated GHR lacking the cytoplasmic domain of the receptor acts as a dominant negative regulator of receptor signalling. Glycosylphosphatidylinositol (GPI) anchors are a common mechanism by which cells direct and anchor proteins in the cell membrane. We have generated a GHR extracellular domain-GPI fusion protein and tested its biological activity as a receptor antagonist. The extracellular domain of GHR (growth hormone binding protein-GHBP) was cloned upstream of a mammalian GPI signal sequence. This vector, GHBP-GPI, and GHRwt were transfected into CHO and HEK293 cells. In CHO cells GHBP-GPI showed high level expression compared to GHRwt; mean plus/minus sem specific binding of 125I-GH was 57.0 plus/minus 0.9 vs 18.7 plus/minus 1.2 percent. This high level expression was confirmed on FACS with a GHR specific antibody, the mean fluorescence being 2473a.u in CHO cells transfected with GHBP-GPI and probed with 2C8 antibody, whereas the negative control showed a mean fluorescence of only 9.4a.u. Functional assays show that GHBP-GPI acts as a potent antagonist when it is co-expressed with GHRwt. HEK293 cells were transfected with GHBP-GPI and GHRwt at a 1:1 ratio, and GH stimulation of the transcriptional assay was inhibited by 72.0 plus/minus 7.2 percent. In conclusion, a truncated GHR anchored in the membrane by GPI shows high level expression on the cell surface and acts as a GHR antagonist.
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Endocrine Abstracts
ISSN 1470-3947 (print) | ISSN 1479-6848 (online)
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