Endocrine Abstracts (2006) 11 P237

Growth hormone receptor extracellular domain linked to glycophosphatidyl inositol (GHR-GPI); a potential growth hormone receptor antagonist

CE Bowles1, I Wilkinson1, N Gavalas1, P Watson1, D Watts2 & R Ross1

1University of Sheffield, Clinical Sciences, Northern General Hospital, Sheffield, United Kingdom; 2University of Sheffield, Dept. Molecular Biology & Biotechnology, Sheffield, United Kingdom.

Introduction: The growth hormone receptor (GHR) consists of 620 residues and belongs to the class I cytokine receptor family. It is a single membrane spanning protein that binds its ligand, GH, via the extracellular domain. GH binding to GHR induces a conformational change in the preformed receptor dimer, which leads to intracellular signalling. Correct functional dimerisation of two GHR molecules is essential for GH signalling. We have previously shown that membrane bound truncated extracellular GHR can act as a dominant negative inhibitor of GHR signalling. We have now generated a GHR extracellular molecule linked to a GPI lipid moiety, to insert into membranes, in order to produce a potential cytokine receptor antagonist.

Methods: The GHR-GPI was cloned and expressed in Dictyostelium discoideum strain AX-2, generating 5×106 amoebae ml−1 in the late exponential phase of growth. The harvested cells were used to produce cell membranes containing GHR-GPI. The membranes were then solubilised using n-octylglucoside and the GHR-GPI purified using affinity chromatography involving GH covalently linked to activated sepharose. Elutions were analysed by western blotting to determine the presence of GHR-GPI and assayed using the Bradford protein assay. Efficiency of insertion of GHR-GPI into cell membranes was measured using flow cytometry techniques.

Results: Western blotting results showed that the GHR-GPI containing membranes could be extracted, solubilised and the GHR-GPI protein purified. Flow cytometry analysis showed that the GHR-GPI protein inserted into the cell membrane to high levels.

Conclusions: The GHR-GPI protein can insert into cell membranes, where it may potentially disrupt the preformed GHR dimer, and theoretically block GH signalling.

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