Endocrine Abstracts (2004) 7 P26

GHR-GPI insertion into target CHO cell membranes

NG Gavalas1, SH Zarkesh-Esfahani1, IR Wilkinson1, SL Pradhananga1, C Beghadi2, GP Baumann3, Z Wu4, PJ Artymiuk5, JR Sayers6 & RJM Ross1

1University of Sheffield, Medical School, Department of Clinical Sciences (North), Northern General Hospital, Sheffield, UK; 2University of Lausanne, Lausanne, Switzerland; 3Northwestern University, Medical School, Chicago, IL, USA; 4Division of Medicine, Berlin, Germany; 5University of Sheffield, Department of Molecular Biology and Biotechnology, Sheffield, UK; 6University of Sheffield, Medical School, Division of Genomic Medicine, Sheffield, UK.

Introduction: We have generated a truncated Growth Hormone Receptor (GHR) fused to the cell membrane by a glycosyl phosphatidylinositol anchor (GHR-GPI). The protein consists of the complete extracellular domain of the GHR, but lacks the intracellular domain, and we predict it would act as a GH antagonist. Aim: To test if partially-purified soluble GHR-GPI could re-insert into CHO cell membranes. Material and methods: Membrane preparations were made from CHO cells stably expressing GHR-GPI. As controls, membrane preparations were also made from non-transfected CHO cells and from HEK293 cells that express full length GHR (GHRfl). Non-transfected CHO cells were incubated with different amounts of the above membrane preparations for 4 hours. The insertion of GHR into the non-transfected CHO cells was determined using FACS analysis and a specific antibody against the extracellular domain of the GHR. Results: CHO cells incubated with GHR-GPI showed high levels of specific binding to the GHR antibody in a dose dependent manner compared to control antibody, confirming the insertion of GHR-GPI into the membranes of non-transfected CHO cells (Mean channel fluorescence (arbitrary units): GHR-GPI=330 vs Control=70). CHO cells incubated with the membrane preparation of GHRfl did not show any specific binding to GHR antibody (Mean channel fluorescence (arbitrary units): GHRfl=48) indicating that only GHR-GPI can re-insert into the membranes of CHO cells. Conclusion: Addition of GPI at the end of extracellular domain of the GHR enables the GHR to reinsert itself into the cell membrane.

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