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Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum
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Coupling of heterotrimeric Gi proteins to the erythropoietin receptor

C Guillard1, S Chrétien, R Jockers

  • 1INSERM, U 363 and CNRS-UPR 0415, Institut Cochin de Génétique Moléculaire, 75014 Paris, France.

The Journal of Biological Chemistry
|October 29, 2000
PubMed

To identify new proteins involved in erythropoietin (Epo) signal transduction, we purified the entire set of proteins reactive with anti-phosphotyrosine antibodies from Epo-stimulated UT7 cells. Antisera generated against these proteins were used to screen a lambdaEXlox expression library. One of the isolated cDNAs encodes Gbeta2, the beta2 subunit of heterotrimeric GTP-binding proteins. Gbeta and Galpha(i) coprecipitated with the Epo receptor (EpoR) in extracts from human and murine cell lines and from normal human erythroid progenitor cells. In addition, in vitro Gbeta associated with a fusion protein containing the intracellular domain of the EpoR. Using EpoR mutants, we found that the distal part of the EpoR (between amino acids 459-479) was required for Gi binding. Epo activation of these cells induced the release of the Gi protein from the EpoR. Moreover in isolated cell membranes, Epo treatment inhibited ADP-ribosylation of Gi and increased the binding of GTP. Our results show that heterotrimeric Gi proteins associate with the C-terminal end of the EpoR. Receptor activation leads to the activation and dissociation of Gi from the receptor, suggesting a functional role of Gi protein in Epo signal transduction.

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