Coupling of heterotrimeric Gi proteins to the erythropoietin receptor

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 mere used to screen a lambda EXlox expression library. One of the isolated cDNAs encodes G beta (2), the beta (2) subunit of heterotrimeric GTP-binding proteins. G beta and G alpha (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 G beta 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 G(i) binding. Epo activation of these cells induced the release of the G(i) protein from the EpoR. Moreover in isolated cell membranes, Epo treatment inhibited ADP-ribosylation of G(i) and increased the binding of GTP. Our results show that heterotrimeric G(i) proteins associate with the C-terminal end of the EpoR. Receptor activation leads to the activation and dissociation of G(i) from the receptor, suggesting a functional role of G(i) protein in Epo signal transduction.