To identify new proteins involved in erythropoietin (Epo) signal transducti
on, we purified the entire set of proteins reactive with anti-phosphotyrosi
ne antibodies from Epo-stimulated UT7 cells. Antisera generated against the
se proteins mere used to screen a lambda EXlox expression library. One of t
he isolated cDNAs encodes G beta (2), the beta (2) subunit of heterotrimeri
c 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 norma
l human erythroid progenitor cells. In addition, in vitro G beta associated
with a fusion protein containing the intracellular domain of the EpoR. Usi
ng EpoR mutants, we found that the distal part of the EpoR (between amino a
cids 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 incre
ased the binding of GTP. Our results show that heterotrimeric G(i) proteins
associate with the C-terminal end of the EpoR. Receptor activation leads t
o the activation and dissociation of G(i) from the receptor, suggesting a f
unctional role of G(i) protein in Epo signal transduction.