Coupling of Heterotrimeric Gi Proteins to the Erythropoietin Receptor

Christine Guillard,Stany Chrétien,Ralf Jockers,Serge Fichelson,Patrick Mayeux,Véronique Duprez

doi:10.1074/jbc.m003527200

Abstract

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.

Highlights

Activation of the EpoR1 elicits multiple intracellular signals that lead to cell division and differentiation of erythroid progenitor and precursor cells
UT7 cells were selected for their high surface Epo receptor (EpoR) expression (ϳ7000 receptors/cell) and their ability to proliferate in response to Epo
Proteins recovered from the column represented about 0.06% of solubilized proteins, a value in good agreement with epidermal growth factor (EGF) receptor substrates previously isolated with a similar procedure [20]

Summary

The abbreviations used are

Erythropoietin; EpoR, erythropoietin receptor; EGF, epidermal growth factor; IGF, insulin-like growth factor; G protein, heterotrimeric GTP-binding protein; GPCR, G protein-coupled receptor; PT, pertussis toxin; PAGE, polyacrylamide gel electrophoresis; IL, interleukin; GM-CSF, granulocyte-macrophage colony-stimulating factor; CSF-1, colony-stimulating factor-1; GTP␥S, guanosine 5Ј-3-O-(thio)triphosphate; CHO, Chinese hamster ovary. Antisera generated against the entire pool of purified proteins were subsequently used for the screening of cDNAs expression libraries We applied this methodology to identify new cDNAs encoding signaling proteins involved in Epo activation, either tyrosine phosphorylated proteins or proteins bound to these proteins. G proteins function as intermediates that couple cell surface receptors to intracellular effectors. In some cases a physical association, in addition to a functional coupling, has been demonstrated between a single-spanning membrane receptor and G proteins (29 –32). Gi Coupling to Epo Receptor macrophage colony-stimulating factor (GM-CSF) and colonystimulating factor-1 (CSF-1) [35,36,37,38] in hematopoietic cells. Epo activates G protein in cell membranes and induces the release of Gi bound to the EpoR in hematopoietic cells. EpoR appears to be physically and functionally coupled to G proteins

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION