LOCALIZATION OF AN ESSENTIAL LIGAND-BINDING DETERMINANT OF THE HUMAN ERYTHROPOIETIN RECEPTOR TO A DOMAIN N-TERMINAL TO THE WSXWS MOTIF - IMPLICATIONS FOR SOLUBLE RECEPTOR FUNCTION

The interaction of erythropoietin (Epo) with the erythropoietin recept or (EpoR) supports erythropoiesis. The EpoR is a member of the well-re cognized cytokine receptor superfamily characterized by four conserved cysteines and a WSXWS domain in the extracellular portion of the mole cule. To localize ligand-binding determinants of the EpoR near the WSX WS domain, we tested the ligand-binding ability of the wild-type human EpoR extracellular domain (EREx), two truncated and three chimeric co nstructs with the interleukin-2 receptor beta subunit (IL2R beta). Con structs were expressed in E. coli as GST fusion proteins linked to a s olid-phase support and assayed for binding to I-125 Epo. AS previously shown, Epo bound specifically to the expressed extracellular domain, EREx. Epo did not bind to truncated receptors lacking either the entir e fifth exon or the WSXWS domain. Epo also did not bind to chimeric re ceptors that had the amino acids encoded by the fifth exon replaced by IL2R beta or that had the amino acids subsequent to asparagine residu e 209 replaced by IL2R beta. Specific binding was demonstrated for a c onstruct in which the WSXWS was replaced by that of IL2R beta. We conc lude that the amino acids encoded by this 5' portion of exon 5 of the EpoR are necessary for ligand binding and that the WSXWS domain is nec essary for Epo binding but is not involved in ligand-binding specifici ty. We also speculate that if the putative soluble form of the EpoR is expressed (predicted to lack exon 5), it does not bind Epo and theref ore may serve a physiologic purpose other than ligand binding.