Mutagenesis Studies of the Human Erythropoietin Receptor

Abstract

Mutagenesis of the erythropoietin receptor (EPOR)permits analysis of the contribution that individual amino acid residues make to erythropoietin (EPO) binding. We employed both random and site-specific mutagenesis to determine the function of amino acid residues in the extracellular domain (referred to as EPO binding protein, EBP) of the EPOR. Residues were chosen for site-specific alanine substitution based on the results of the random mutagenesis or on their homology to residues that are conserved or have been reported to be involved in ligand binding in other receptors of the cytokine receptor family. Site-specific mutants were expressed in Escherichia coli as soluble EBP and analyzed for EPO binding in several different assay formats. In addition, selected mutant proteins were expressed as full-length EPOR on the surface of COS cells and analyzed for 125 I-EPO binding in receptor binding assays. Using these methods, we have identified residues that appear to be involved in EPO binding as well as other residues, most of which are conserved in receptors of the cytokine receptor family, that appear to be necessary for the proper folding and/or stability of the EPOR. We present correlations between these mutagenesis data and the recently solved crystal structure of the EBP with a peptide ligand.Erythropoietin (EPO) 1 is a glycoprotein hormone that functions as the primary regulator of erythropoiesis by binding a specific receptor (EPOR) on the surface of erythrocyte precursor cells, signaling their proliferation and differentiation into mature red blood cells (reviewed in Ref. 1). The human EPOR is a 484-amino acid glycoprotein comprised of extracellular and cytoplasmic domains of nearly equal size and a single transmembrane domain (2). The extracellular domain of the EPOR contains a 225-amino acid region referred to as the cytokine receptor homology (CRH) domain that shares conserved features with an expanding family of cytokine and growth factor receptors (3, 4) including the receptors for many interleukins (IL), colony stimulating factors, growth hormone (GH), thrombopoietin, leptin, interferons (IFN), and tissue factor among others. The CRH domains consist of two motifs of approximately 100 amino acids each that are structurally related to fibronectin type III domains. Based on this homology, Bazan (3, 4) proposed that the CRH domains consist of two motifs of seven ␤-strands each which adopt ␤-sheet structures with fibronectin type III-like or immunoglobulin (Ig)-like folds. These structural predictions have been confirmed by the solution of the crystal structures of the ligand-bound extracellular domains of the GH receptor and IFN-␥ receptor ␣ (IFN-␥R␣), the GH bound extracellular domain of the prolactin receptor, the extracellular domain of tissue factor, and most recently, the extracellular domain of the EPOR with a peptide ligand (5-10). Alignment of the CRH domains of this family of receptors based on their predicted ␤-strand secondary structural elements reveals several conserved characteristics (3, 4,...

A High-Throughput Assay to Identify Compounds That Can Induce Dimerization of the Erythropoietin Receptor

“…When expressed in E. coli, much like the wild-type rEBP, the rEBP-kemp also partitioned into the insoluble fraction and was purified using the procedure developed for the production of the wild-type rEBP. When tested in an antagonistic version of an erythropoietin-dependent luciferase assay (25), both the rEBP and the rEBPkemp inhibited erythropoietin-induced increases in the luciferase activity with a IC 50 of 5-10 nM (Table 1), which is in accord with published IC 50 for this protein (29). Moreover, both proteins exhibited similar affinity of binding EPO (k D ϳ 5 nM) in a EPO receptor binding assay (data not shown).…”

Shared and Unique Determinants of the Erythropoietin (EPO) Receptor Are Important for Binding EPO and EPO Mimetic Peptide

“…44 -46). In the GH-GHR interaction, two tryptophans (Trp 104 and Trp 169 ) accounted for the majority of the binding free energy of the interaction, constituting a hot spot for GH binding to GHR (44 (20), we found we had already examined the function of 40% of the residues involved in H-bonds or salt bridges and nearly 50% of the residues involved in non-polar interactions with EPO (29,43). The data presented here, combined with the fact that Phe 93 and Phe 205 dominate the non-polar contacts with EPO site 1 and site 2 (20), indicate that these residues are an important component of the hot spot for EPO binding.…”

“…Of the 45 amino acid residues (20%) of the EBP that we have examined by alanine substitution to date (29,43), only Phe 93 and Phe 205 were found to be critical for EPO binding. The remaining residues had relatively little or no role in EPO binding or were important for structure.…”