Frank J. McMahon

Amino-terminal dimerization of an erythropoietin mimetic peptide results in increased erythropoietic activity

BACKGROUND Erythropoietin (EPO), the hormone involved in red blood cell production, activates its receptor by binding to the receptors extracellular domain and presumably dimerizing two receptor monomers to initiate signal transduction. EPO-mimetic peptides, such as EMP1, also bind and activate the receptor by dimerization. These mimetic peptides are not as potent as EPO, however. The crystal structure of the EPO receptor (EBP) bound to EMP1 reveals the formation of a complex consisting of two peptides bound to two receptors, so we sought to improve the biological activity of EPO-mimetic peptides by constructing covalent dimers of EMP1 and other peptide mimetics linked by polyethylene glycol (PEG). RESULTS The potency of the PEG-dimerized EPO peptide mimetics both in vitro and in vivo was improved up to 1,000-fold compared to the corresponding peptide monomers. The dimers were constructed using peptide monomers which have only one reactive amine per molecule, allowing us to conclude that the increase in potency can be attributed to a structure in which two peptides are linked through their respective amino termini to the difunctional PEG molecule. In addition, an inactive peptide was converted into a weak agonist by PEG-induced dimerization. CONCLUSIONS The potency of previously isolated peptides that are modest agonists of the EPO receptor was dramatically increased by PEG-induced dimerization. The EPO receptor is thought to be dimerized during activation, so our results are consistent with the proposed 2:2 receptor : peptide stoichiometry. The conversion of an inactive peptide into an agonist further supports the idea that dimerization can mediate receptor activation.

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

We have shown previously that Phe93 in the extracellular domain of the erythropoietin (EPO) receptor (EPOR) is crucial for binding EPO. Substitution of Phe93 with alanine resulted in a dramatic decrease in EPO binding to the Escherichia coli-expressed extracellular domain of the EPOR (EPO-binding protein or EBP) and no detectable binding to full-length mutant receptor expressed in COS cells. Remarkably, Phe93 forms extensive contacts with a peptide ligand in the crystal structure of the EBP bound to an EPO-mimetic peptide (EMP1), suggesting that Phe93 is also important for EMP1 binding. We used alanine substitution of EBP residues that contact EMP1 in the crystal structure to investigate the function of these residues in both EMP1 and EPO binding. The three largest hydrophobic contacts at Phe93, Met150, and Phe205 and a hydrogen bonding interaction at Thr151 were examined. Our results indicate that Phe93 and Phe205 are important for both EPO and EMP1 binding, Met150 is not important for EPO binding but is critical for EMP1 binding, and Thr151 is not important for binding either ligand. Thus, Phe93 and Phe205 are important binding determinants for both EPO and EMP1, even though these ligands share no sequence or structural homology, suggesting that these residues may represent a minimum epitope on the EPOR for productive ligand binding.

Mutagenesis Studies of the Human Erythropoietin Receptor ESTABLISHMENT OF STRUCTURE-FUNCTION RELATIONSHIPS

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 125I-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.