Activation of erythropoietin receptor in the absence of hormone by a peptide that binds to a domain different from the hormone binding site

Abstract

Applying a homology search method previously described, we identified a sequence in the extracellular dimerization site of the erythropoietin receptor, distant from the hormone binding site. A peptide identical to that sequence was synthesized. Remarkably, it activated receptor signaling in the absence of erythropoietin. Neither the peptide nor the hormone altered the affinity of the other for the receptor; thus, the peptide does not bind to the hormone binding site. The combined activation of signal transduction by hormone and peptide was strongly synergistic. In mice, the peptide acted like the hormone, protecting against the decrease in hematocrit caused by carboplatin.We previously had described a method for identifying peptides that would selectively inhibit internalization of insulin, insulinlike growth factor 1, and other receptors (1-3). We used the same MHC class I protein (MHC-I) sequence D k -(61-85) as probe in a homology search with the erythropoietin (EPO) receptor (EPO-R) as target. We identified a homologous sequence in the extracellular domain of the receptor, distant from the hormone binding site, in a region suggested to participate in dimerization (4). An EPO-R-derived peptide (ERP) was synthesized identical to that sequence. Now we describe the wholly unexpected finding that EPO-R, which normally is activated by hormone-induced dimerization, can be activated by ERP in the absence of hormone. The peptide does not bind to the hormone binding site; it apparently complexes with its identical sequence in the homology domain. Interestingly, EPO and ERP together produce a dramatic synergy of effect. Finally, we show that in mice ERP has the same effect as EPO.

Recombinant human thrombopoietin: basic biology and evaluation of clinical studies

“…An analogous peptide that binds the erythropoietin receptor and potentiates erythropoietin has also recently been described. 97 …”

Small-molecule agonists for the glucagon-like peptide 1 receptor

“…Glucagon binds to the GLP-1 receptor with low affinity, whereas GLP-1 does not bind to the glucagon receptor (17). In recent years, smallmolecule agonists have been described, even for receptors for larger hormones like insulin, TPO, and EPO (18)(19)(20), but none of these receptors belong to the GPCR superfamily. Within the GPCRs, small-molecule agonists have been described, e.g., for the arginine vasopressin V-2 receptor, the somatostatin receptor, the bradykinin receptor, the cholecystokinin receptor, the angiotensin II receptor, and the growth hormone secretagogue receptor (21)(22)(23)(24)(25)(26).…”

Discovery and Characterization of Nonpeptidyl Agonists of the Tissue-Protective Erythropoietin Receptor

“…The design of the initial library was facilitated by the structureactivity relationship (SAR) information available from small molecule (Qureshi et al, 1999;Goldberg et al, 2002) and allosteric peptide (Naranda et al, 1999(Naranda et al, , 2002 agonists of the EPO receptor, and allosteric small molecule agonists of the structurally related TPOR (Erickson-Miller et al, 2005;Desjardins et al, 2006;Dziewanowska et al, 2007;Kim et al, 2007;Kalota and Gewirtz, 2010;Abe et al, 2011) as well as structural data on the receptors (Livnah et al, 1996(Livnah et al, , 1999Carr et al, 2001). An initial hit was identified based on receptor interaction and EPO-like functional activity, and subsequent compound designs for developing an SAR for in vitro neuroprotection focused on 1,2,4-triazolo[1,5-a]pyrimidines.…”

“…The allosteric TPOR agonists described to date are also smaller than the previously described orthosteric EPOR agonists, supporting the potential to design compounds that cross the BBB. Allosteric small peptide agonists of the EPO receptor have been described, but no tissue-protective effects were reported (Naranda et al, 1999(Naranda et al, , 2002. Small molecule compounds selective for the tissue-protective EPOR/CD131 heterodimer have the potential to overcome the limitations of EPO regarding tissue availability and may improve safety by avoiding the hematopoietic side effects.…”

“…Agonists of several cytokine receptors, including the receptors for granulocyte colony-stimulating factor (G-CSF) (Tian et al, 1998) and thrombopoietin (TPO) (Erickson-Miller et al, 2005;Desjardins et al, 2006;Dziewanowska et al, 2007;Kim et al, 2007;Kalota and Gewirtz, 2010;Abe et al, 2011) have been described, and the TPO receptor (TPOR) agonist eltrombopag is approved by the U.S. Food and Drug Administration for the treatment of thrombocytopenia (Kuter, 2013). The feasibility of discovering small molecule agonists of receptors for EPO was also supported by the small allosteric peptide agonists that activated the EPOR/EPOR homodimer and triggered EPO-like biologic activity (Naranda et al, 1999;Naranda et al, 2002).…”