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| - Dimerization of the Extracellular Domain of the Erythropoietin (EPO) Receptor byEPO: One High-Affinity and One Low-Affinity Interaction
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| - Although there is considerable evidence that signaling by theerythropoietin (EPO) receptor isinitiated when it is dimerized by binding EPO, it has been previouslyreported that the soluble extracellulardomains of the EPO receptor (sEPOR) are not dimerized in the presenceof EPO and are able to formonly 1:1 complexes with EPO. We have now shown unambiguously bylight scattering, sedimentationequilibrium, and titration calorimetry that two molecules of sEPOR canbind to a single EPO monomerbut that the binding of the second sEPOR is ∼1000-fold weaker thanthat of the first. Because thissecond binding interaction is quite weak (Kd of∼1 μM), the 2:1 sEPOR·EPO complexes are easilydissociated during chromatography (forming the 1:1 complexes reportedpreviously) and cannot be isolatedin pure form. Global analysis of the sedimentation equilibriumdata has enabled us to determine thebinding constants and is consistent with a model in which EPO has twoindependent binding sites forsEPOR but cannot exclude anticooperative or sequential binding models.The influence of glycosylationof EPO and/or sEPOR on the binding affinities has also beeninvestigated. Titration calorimetry is consistentwith the sedimentation data and shows that the weaker binding site hasa more negative ΔH. The relationof these results to the binding of EPO to membrane-bound receptors andto the phenomenon of apparenthigh-affinity and low-affinity classes of receptors isdiscussed.
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