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J. Biol. Chem., Vol. 281, Issue 11, 7002-7011, March 17, 2006

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Active Conformation of the Erythropoietin Receptor

RANDOM AND CYSTEINE-SCANNING MUTAGENESIS OF THE EXTRACELLULAR JUXTAMEMBRANE AND TRANSMEMBRANE DOMAINS^*

Xiaohui Lu, Alec W. Gross, and Harvey F. Lodish¹

From the Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142 and the Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

In the absence of erythropoietin (Epo) cell surface Epo receptors (EpoR) are dimeric; dimerization is mediated mainly by the transmembrane domain. Binding of Epo changes the orientation of the two receptor subunits. This conformational change is transmitted through the juxtamembrane and transmembrane domains, leading to activation of JAK2 kinase and induction of proliferation and survival signals. To define the active EpoR conformation(s) we screened libraries of EpoRs with random mutations in the transmembrane domain and identified several point mutations that activate the EpoR in the absence of ligand, including changes of either of the first two transmembrane domain residues (Leu²²⁶ and Ile²²⁷) to cysteine. Following this discovery, we performed cysteine-scanning mutagenesis in the EpoR juxtamembrane and transmembrane domains. Many mutants formed disulfide-linked receptor dimers, but only EpoR dimers linked by cysteines at positions 223, 226, or 227 activated EpoR signal transduction pathways and supported proliferation of Ba/F3 cells in the absence of cytokines. These data suggest that activation of dimeric EpoR by Epo binding is achieved by reorienting the EpoR transmembrane and the connected cytosolic domains and that certain disulfide-bonded dimers represent the activated dimeric conformation of the EpoR, constitutively activating downstream signaling. Based on our data and the previously determined structure of Epo bound to a dimer of the EpoR extracellular domain, we present a model of the active and inactive conformations of the Epo receptor.

Received for publication, November 28, 2005 , and in revised form, January 11, 2006.

^* This work was supported by National Institutes of Health Grant P01 HL 32262 and a research grant from Amgen, Inc. (to H. F. L.) and National Institutes of Health NHLBI Grant 5F32HL077036 (to A. W. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142. Tel.: 617-258-5216; Fax: 617-258-6768; E-mail: lodish{at}wi.mit.edu.

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