Active Conformation of the Erythropoietin Receptor: RANDOM AND CYSTEINE-SCANNING MUTAGENESIS OF THE EXTRACELLULAR JUXTAMEMBRANE AND TRANSMEMBRANE DOMAINS

Xiaohui Lu,Alec W Gross,Harvey F Lodish

doi:10.1074/jbc.m512638200

Abstract

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 (Leu(226) and Ile(227)) 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.

Highlights

The cytokine erythropoietin (Epo)2 is the primary regulator of mammalian erythropoiesis
These results suggest that activation of dimeric erythropoietin receptor (EpoR) by Epo binding is achieved by reorienting the EpoR transmembrane domains and that certain disulfide-bonded dimers represent the activated dimeric conformation of the EpoR
These results demonstrate that mutants L223C, L226C, and I227C are active in the absence of Epo, as they support activation by phosphorylation of three major signaling proteins normally activated by Epo addition, JAK2, the EpoR itself, and STAT5

Summary

Active Conformation of the Erythropoietin Receptor

RANDOM AND CYSTEINE-SCANNING MUTAGENESIS OF THE EXTRACELLULAR JUXTAMEMBRANE AND TRANSMEMBRANE DOMAINS*. Because the EpoR transmembrane and juxtamembrane domains form a rigid ␣-helix that locks it in an inactive configuration in the absence of ligand, mutations in this region may mimic the conformational changes that the Epo binding imposes on the EpoR, causing the realignment of the intracellular domains and the associated JAK2 kinases, and leading to activation of JAK2. Many mutants form disulfide-linked dimers, but only EpoR dimers linked by cysteine at positions 223, 226, or 227 activate all of the EpoR signal transduction pathways we tested and support proliferation of Ba/F3 cells in the absence of cytokines These results suggest that activation of dimeric EpoR by Epo binding is achieved by reorienting the EpoR transmembrane domains and that certain disulfide-bonded dimers represent the activated dimeric conformation of the EpoR. Structural analysis of disulfide-linked synthetic EpoR transmembrane domains, locked in either an inactive or an active state as defined by these mutations, will elucidate how the conformational changes in a dimeric EpoR activates JAK2 and downstream signaling pathways

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION