Active conformation of the erythropoietin receptor: random and cysteine-scanning mutagenesis of the extracellular juxtamembrane and transmembrane domains.
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.
Lu
Xiaohui
X
Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142, USA.
Gross
Alec W
AW
Lodish
Harvey F
HF
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D049109:Cell Proliferation; D003545:Cysteine / Q000737:chemistry; D003600:Cytosol / Q000378:metabolism; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D015723:Gene Library; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000378:metabolism; D015151:Immunoblotting; D051379:Mice; D008954:Models, Biological; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017467:Receptors, Erythropoietin / Q000737:chemistry*; D015398:Signal Transduction; D013997:Time Factors
D004220:Disulfides; D017467:Receptors, Erythropoietin; D006026:Glycoside Hydrolases; D003545:Cysteine