Abstract
The erythropoietin receptor (EpoR) is crucial for erythrocyte formation. The x-ray crystal structures of the EpoR extracellular domain lack the juxtamembrane (JM) region and the junction to the transmembrane (TM) domain. Yet the JM-TM regions are important for transmitting the conformational change imposed on the receptor dimer by Epo binding. Cysteine-scanning mutagenesis of the JM-TM regions identified three novel constitutively active mutants, demonstrating close disulfide-bonded juxtapositioning of these residues in the JM (L223C) and N-terminal TM domain (L226C, I227C). Chemical cross-linking defined the interface of the active helical TM dimer and revealed that the JM-TM segment encompassing Leu(226)-Leu(230) is non-helical. Molecular dynamics and NMR studies indicated that the TM-JM junction forms an N-terminal helix cap. This structure is important for EpoR function because replacement of this motif by consecutive leucines rendered the receptor constitutively active.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Amino Acid Motifs
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Animals
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Blotting, Western
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Cell Line
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Cell Membrane / metabolism
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Cell Proliferation
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Cell Separation
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Cross-Linking Reagents / pharmacology
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Crystallography, X-Ray
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Cysteine / chemistry
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DNA Mutational Analysis
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DNA, Complementary / metabolism
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Dimerization
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Disulfides / chemistry
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Flow Cytometry
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Glycine / analogs & derivatives*
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Glycine / chemistry
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Growth Substances / metabolism
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Humans
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Leucine / chemistry
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Luciferases / metabolism
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Magnetic Resonance Spectroscopy
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Mice
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Mutagenesis
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Mutagenesis, Site-Directed
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Mutation
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Peptides / chemistry
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Phosphorylation
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Protein Binding
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Protein Conformation
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Protein Structure, Secondary
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Protein Structure, Tertiary
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Receptors, Erythropoietin / chemistry*
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Receptors, Erythropoietin / metabolism
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Transfection
Substances
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Cross-Linking Reagents
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DNA, Complementary
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Disulfides
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Growth Substances
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Peptides
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Receptors, Erythropoietin
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Luciferases
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Leucine
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Cysteine
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Glycine
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tricine