The interface between self-assembling erythropoietin receptor transmembrane segments corresponds to a membrane-spanning leucine zipper.
Structural and functional studies recently indicated that the erythropoietin receptor exists as a preassembled homodimer whose activation by ligand binding requires self-interaction of its transmembrane segment. Here, we probed the interface formed by the transmembrane segments by asparagine-scanning mutagenesis in a natural membrane. We show that this interface is based on a leucine zipper-like heptad repeat pattern of amino acids. The strongest impact of asparagine was observed at position 241, suggesting the highest packing density around this position, which is in agreement with results obtained upon mutation to alanine. Interestingly, the same face of the transmembrane helix had previously been shown to enter a heterophilic interaction with the transmembrane segment of gp55-P, a viral membrane protein that leads to ligand-independent receptor activation in infected cells. Further, functional characterization of an erythropoietin receptor mutant with asparagine at position 241 in a hematopoietic cell line showed that this protein could still be activated by erythropoietin yet was not constitutively active. This suggests that forced self-interaction of the transmembrane segments does not suffice to induce signaling of the erythropoietin receptor.
Ruan
Weiming
W
Lehrstuhl Chemie der Biopolymere, Technische Universität Munich, Weihenstephaner Berg 3, 85354 Freising, Germany.
Becker
Verena
V
Klingmüller
Ursula
U
Langosch
Dieter
D
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000737:chemistry; D001426:Bacterial Proteins; D015153:Blotting, Western; D002455:Cell Division; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D015151:Immunoblotting; D016350:Leucine Zippers; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017467:Receptors, Erythropoietin / Q000737:chemistry* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000737:chemistry
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D008024:Ligands; D017467:Receptors, Erythropoietin; D014157:Transcription Factors; D014759:Viral Envelope Proteins; C078780:glycoprotein gp55, Friend spleen focus-forming virus; C066719:toxR protein, bacteria; D001216:Asparagine; D000409:Alanine