Abstract
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.
Highlights
Sequence-specific interactions between ␣-helical transmembrane segments (TMSs)1 support assembly of many integral membrane proteins [1,2,3]
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
The phase of the TMS appears to be optimal within mEpoR4.16, and this construct was used for further analysis
Summary
Plasmid Constructs—Construction of plasmids pToxRmEpoR16 and its mutant L240G/L241P was described previously [5]. Plasmids mEpoR3.16.1, mEpoR4.16, mEpoR5.15, and mEpoR6.14 were constructed by ligating synthetic oligonucleotide cassettes encoding the desired sequences into the plasmid pHKToxR(TMIl4)MalE [20] or pToxR IV [21] previously cut with NheI and BamHI. Each site-directed mutant was constructed by the Kunkel method [22] using a Bio-Rad T7 mutagenesis kit according to the manufacturer’s instructions. All of the constructs were verified by dideoxy sequencing. The retroviral expression vectors pMOWS-mEpoR and pMOWS-HA-mEpoR were described elsewhere [23]. The construct pMOWS-L241N was established by PCR
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