A Functional Green Fluorescent Protein-tagged Erythropoietin Receptor Despite Physical Separation of JAK2 Binding Site and Tyrosine Residues

Abstract

Signaling through hematopoietic cytokine receptors such as the erythropoietin receptor (EpoR) depends on the activation of a receptor-bound Janus kinase (JAK) and tyrosine phosphorylation of the cytoplasmic domain. To visualize the EpoR and elucidate structural requirements coordinating signal transduction, we probed the EpoR by inserting the green fluorescent protein (GFP) at various positions. We show that insertion of GFP in proximity to the transmembrane domain, either in the extracellular or the cytoplasmic domain, results in EpoR-GFP receptors incompetent to elicit biological responses in a factor-dependent cell line or in erythroid progenitor cells. Surprisingly, a receptor harboring GFP insertion in the middle of the cytoplasmic domain, and thereby separating the JAK2 binding site from the tyrosine residues, is capable of supporting signal transduction in response to ligand binding. Comparable with the wild type EpoR, but more efficient than a C-terminal EpoR-GFP fusion, this chimeric receptor promotes the maturation of erythroid progenitor cells and is localized in punctated endosome-like structures. We conclude that the extracellular, transmembrane, and membrane-proximal segment of the cytoplasmic domain form a rigid structural entity whose precise orientation is essential for the initiation of signal transduction, whereas the cytoplasmic domain possesses flexibility in adopting an activated conformation.

Examining the Involvement of Erythropoiesis-Stimulating Agents in Tumor Proliferation (Erythropoietin Receptors, Receptor Binding, Signal Transduction), Angiogenesis, and Venous Thromboembolic Events

“…If those proteins detected within the cells were indeed EPORs, it is unlikely that they would confer signaling upon EPO treatment. In addition, for erythropoietic progenitors, which undoubtedly can express EPORs, it was shown that cytoplasmic EPOR immunoreactivity can be caused by immature forms of the receptor that do not contribute to EPOR signaling [6].…”

A mouse model for visualization and conditional mutations in the erythroid lineage

“…To establish a fluorescent-labeled Cre recombinase detectable in living cells, we fused a thermostable EGFP 23 in-frame to the Nterminal coding sequence of a codon-improved Cre recombinase. 29 As linker sequence between the 2 functional domains, a dodecameric peptide was introduced to allow flexibility.…”

“…The enhanced GFP with improved thermostability (tEGFP) has been described elsewhere. 23 To eliminate a potential splice donor site in iCre, codon V343 was altered by site-directed mutagenesis from GTG to GTC. A DNA fragment of pEPOR encompassing parts of exon 2 as well as exons 3 to 8 of the EpoR gene was replaced by a neomycin selection cassette flanked by 2 FLP recombinase recognition (FRT) sites, thereby leaving the splice acceptor site of exon 2 upstream and the authentic poly-A signal downstream of the selection cassette ( Figure 1A).…”

Kinase activity and subcellular distribution of a chimeric green fluorescent protein-tagged Janus kinase 2

“…Derivatives of the green fluorescent protein cloned from Aequora victoria have been used extensively in biological research based on the assumption that it does not significantly alter the function of its fusion partner [37,38]. Yet there are occasional reports of functional changes arising in proteins as a consequence of the relative placement of the GFP fusion partner [39,40]. When we began this study of EGFP-fused JAK2 chimeras, we noticed a change in the fluorescence intensities of the chimeric fluorescent proteins.…”