title: A functional green fluorescent protein-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.
authors: Max-Planck-Institute of Immunobiology, 79108 Freiburg, Germany.
authors: Robin
authors: R
authors: Ketteler
authors: Achim C
authors: AC
authors: Heinrich
authors: Julia K
authors: JK
authors: Offe
authors: Verena
authors: V
authors: Becker
authors: Jacob
authors: J
authors: Cohen
authors: Drorit
authors: D
authors: Neumann
authors: Ursula
authors: U
authors: Klingmüller
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D049452:Green Fluorescent Proteins; D053614:Janus Kinase 2; D008164:Luminescent Proteins; D051379:Mice; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins; D017467:Receptors, Erythropoietin / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D008164:Luminescent Proteins; D011518:Proto-Oncogene Proteins; D017467:Receptors, Erythropoietin; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; C507926:Jak2 protein, mouse; D053614:Janus Kinase 2