title: Tyrosine 462 of the membrane-proximal F'-G' loop of murine Mpl is not essential for high-affinity binding of thrombopoietin.
abstract: The ligand binding site of Mpl, the thrombopoietin (Tpo) receptor, has not been determined. Tyr(462)of murine Mpl corresponds to Tyr(421)of the common beta chain of the human IL-3, IL-5 and GM-CSF receptors. Tyr(421)has been identified as essential for high-affinity ligand binding. To determine whether Tyr(462)is similarly required for Tpo binding, wild-type murine Mpl (Mpl-WT) or mutant receptors containing an alanine (Y462A) or lysine (Y462K) in place of Tyr(462)were expressed in BaF3 cells. In proliferation studies, the Y462A mutation had no effect on Tpo-induced growth. In contrast, the Y462K mutation led to an attenuated proliferative response to Tpo. In single-point binding studies, both Mpl-WT and Y462A cells were able to bind [(125)I]Tpo in a specific manner. In contrast, there was a marked reduction in binding of [(125)I]Tpo by Y462K cells. Mpl-WT cells bound Tpo with a K(d)of approximately 330 pM, while Y462A cells bound Tpo with a K(d)of approximately 268 pM. The binding affinity of Y462K cells was below that quantifiable by Scatchard analysis. This study suggests that unlike the corresponding Tyr(421)of the common human beta chain, Tyr(462)of murine Mpl is not required for high-affinity ligand binding, although it may be located in proximity to the ligand binding site.
authors.affiliation: Department of Medicine, University of Washington, Seattle, WA 98195, USA. dfsabath@u.washington.edu
authors.forename: D F
authors.initials: DF
authors.lastname: Sabath
authors.forename: N
authors.initials: N
authors.lastname: Lin
authors.forename: D E
authors.initials: DE
authors.lastname: Sabath
authors.forename: K
authors.initials: K
authors.lastname: Kaushansky
authors.forename: V C
authors.initials: VC
authors.lastname: Broudy
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002455:Cell Division / Q000235:genetics; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins; D017354:Point Mutation; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018121:Receptors, Cytokine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D053628:Receptors, Thrombopoietin; D017386:Sequence Homology, Amino Acid; D013926:Thrombopoietin / Q000378:metabolism*; D014162:Transfection; D014443:Tyrosine / Q000737:chemistry
chemical_list: D017931:DNA Primers; D008024:Ligands; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D018121:Receptors, Cytokine; D053628:Receptors, Thrombopoietin; C082218:MPL protein, human; D014443:Tyrosine; D013926:Thrombopoietin