Divergent Pathways in COS-7 Cells Mediate Defective Internalization and Intracellular Routing of Truncated G-CSFR Forms in SCN/AML

Expression of truncated G-CSFR forms in patients with SCN/AML induces hyperproliferation and prolonged cell survival. Previously, we showed that ligand internalization is delayed and degradation of truncated G-CSFR forms is defective in patients with SCN/AML.In this study, we investigated the potential roles of dileucine and tyrosine-based motifs within the cytoplasmic domain of the G-CSFR in modulating ligand/receptor internalization. Using standard binding assays with radiolabeled ligand and COS-7 cells, substitutions in the dileucine motif or deletion of tyrosine residues in the G-CSFR did not alter internalization. Attachment of the transferrin receptor YTRF internalization motif to a truncated G-CSFR form from a patient with SCN/AML corrected defective internalization, but not receptor degradation suggesting that receptor internalization and degradation occur independently via distinct domains and/or processes.Our data suggest that distinct domains within the G-CSFR mediate separate processes for receptor internalization and degradation. Our findings using standard binding assays differ from recently published data utilizing flow cytometry

Spatiotemporal Quantification of Local Drug Delivery Using MRI

Controlled release formulations for local, in vivo drug delivery are of growing interest to device manufacturers, research scientists, and clinicians; however, most research characterizing controlled release formulations occurs in vitro because the spatial and temporal distribution of drug delivery is difficult to measure in vivo. In this work, in vivo magnetic resonance imaging (MRI) of local drug delivery was performed to visualize and quantify the time resolved distribution of MRI contrast agents. Three-dimensional maps (generated from -weighted images with varied ) were processed using noise-reducing filtering. A segmented region of contrast, from a thresholded image, was converted to concentration maps using the equation , where and are the precontrast and postcontrast map values, respectively. In this technique, a uniform estimated value for was used. Error estimations were performed for each step. The practical usefulness of this method was assessed using comparisons between devices located in different locations both with and without contrast. The method using a uniform , requiring no registration of pre- and postcontrast image volumes, was compared to a method using either affine or deformation registrations

Src family kinases are important negative regulators of G-CSF-dependent granulopoiesis

Granulocyte colony-stimulating factor (G-CSF) is the principal cytokine regulating granulopoiesis. Truncation mutations of the G-CSF receptor (G-CSFR) are associated with the development of acute myeloid leukemia in patients with severe congenital neutropenia. Although increased proliferative signaling by a representative G-CSFR truncation mutation (termed d715) has been documented, the molecular basis for this hyperproliferative phenotype has not been fully characterized. Given the accumulating evidence implicating Src family kinases in the transduction of cytokine receptor signals, the role of these kinases in the regulation of G-CSF signaling was examined. We show that Hck and Lyn, Src family kinases expressed in myeloid cells, are negative regulators of granulopoiesis that act at distinct stages of granulocytic differentiation. Whereas Hck regulates the G-CSF-induced proliferation of granulocytic precursors, Lyn regulates the production of myeloid progenitors. Interestingly, d715 G-CSFR myeloid progenitors were resistant to the growth-stimulating effect of treatment with a Src kinase inhibitor. Together, these data establish Lyn and Hck as key negative regulators of granulopoiesis and raise the possibility that loss of Src family kinase activation by the d715 G-CSFR may contribute to its hyperproliferative phenotype

Schematic diagram of G-CSFR forms.

<p>The extracellular (EX), transmembrane (TM), and intracellular (ID) domains of the various G-CSFR forms are shown with the conserved box 1, 2, and 3 regions indicated. The full-length wild-type (WT) G-CSFR contains four cytoplasmic tyrosine residues at a.a. positions 704, 729, 744, and 764. The location of the dileucine motif in the WT G-CSFR at residues 749-754, which is deleted in Δ716, where Leu to Ala substitutions were introduced to generate the L753A, L754A, and the L753/754A G-CSFR is shown. Tyr to Phe substituitions at either Tyr⁷²⁹, Tyr⁷⁴⁴, or Tyr⁷⁶⁴ were also introduced and the corresponding G-CSFR mutants designated Y729F, Y744F, and Y764F, respectively. The Δ716 G-CSFR was isolated from a patient with SCN/AML and contains a premature stop codon resulting in a truncated G-CSFR. The Δ716 -YTRF mutant was generated by attachment of the transferrin receptor YTRF internalization motif 5′ to the stop codon of the Δ716 G-CSFR.</p

Binding and internalization of [¹²⁵I]-G-CSF in cells transfected with WT, Y729F or Y764F G-CSFR forms.

<p>Mutations in the G-CSFR were introduced by subsititution of Phe for either Tyr⁷²⁹ (Y729F) or Tyr⁷⁶⁴ (Y764F). COS-7 cells were transfected with each G-CSFR form and ligand binding analyzed. Surface bound ligand was quantitated by acid stripping in 0.5M NaCl (pH 1.0). Internalized ligand was measured after lysis of the cells in 1M NaOH. Data are expressed as a percentage of initial binding at time 0 at 4°C. Values represent the average ±S.E.M (n = 2).</p