Figure 1.
Schematic diagram of G-CSFR forms.
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.
Figure 2.
Leucine residues in the STPQLL dileucine motif do not mediate G-CSFR internalization.
Mutations in the cytoplasmic dileucine motif of the G-CSFR that is identical to gp130 were introduced by substituting Ala for either Leu753 (L753A) or Leu754 (L754A) or both Leu753 and Leu754 (L753/754A). Binding and internalization of [125I]-G-CSF were analyzed in COS-7 cells transfected with the WT G-CSFR, Δ716, or G-CSFR forms with Leu to Ala mutations in the dileucine motif. Average data±S.E.M (n = 4) are shown and are expressed as the percent of initial binding at time 0 at 4°C.
Figure 3.
Binding and internalization of [125I]-G-CSF in cells transfected with WT, Y729F or Y764F G-CSFR forms.
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).
Figure 4.
Effect of attachment of an YTRF internalization motif to the Δ716 G-CSFR on ligand/receptor internalization and degradation.
(A) Cells expressing the WT, Δ716, or Δ716-YTRF G-CSFR forms were incubated with [125I]-labeled G-CSF and subjected to temperature shift and acid washing to determine surface-bound and internalized ligand. Average data±S.E.M (n = 4) are shown as a percentage of initial binding of G-CSF at time 0 at 4°C. (B) Cells expressing the WT, Δ716 or Δ716-YTRF G-CSFR forms were metabolically labeled with [35S]-methionine and [35S]-cysteine for 1 h, washed then incubated in media containing 1 nM G-CSF and unlabeled amino acids at 37°C. At the indicated times, the cells were lysed, immunoprecipitated with antibody recognizing the N-terminus of the human G-CSFR and analyzed by SDS-PAGE. The blots were subjected to densitometry and the average pixels±S.E.M are shown (n = 2). A representative blot from two independent experiments is shown.