Contacts between membrane proximal regions of the PDGF receptor ectodomain are required for receptor activation but not for receptor dimerization

Abstract

The mechanism of PDGF-receptor beta (PDGFRbeta) activation was explored by analyzing the properties of mutant receptors designed based on the crystal structure of the extracellular region of the related receptor tyrosine kinase KIT/stem cell factor receptor. Here, we demonstrate that PDGF-induced activation of a PDGFRbeta mutated in Arg-385 or Glu-390 in D4 (the fourth Ig-like domain of the extracellular region) was compromised, resulting in impairment of a variety of PDGF-induced cellular responses. These experiments demonstrate that homotypic D4 interactions probably mediated by salt bridges between Arg-385 and Glu-390 play an important role in activation of PDGFRbeta and all type III receptor tyrosine kinases. We also used a chemical cross-linking agent to covalently cross-link PDGF-stimulated cells to demonstrate that a Glu390Ala mutant of PDGFRbeta undergoes typical PDGF-induced receptor dimerization. However, unlike WT PDGFR that is expressed on the surface of ligand-stimulated cells in an active state, PDGF-induced Glu390Ala dimers are inactive. Although the conserved amino acids that are required for mediating D4 homotypic interactions are crucial for PDGFRbeta activation, these interactions are dispensable for PDGFRbeta dimerization. Moreover, PDGFRbeta dimerization is necessary but not sufficient for tyrosine kinase activation.

Fig. 1.

Homology modeling of membrane proximal region of PDGFRs. The membrane proximal region of PDGFRβ ectodomain is shown as ribbons with transparent molecular surface (D4 colored in gold and D5 in magenta) (Left). A closer view (Right) of the D4–D4 interface of two neighboring PDGFRβ molecules demonstrates that interactions between D4 are mediated by residues Arg-385 and Glu-390 projected from two adjacent EF loop. Key amino acids are labeled and shown as a stick model.

Fig. 2.

PDGF-induced PDGFR activation is compromised by mutations in D4. (A) PDGFRα/β−/− MEFs expressing WT PDGFRβ and various PDGFRβ D4 mutants (R385A, E390A, RE/AA, and RKE/AAA) were serum-starved overnight and stimulated with indicated PDGF concentration for 5 min at 37°C. Cell lysates were immunoprecipitated with anti-PDGFR antibodies, followed by immunoblotting with antiphosphotyrosine antibodies 4G10. Membranes were stripped off and reblotted with anti-flag tag antibodies. (B) PDGFRα/β−/− MEFs expressing WT (■), R385A (▴), E390A (▾), and RE/AA (♦) PDGFRβ were incubated with 5 ng/ml ¹²⁵I-PDGF at 4°C for 90 min in the presence of increasing concentration of native PDGF. Cell-associated ¹²⁵I-PDGF were collected with 0.5 M NaOH solution and quantitated with a scintillation counter. The IC₅₀ values were determined by curve fitting with Prism4 (GraphPad). (C) MEFs expressing WT, R385A, E390A, RE/AA, and RKE/AAA PDGFR were serum-starved overnight and lysed. Cell lysates were immunoprecipitated with anti-PDGFR antibodies, and immunopellets were subjected to in vitro autophosphorylation assay in the absence (−) or presence (+) of 1 mM ATP and 10 mM Mg²⁺ for 10 min at room temperature. Pellets were resolved with SDS/PAGE followed by immunoblotting with antiphosphotyrosine antibodies and antiflag antibodies.

Fig. 3.

PDGF-stimulated PDGFRβ mutated in D4 are expressed on the cell surface in the form of inactive dimers. PDGFRα/β−/− MEFs expressing WT PDGFRβ or E390A mutant were serum-starved overnight, followed by incubation with the indicated amount of PDGF at 4°C for 90 min. After removing the unbound ligand, cells were incubated with 0.5 mM DSS in PBS for 30 min. Cell lysates were immunoprecipitated with anti-PDGFR antibodies, and immunopellets were analyzed by SDS/PAGE and immunoblotted with anti-flag antibodies (Left) and antiphosphotyrosine antibodies (Right), respectively.

Fig. 4.

PDGF-induced cellular responses are compromised by mutations in PDGFRβ D4 mutant. (A) Cells were stimulated with 10 ng/ml PDGF for 5 min, as described above. Total lysates were subjected to SDS/PAGE and analyzed by immunoblotting with antiphospho-MAPK, MAPK, phospho-Akt, and Akt antibodies. (B) Cells seeded on coverslips were serum-starved for 16 h and either left untreated or stimulated with 50 ng/ml PDGF for 2, 5, 10, or 30 min. Coverslips were stained with FITC-phalloidin, and the percentage of cells showing dorsal actin rings were quantitated and presented linearly in C. WT (■), R385A (□), E390A (▴), and RE/AA (Δ).

Fig. 5.

Altered kinetics of the ligand–receptor complex internalization and receptor degradation in cells expressing PDGFR D4 mutants. (A) Cells were incubated with 5 ng/ml ¹²⁵I-PDGF for 90 min, and unbound ligand was removed. Cells were transferred to 37°C for the indicated time intervals. Cell surface receptor-associated (■), internalized (▴), and degradation product (▾) of ¹²⁵I-PDGF were determined and expressed as percentage of total binding at t = 0 min. Each point was performed in triplicates and presented as mean ± standard error. (B) Cells were pretreated with 10 μg/ml cycloheximide for 30 min before PDGF stimulation. PDGF (20 ng/ml) was added for the indicated time. Cell lysates were immunoprecipitated with PDGFR antibodies and immunoblotted with anti-flag antibodies. Total cell lysates were immunoblotted with anti-actin antibodies as control.