Constitutive complex formation between FGFR1 and FRS2α. (A) L6 myoblasts (L6 Flg) stably transfected with FGFR1 were stimulated with aFGF and heparin (+) (100 ng/ml and 5 μg/ml, respectively) or with medium alone (−) for 5 min at 37°C. The lysates were immunoprecipitated (IP) with anti-FGFR1 or anti-Grb2 antibodies. The immunoprecipitates were analyzed by SDS-PAGE followed by immunoblotting (IB) with anti-P-Tyr (left) or anti-FRS2α (right) antibodies. (B) NIH 3T3 (mouse fibroblast), L6 (rat myoblast), and PC12 (rat pheochromocytoma) cells stably transfected with FGFR1 were stimulated (+) with aFGF and heparin (100 ng/ml and 5 μg/ml, respectively) or with medium alone (−) for 5 min at 37°C. The lysates were immunoprecipitated with anti-FGFR1 or anti-Grb2 antibodies. The immunoprecipitates were analyzed by SDS-PAGE followed by immunoblotting with anti-FRS2α antibodies. (C) 293 cells were transiently transfected with FRS2α alone (lane 1), together with the wild-type FGFR1 (lane 2) or with the kinase-inactive FGFR1 (KM; lane 3). The lysates were immunoprecipitated with anti-FGFR1 antibodies, followed by SDS-PAGE and immunoblotting with anti-FRS2α (top), anti-P-Tyr (middle), or anti-FGFR1 antibodies (bottom).

Association between FGFR1 and the PTB domain of FRS2α in transfected cells and in vitro. (A) N-myristylation of FRS2α enhances complex formation with FGFR1 in transfected cells. 293 cells were transiently transfected with wild-type FGFR1 (lanes 1 to 4) or the kinase-inactive (KM; lanes 5 to 8) FGFR1 mutant together with the full-length FRS2α (lanes 2 and 6), the N-terminal region of FRS2α consisting of the N-myristylation signal and the PTB domain (lanes 3 and 7), or the G2A mutant with a mutation in the N-myristylation sequence and the PTB domain of FRS2α (lanes 4 and 8). The expression level of FGFR1 and its kinase activity were analyzed by immunoprecipitation (IP) with anti-FGFR1 followed by immunoblotting (IB) with anti-FGFR1 and anti-P-Tyr antibodies. The expression levels of the various FRS2α constructs were analyzed by immunoprecipitation and immunoblotting with anti-FRS2 antibodies which were directed against the N-terminal region of FRS2α. The interactions between wild-type FGFR1 or its kinase-inactive mutant and the various FRS2 mutants were analyzed by immunoprecipitating the lysates with anti-FGFR1 antibodies followed by immunoblotting with anti-FRS2 antibodies. (B) The PTB domains of the FRS2 proteins interact with FGFR1. Lysates from 293 cells transiently transfected with the wild-type (lanes 1 to 3) or kinase-inactive (lanes 4 to 6) FGFR1 were subjected to a pulldown assay with the PTB domains of FRS2α (lanes 2 and 5) or FRS2β (lanes 3 and 6) expressed as GST fusion proteins and immobilized on glutathione-agarose beads. GST alone was used as a control (lanes 1 and 4). The specifically bound proteins were eluted and resolved by SDS-PAGE, and the presence of FGFR1 was detected by immunoblotting with anti-FGFR1 antibodies. (C) Alignment of the PTB domains of FRS2α and FRS2β by the Clustal method with DNASTAR. The two PTB domains exhibit 75% sequence identity. Identical residues are shaded.

The PTB domains of the FRS2 proteins bind to the juxtamembrane region of FGFR1. (A) Mapping of the binding site of FRS2α on FGFR1 by deletion mutagenesis. Deletion mutagenesis of FGFR1 was carried out by insertion of the DNA sequence encoding the HA epitope, followed by the stop codon after codons for amino acids 410, 420, 432, 445, 469, 578, 594, and 765 (lanes 1 to 8). Each of these deletion mutants and the full-length FGFR1 were tagged with the HA epitope at the C-terminal end. 293 cells were transiently transfected with FRS2α either alone (lane 10) or together with each of the HA-tagged FGFR1s (wild type [lane 9] or the deletion mutants [lanes 1 to 8]). The lysates were subjected to immunoprecipitations (IP) with anti-HA (top) or anti-FRS2α (bottom) antibodies followed by SDS-PAGE and immunoblotting (IB) with anti-HA antibodies. (B) Alignment of amino acid sequences of the juxtamembrane domains of FGF receptors. The primary sequences of human FGFR1, FGFR2, FGFR3, and FGFR4 were aligned by the Clustal method with DNASTAR. Identical residues are shaded. ∗, amino acids critical for the interaction between the PTB domains of the FRS2 proteins and FGFR1. (C) FRS2α binds to the juxtamembrane region of FGFR1. 293 cells were transiently transfected with FGFR1. The lysates were subjected to a pulldown assay with the GST-PTB-FRS2α beads either alone (lane 1) or in the presence of the recombinant protein of the juxtamembrane of FGFR1 (amino acids 399 to 470) expressed as a histidine-tagged fusion protein (1 μM; lane 2), a bovine serum albumin control (1 μM; lane 3), FGFR1 juxtamembrane peptide (MAVHKLAKSIPLRRQVTVSADS; 1 and 10 μM; lanes 4 and 5, respectively), FGFR1 C-terminal peptide (PRHPAQLANGGKLRR; 1 and 10 μM; lanes 6 and 7, respectively), and the peptide solvent dimethyl sulfoxide (1 and 10 μl; lanes 8 and 9, respectively). The specifically bound proteins were resolved by SDS-PAGE, and the presence of FGFR1 was detected by immunoblotting with anti-FGFR1 antibodies. Similar results were observed when the PTB domain of FRS2α was used for the pulldown experiments (data not shown). (D) The interaction between the PTB domain of FRS2 and the juxtamembrane region of FGFR1 is direct. Equivalent amounts of the PTB domain of FRS2α expressed as a GST fusion protein (GST-PTB-FRS2α) and immobilized on glutathione-agarose beads were incubated with increasing concentrations (2.5, 5, 10 and 20 μM; lanes 5 to 8, respectively) of the juxtamembrane region of FGFR1 expressed as a hexahistidine-tagged fusion protein (Juxt. D; amino acids 399 to 470). As a control, similar concentrations of the Juxt. D protein were incubated with GST beads (lanes 1 to 4). Specifically bound proteins were eluted and resolved by SDS-PAGE, followed by detection and Western blotting with antibodies against the hexahistidine epitope.

FRS2 proteins bind to similar regions on FGFR1 via their PTB domains. (A) Binding of FRS2 proteins to deletion mutants of FGFR1. HA-tagged deletion mutants of FGFR1 truncated at amino acids 410, 420, 421, 423, 427, or 432 (lanes 1 to 6, respectively) were cotransfected with FRS2α expression plasmid in 293 cells. The lysates were subjected to immunoprecipitations (IP) with anti-HA or anti-FRS2α antibodies, followed by SDS-PAGE and immunoblotting (IB) with anti-HA antibodies. The same series of deletion mutants were expressed in 293 cells, and the lysates were subjected to a pulldown assay with GST-PTB-FRS2α or GST-PTB-FRS2β beads. The specifically bound proteins were eluted and resolved by SDS-PAGE, and the presence of various deletion mutants of FGFR1 was detected by immunoblotting with anti-HA antibodies. (B) Alanine scanning of the PTB domain binding site on human FGFR1. 293 cells were transfected with FGFR1 mutants, each with a single alanine substitution at amino acids 419 to 430 (lanes 1 to 12, respectively), or with wild-type FGFR1 (lane 13). The expression levels of the alanine mutants were shown to be equivalent by immunoblotting total cell lysate with anti-FGFR1 antibodies. The lysates were subjected to a pulldown assay with GST-SH2-PLC-γ or GST-PTB-FRS2α beads. The specifically bound proteins were eluted and resolved by SDS-PAGE, and the presence of alanine mutants of FGFR1 was detected by immunoblotting with anti-FGFR1 antibodies. (C) The PTB domains of the FRS2 proteins bind to similar binding sites on FGFR1. 293 cells were transiently transfected with FGFR1 mutants, each with a single alanine substitution at amino acids 419 to 430 (lanes 1 to 12, respectively), or with wild-type FGFR1 (lane 13). The expression levels of the alanine mutants were shown to be equivalent by immunoblotting the total cell lysate with anti-FGFR1 antibodies. The lysates were subjected to a pulldown assay with GST-PTB-FRS2β beads. The specifically bound proteins were resolved by SDS-PAGE, and the presence of the alanine mutants of FGFR1 was detected by immunoblotting with anti-FGFR1 antibodies.

Alanine substitutions of amino acids in the FRS2 binding site on FGFR1 abrogates FRS2 phosphorylation and MAPK activation. (A) 293 cells were transfected with FGFR1 with combined alanine substitutions at amino acids 422, 423, and 425 (lane 3); 419, 422, 423, and 425 (lane 4); 427 and 429 (lane 5); 419, 422, 423, 425, 427, and 429 (lane 6); and wild-type FGFR1 (lane 1) or its kinase-inactive mutant (lane 2) as a control. The expression of each receptor was shown to be equivalent by immunoblotting (IP) the total cell lysate with anti-FGFR1 antibodies. The lysates were subjected to a pulldown assay with GST-SH2-PLC-γ, GST alone, or GST-PTB-FRS2α beads. The specifically bound proteins were resolved by SDS-PAGE, and the presence of the alanine mutants of FGFR1 was detected by immunoblotting with anti-FGFR1 antibodies. (B) 293 cells were transfected with FRS2α (lanes 1 to 7) and FGFR1 with combined alanine substitutions as described for panel A. The expression levels of the FGF receptors were shown to be equivalent by immunoblotting total cell lysate with anti-FGFR1 and anti-FRS2α antibodies. The lysates were subjected to immunoprecipitation (IP) with anti-FGFR1 antibodies or anti-FRS2α antibodies followed by immunoblotting with anti-P-Tyr antibodies. (C) 293 cells were transfected with FRS2α and ERK1 (lanes 1 to 5) and FGFR1 with the indicated combined alanine substitutions (lanes 1 to 4) or the wild-type FGFR1 (lane 5). The cells were lysed, and equivalent amounts of the total cell lysates were resolved by SDS-PAGE and immunoblotted with anti-FRS2, anti-FGFR1, anti-P-Tyr, anti-MAPK, or anti-phospho-MAPK (P-MAPK) antibodies. (D) Densitometric quantitation of MAPK phosphorylation. Standard deviations from two separate experiments are shown. WT, wild type.

FRS2 binds via its PTB domain to Y490 site on NGF receptor in a phosphorylation-dependent manner. (A) Ternary complex of FRS2α–Grb2-Sos1 and tyrosine-phosphorylated TrkA. PC12 cells stably transfected with TrkA and FRS2α were stimulated (+) with 100 ng of NGF/ml or with medium alone (−) for 5 min at 37°C. The lysates were subjected to immunoprecipitations (IP) with anti-Grb2, anti-Sos1, anti-Shc, anti-FRS2, or anti-TrkA antibodies. The immunoprecipitates were resolved by SDS-PAGE followed by immunoblotting (IB) with anti-P-Tyr antibodies. (B) FRS2α binds to tyrosine-phosphorylated TrkA. PC12 cells stably transfected with TrkA and FRS2α were stimulated with 100 ng of NGF/ml or with medium alone for 5 min at 37°C. The lysates were subjected to immunoprecipitations with anti-FRS2α or anti-TrkA antibodies. The immunoprecipitates were resolved by SDS-PAGE followed by immunoblotting with anti-TrkA antibodies. (C) FRS2α binds to tyrosine-phosphorylated TrkA via the PTB domain. PC12 cells stably transfected with TrkA and FRS2α were stimulated with 100 ng of NGF/ml or medium alone for 5 min at 37°C. The lysates were subjected to a pulldown assay with immobilized GST (control), GST-PTB-FRS2α, or GST-PTB-FRS2β. The specifically bound proteins were resolved by SDS-PAGE, and the associated TrkA was detected by immunoblotting with anti-TrkA antibodies. (D) The PTB domains of FRS2 proteins bind to the NPQpY sequence of TrkA. PC12 cells stably transfected with TrkA and FRS2α were stimulated with 100 ng of NGF/ml or medium alone for 5 min at 37°C. The lysates were subjected to a pulldown assay with immobilized GST-PTB-FRS2β. The phosphopeptide encompassing the NPQpY motif of TrkA was added at 10 or 1 μM. A control peptide corresponding to the C-terminal region of human FGFR1 (C-ter-P) was similarly added at 10 or 1 μM. The specifically bound proteins were resolved by SDS-PAGE, and the associated TrkA was detected by immunoblotting with anti-TrkA antibodies.

FGFR1 and TrkA interact with similar sites on the PTB domain of FRS2. (A) 293 cells were transiently transfected with FGFR1. The lysates were subjected to a pulldown assay with the GST-PTB-FRS2β beads either alone (lane 1) or in the presence of the recombinant protein of the juxtamembrane of FGFR1 (amino acids 399 to 470) expressed as a histidine-tagged fusion protein (1 μM; lane 2), bovine serum albumin (control; 1 μM; lane 3), FGFR1 juxtamembrane peptide (1 and 10 μM; lanes 4 and 5, respectively), TrkA phosphopeptide (1 and 10 μM; lanes 6 and 7, respectively), and a control peptide (1 and 10 μM; lanes 8 and 9, respectively). The specifically bound proteins were resolved by SDS-PAGE, and the presence of FGFR1 was detected by immunoblotting (IB) anti-FGFR1 antibodies. (B) A similar experiment was carried out with lysates of PC12 cells stably transfected with TrkA and FRS2α. Cells were stimulated (+) with 100 ng of NGF/ml or medium alone (−) for 5 min at 37°C. The specifically bound proteins were resolved by SDS-PAGE, and the associated TrkA was detected by immunoblotting with anti-TrkA antibodies.

Inhibition of TrkA-mediated FRS2α phosphorylation by overexpression of kinase-inactive FGFR1 mutant. 293 cells were transfected with constant levels of TrkA and FRS2α expression plasmids (lanes 1 to 6; 1 and 0.5 μg of the respective plasmids) and increasing levels of expression plasmid encoding the kinase-inactive FGFR1 mutant (0, 0.5, 1, 3, 6, and 18 μg of plasmids in lanes 1 to 6, respectively). The cells were lysed, and equivalent amounts of the total cell lysates were resolved by SDS-PAGE and immunoblotted (IB) with anti-TrkA, anti-FRS2α, or anti-FGFR1 antibodies, respectively. Immunoprecipitation (IP) of FRS2α was followed by immunoblotting with anti-P-Tyr antibodies.