Controlled dimerization of ErbB receptors provides evidence for differential signaling by homo- and heterodimers

Abstract

The four members of the ErbB family of receptor tyrosine kinases are involved in a complex array of combinatorial interactions involving homo- and heterodimers. Since most cell types express more than one member of the ErbB family, it is difficult to distinguish the biological activities of different homo- and heterodimers. Here we describe a method for inducing homo- or heterodimerization of ErbB receptors by using synthetic ligands without interference from the endogenous receptors. ErbB receptor chimeras containing synthetic ligand binding domains (FK506-binding protein [FKBP] or FKBP-rapamycin-binding domain [FRB]) were homodimerized with the bivalent FKBP ligand AP1510 and heterodimerized with the bifunctional FKBP-FRB ligand rapamycin. AP1510 treatment induced tyrosine phosphorylation of ErbB1 and ErbB2 homodimers and recruitment of Src homology 2 domain-containing proteins (Shc and Grb2). In addition, ErbB1 and ErbB2 homodimers activated downstream signaling pathways leading to Erk2 and Akt phosphorylation. However, only ErbB1 homodimers were internalized upon AP1510 stimulation, and only ErbB1 homodimers were able to associate with and induce phosphorylation of c-Cbl. Cells expressing AP1510-induced ErbB1 homodimers were able to associate with and induce phosphorylation of c-Cbl. Cells expressing AP1510-induced ErbB1 homodimers were able to form foci; however, cells expressing ErbB2 homodimers displayed a five- to sevenfold higher focus-forming ability. Using rapamycin-inducible heterodimerization we show that c-Cbl is unable to associate with ErbB1 in a ErbB1-ErbB2 heterodimer most likely because ErbB2 is unable to phosphorylate the c-Cbl binding site on ErbB1. Thus, we demonstrate that ErbB1 and ErbB2 homodimers differ in their abilities to transform fibroblasts and provide evidence for differential signaling by ErbB homodimers and heterodimers. These observations also validate the use of synthetic ligands to study the signaling and biological specificity of selected ErbB dimers in any cell type.

FIG. 1

Synthetic ligands for ErbB dimerization. The ligand-binding domains FKBP12 and FRB (A) were subcloned as single or double copies to generate the expression vectors shown in panel B. The intracellular domains of ErbB receptors were PCR amplified and subcloned into the expression vectors as described in Materials and Methods. The addition of AP1510 to cells expressing FKBP-fused ErbB receptors (C) will result in the generation of homodimers (D), while the addition of rapamycin to cells coexpressing the ErbB1-FKBP chimera (p75.B1.F1.HA) and ErbB2-FRB chimera (p75.B2.R1.Flag) will result in a p75.B1.F1.HA-p75.B2.R1.Flag heterodimer (D).

FIG. 2

Dimerization of ErbB1 cytoplasmic domain with synthetic ligands results in a dose-dependent stimulation of receptor and substrate phosphorylation. (A) Rat1 fibroblasts expressing ErbB1 fused to one copy of FKBP (p75.B1.F1.HA) were stimulated with increasing amounts of AP1510 (nanomolar) (lanes 2 to 6) or FK506 (lanes 7 and 8) for 15 min or stimulated with 50 ng of EGF per ml for 5 min. Cell lysates were collected, and 45 μg of protein was resolved and immunoblotted with anti-phosphotyrosine (anti-pTyr) antibodies. The blot was stripped and reprobed with anti-Shc antibodies, and the p46 and p52 isoforms of Shc and other cellular proteins that were tyrosine phosphorylated by ligand stimulation are indicated by asterisks. (B) Seven hundred micrograms of lysate was used for immunoprecipitation with anti-HA antibodies and immunoblotted with anti-pTyr (upper panel). The anti-pTyr blot was subsequently stripped and reprobed with anti-EGFR antibodies (lower panel).

FIG. 3

Synthetic ligand-activated receptors are competent in recruiting signaling molecules and activating a downstream target. (A) HA epitope-containing proteins were immunoprecipitated (IP) (lanes 4 to 7) from 500 μg of cell lysate, and the membrane was probed with antibodies against anti-pTyr (first panel), anti-Grb2 (second panel), or anti-Shc (third panel). The blot from the first panel was stripped and reprobed with anti-EGFR (fourth panel). Normal mouse serum (NMS; lanes 1 to 3) was used as a nonspecific control. (B) Total cell lysates from p75.B1.F1.HA or Rat1 cells stimulated with AP1510 (nanomolar) were immunoblotted with anti-Erk2 antibodies.

FIG. 4

Synthetic ligands can activate other members of the ErbB family, and the activated receptors retain their kinase specificity. (A) Total cell lysates from cells stimulated with AP1510 (nanomolar) or EGF (50 ng/ml) were resolved and blotted with anti-pTyr antibodies. The positions of p75.B1.F2.HA, p75.B2.F2.HA, and Shc are indicated. (B) The top two-thirds of the blot in panel A was stripped and reprobed with anti-phospho-473 Akt antibody (upper panel), and the blot was restripped and blotted with anti-Akt antibody (lower panel). (C) The lower third of the blot in panel A was stripped and reprobed with anti-Erk2. (D) c-Cbl was immunoprecipitated (IP) from 750 μg of lysate and immunoblotted with anti-pTyr antibodies (upper panel), and the blot was subsequently stripped and reprobed with anti-Cbl antibodies (lower panel). The position of the coimmunoprecipitated p75.B1.F2.HA is indicated.

FIG. 5

ErbB1 but not ErbB2 chimeras are internalized after AP1510 stimulation. Cell lines expressing either ErbB1 or ErbB2 chimeras were stimulated with carrier alone (ethanol [EtOH]) or with 500 nM AP1510 for the indicated lengths of time. The cell surface proteins were subsequently biotinylated by incubating with NHS-S-S-Biotin at 4°C for 1 h. The biotinylated proteins were immunoprecipitated (IP) with Sepharose-conjugated NeutrAvidin beads and immunoblotted with either anti-HA (A) or anti-beta1 integrin (B) antibodies.

FIG. 6

Activation of ErbB homodimers results in induction of cell cycle progression. The parental Rat1 cells or cell lines expressing different ErbB chimeras were serum starved, stimulated, and analyzed by FACS as described in Materials and Methods. The graph shows the fold increase ± standard deviations (error bars) in the percentages of cells that have left the G₀-G₁ stage of the cell cycle. The parental Rat1 cells (open squares), cells expressing ErbB1 with one copy FKBP (p75.B1.F1) (closed squares), cells expressing ErbB1 with two copies of FKBP (p75.B1.F2) (open circles), and cells expressing ErbB2 with two copies of FKBP (p75.B2.F2) (open triangles) were used for the analysis. EGF was used at a 10-ng/ml concentration.

FIG. 7

ErbB1 and ErbB2 homodimers differ in their abilities to induce focus formation in rat fibroblasts. Rat1 cells were infected with retroviruses containing different ErbB fusions. The cells were maintained in media containing the indicated amounts of AP1510 (nanomolar) for 14 days, fixed, and stained with Giemsa stain. One set of infected cells were trypsinized, and 1/10 of the cells were replated in media containing G418 to ascertain the number of CFU infected per plate.

FIG. 8

Both ErbB1 and ErbB2 homodimers are able to induce reversible morphological transformation of fibroblasts. The cells were plated in the presence of AP1510 (concentrations shown are nanomolar) and allowed to grow for 48 h. The morphologies of the cells were recorded (a to i and m to o). The cells in panels i and o were trypsinized and replated either in media without (j and p) or with AP1510 (k, l, q, and r). P75.F1 (Fig. 1B) corresponds to cells expressing the chimera without the ErbB cytoplasmic domain.

FIG. 9

Synthetic ligand-induced heterodimerization between ErbB receptors. (A) P75.B1.F2.HA- and p75.B2.F2.HA-expressing cells were transfected with p75.kdB1.R1.Flag, and stable pools containing B1.F2.HA plus kdB1.R1.Flag and B2.F2.HA plus kdB1.R1.Flag were selected. The relative expression levels of each chimera in both pools were examined by immunoblotting cell lysates with either anti-HA or anti-Flag antibodies. The parental Rat1 cell lysate was used as a negative control. The pools were stimulated with either AP1510 (concentrations shown are nanomolar) (lanes 2, 3, 8, and 9) or with rapamycin (nanomolar) (lanes 4 to 6 and 10 to 12), immunoprecipitated (IP) with either anti-HA (B) or anti-Flag (C) antibodies, and immunoblotted with anti-pTyr antibodies. The blots in the upper panels (B and C) were stripped and reprobed with anti-HA and anti-Flag antibodies, respectively (lower panels).

FIG. 10

c-Cbl can differentiate ErbB1 in homodimers from ErbB1 in ErbB1-ErbB2 heterodimers. COS7 cells were cotransfected with p75.kdB1.R1.Flag and p75.B1.F2.HA (lanes 1 to 3 and 7 to 9) or p75.B2.F2.HA (lanes 4 to 6 and 10 to 12). The cells were stimulated with indicated amounts of rapamycin (nanomolar), and 1.5 mg of lysate was used for immunoprecipitation (IP) with anti-Flag antibodies (lanes 1 to 6) or anti-Cbl antibodies (lanes 7 to 12) and immunoblotted with anti-pTyr antibodies (upper panel). The c-Cbl portion of the blot (lanes 7 to 12) was stripped and reprobed with anti-Cbl antibodies (lower panel).