GRP78 and Cripto form a complex at the cell surface and collaborate to inhibit transforming growth factor beta signaling and enhance cell growth

Abstract

Cripto is a multifunctional cell surface protein with important roles in vertebrate embryogenesis and the progression of human tumors. While Cripto has been shown to modulate multiple signaling pathways, its binding partners do not appear to fully explain its molecular actions. Therefore, we conducted a screen aimed at identifying novel Cripto-interacting proteins. This screen led to our identification of glucose-regulated protein 78 (GRP78), an endoplasmic reticulum (ER) chaperone that is also expressed at the surfaces of tumor cells. Here we demonstrate that Cripto and GRP78 interact at the cell surfaces of multiple cell lines and that their interaction is independent of prior association within the ER. Interestingly, short hairpin RNA knockdown of endogenous GRP78 resulted in enhanced transforming growth factor beta (TGF-beta) signaling, indicating that like Cripto, GRP78 inhibits this pathway. We further show that when coexpressed, GRP78 and Cripto collaborate to antagonize TGF-beta responses, including Smad phosphorylation and growth inhibition of prostate cancer cells grown under anchorage-dependent or -independent conditions. Finally, we provide evidence that cells coexpressing GRP78 and Cripto grow much more rapidly in soft agar than do cells expressing either protein individually. Together, our results indicate that these proteins bind at the cell surface to enhance tumor growth via the inhibition of TGF-beta signaling.

FIG. 1.

Identification of novel Cripto binding proteins. 293T cells were transfected with empty vector or Cripto-Flag, subjected to immunoprecipitation on anti-Flag beads, and then eluted with Flag peptide. Cripto-associated proteins were separated by SDS-PAGE and then either silver stained (A) or immunoblotted to nitrocellulose and probed with anti-GRP78 or anti-Cripto antibodies (C) as described in Materials and Methods. Bands a and b in panel A were excised and subjected to mass spectrometric analysis as described in Materials and Methods. Mass fingerprint data corresponding to band a identified as GRP78 are shown in panel B. IB, immunoblot; IP, immunoprecipitate; −, absence of; +, presence of.

FIG. 2.

Cripto binds GRP78 at the cell surface. 293T cells transfected with the indicated constructs (A to D) and P19 cells (D) were labeled with cell-impermeable NHS-LC-biotin. Cell lysates were subjected to immunoprecipitation by using the indicated antibodies and then eluted with Flag peptide (peptide elute) or by heating the beads in sample buffer. Samples were resolved via SDS-PAGE and blotted with avidin-HRP or the indicated antibodies as described in Materials and Methods. In some cases (B and C), 293T cells overexpressing GRP78 were subjected to cell surface biotinylation and then resulting cell lysates were incubated with vector or Cripto-Flag beads. IB, immunoblot; IgG, immunoglobulin G; IP, immunoprecipitate; +, presence of.

FIG. 3.

Cripto and GRP78 colocalize at the cell surface. (A) 293T cells stably infected with vector or GRP78 and Cripto (GRP78 + Cripto) were stained with anti-Cripto antibody (red) and anti-GRP78 antibody (green) as described in Materials and Methods. Scale bar = 20 μm. (B) P19 cells were stained with the same anti-Cripto (red) and anti-GRP78 (green) antibodies as in panel A and anti-pan-cadherin antibody (blue) as described in Materials and Methods. Magnified images corresponding to the boxed region are presented to highlight colocalization of Cripto, GRP78, and cadherin proteins. Scale bar = 10 μm. All cells were analyzed using confocal microscopy as described in Materials and Methods.

FIG. 4.

Targeted reduction of GRP78 expression using RNA interference. HeLa cells were infected with lentivirus containing either GRP78 shRNA (G1) or empty vector and were either left untreated or treated with thapsigargin as indicated. (A) Cell lysates were analyzed by Western blotting using anti-GRP78 or anti-actin antibodies as described in Materials and Methods. (B) Bars represent the number of apoptotic cells per 100 GFP-positive cells as described in Materials and Methods. Error bars indicate standard deviations. (C) Cells infected with vector or G1 shRNA virus were reinfected with virus containing Cripto-Flag. Lysates from these cells were subjected to immunoprecipitation by using anti-Flag beads and then eluted with Flag peptide. Eluted proteins were subjected to Western blot analysis using avidin-HRP, anti-GRP78, and anti-Cripto as described in Materials and Methods. IB, immunoblot; IP, immunoprecipitate; TG, thapsigargin; −, absence of; +, presence of.

FIG. 5.

Inhibition of endogenous GRP78 expression enhances TGF-β-induced Smad2 phosphorylation. HeLa cells were infected with lentivirus containing either shRNA (G1) targeted against GRP78 or empty vector and were then left untreated or treated with 5 μM thapsigargin as indicated. Following overnight thapsigargin treatment, cells were treated with the indicated doses of TGF-β1 (A and B) and then phospho-Smad2 and total Smad2 levels were determined by Western blot analysis as described in Materials and Methods. Alternatively, the same cells treated as indicated were labeled with cell-impermeable biotin with resulting lysates subjected to immunoprecipitation with anti-GRP78 antibodies (anti-KDEL), followed by Western blotting using avidin-HRP (C), or lysates were subjected directly to Western blotting using anti-TβRI, anti-TβRII or anti-actin antibodies (D) as described in Materials and Methods. IP, immunoprecipitate; TG, thapsigargin; −, absence of; +, presence of.

FIG. 6.

GRP78 does not bind directly to TGF-β type I and type II receptors. 293T cells were transfected with p26-Flag, Cripto-Flag, TβRI-HA, or TβRII-His and then subjected to immunoprecipitation using anti-Flag, anti-HA, or anti-His antibodies as indicated. Precipitated proteins were analyzed via Western blotting using avidin-HRP or the indicated antibodies as described in Materials and Methods. IB, immunoblot; IP, immunoprecipitate; +, presence of.

FIG. 7.

Cripto and GRP78 cooperate to inhibit TGF-β signaling. (A) PC3 cells infected with empty vector, GRP78, Cripto, or both were either left untreated or treated with TGF-β1 (10 pM) as indicated. Phospho-Smad2 (pSmad2) and total Smad2 (Smad2) levels were determined by Western blot analysis as described in Materials and Methods. (B) Phospho-Smad2 bands from panel A were quantitated using densitometry and normalized relative to corresponding Smad2 bands as described in Materials and Methods. (C) PC3 cell lysates were subjected to Western blotting using anti-TβRII, anti-TβRI, and anti-actin antibodies as indicated and as described in Materials and Methods. (D) Cells were plated on 96-well plates, and then cell proliferation was measured 8 days later as described in Materials and Methods. Error bars indicate standard deviations. −, absence of; +, presence of.

FIG. 8.

GRP78 and Cripto collaborate to inhibit the antiproliferative effects of TGF-β on anchorage-independent growth of prostate carcinoma cells. PC3 cells infected with vector, GRP78, Cripto or both were grown for 15 days under anchorage-independent conditions in soft agar in the presence of either vehicle or escalating doses of TGF-β1 as described in Materials and Methods. Data are presented as the number of colonies counted within a single field in the absence or presence of the indicated doses of TGF-β1 (A) or as the number of colonies counted in the presence of the indicated TGF-β1 concentrations divided by the number of colonies counted in the absence of TGF-β1 treatment (% basal) (B). (C) Photographs taken from the indicated fields either in the presence of 100 pM TGF-β1 or in its absence. (D) Model illustrating oncogenic function of Cripto/GRP78 complex. TGF-β potently inhibits proliferation of many cell types by signaling via the Smad2/3 pathway (left). Cripto and GRP78 interact to form a complex and act cooperatively to attenuate TGF-β-dependent Smad signaling and growth inhibition. In addition, they independently increase cell proliferation/survival. In the presence of Cripto and GRP78, TGF-β can also increase cellular proliferation (dashed arrow). Error bars indicate standard deviations.