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. 1998 Jul 21;95(15):8985-90.
doi: 10.1073/pnas.95.15.8985.

Ligand-independent activation of platelet-derived growth factor receptor is a necessary intermediate in lysophosphatidic, acid-stimulated mitogenic activity in L cells

A Herrlich  1 , H Daub, A Knebel, P Herrlich, A Ullrich, G Schultz, T Gudermann
Affiliations

Ligand-independent activation of platelet-derived growth factor receptor is a necessary intermediate in lysophosphatidic, acid-stimulated mitogenic activity in L cells

A Herrlich et al. Proc Natl Acad Sci U S A. .

Abstract

Growth factor-derived mitogenic signals from the cell surface are transmitted to the nucleus via receptor tyrosine kinases (RTKs), the adaptor proteins Shc and Grb2, and a Ras-dependent protein kinase cascade that activates the extracellular signal regulated kinase (ERK) subfamily of mitogen-activated protein kinases. ERKs also are activated by hormones that stimulate G protein-coupled receptors (GPCRs). We report here that, in agreement with previous data, the epidermal growth factor receptor (EGFR) is a signaling intermediate in ERK activation by GPCRs. Of import, we show that cross-talk between two classes of surface receptors, RTKs and GPCRs, is a general feature. Lysophosphatidic acid not only induces ligand-independent tyrosine autophosphorylation of EGFR but also of platelet-derived growth factor beta receptor (PDGF-beta-R) as shown by detection of tyrosine phosphorylation and by the use of specific inhibitors of RTKs. The cross-talk appears to be cell type-specific: In L cells that lack EGFR, lysophosphatidic acid-induced Shc and ERK activation is prevented completely by specific inhibition of PDGFR, whereas in COS-7 cells expressing only EGFR, the pathway via EGFR is chosen. In Rat-1 cells, however, that express both EGFR and PDGFR, the EGFR pathway dominates.

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Figures

Figure 1
Figure 1
Effect of receptor tyrosine kinase inhibition on LPA-mediated signaling in Rat-1, COS-7, and L cells. (Left, Rat-1) Quiescent Rat-1 cells were pretreated with either AG1296 or AG1478 at the concentrations indicated and stimulated for 5 min with 1% BSA (control, CO), 10 μM LPA, 10 ng/ml EGF, or 30 ng/ml PDGF-BB as indicated. (Top) Lysates were immunoprecipitated (IP) with anti-EGFR (αEGFR) followed by SDS/PAGE and immunoblotting with anti-phosphotyrosine (αPY). The filter then was reprobed with αEGFR. (Middle) Lysates were immunoprecipitated (IP) with anti-Shc (αShc), and proteins were resolved by SDS/PAGE and immunoblotted with anti-phosphotyrosine (αPY). (Bottom) Lysates from Rat-1 cells were immunoprecipitated (IP) with anti-ERK (αERK). In vitro kinase activity of precipitated endogenous ERK with myelin basic protein (MBP) as substrate was assessed as described in Materials and Methods. Phosphorylated myelin basic protein was visualized by autoradiography. (Center, L cells) After pretreatment with solvent (DMSO) or with 1 μM AG1478, quiescent L cells were stimulated for 5 min with 1% BSA (control, CO), 25 μM LPA, 2 units/ml thrombin receptor peptide (TRP), UV light (100 J/m2 UV C), 10 ng/ml EGF, or 10 ng/ml PDGF-BB. An ERK mobility shift assay was used to detect ERK activation (see Materials and Methods). (Right, COS-7) Confluent serum-starved COS-7 cells were treated with agonists as above. (Top) Lysates from COS-7 cells transiently transfected with either vector alone or dominant negative EGFR (HER-CD533) were immunoprecipitated (IP) with anti-EGFR (αEGFR). After SDS/PAGE, immunoblotting with anti-phosphotyrosine (αPY) was performed. The filter was reprobed with anti-EGFR (αEGFR). (Middle) COS-7 cell lysates were processed as described under Rat-1, middle panel. Shc isoforms with different molecular weights are indicated on the right (p66, p52, p46). The filter was reprobed with anti-Shc (αShc). Two different exposures of the same blot are shown. (Lower) COS-7 cells were transfected with HA-ERK2, pretreated with AG1478, or control-treated. HA-ERK2 was immunoprecipitated with anti-HA (αHA), and kinase activity was assessed with an in vitro ERK assay (see Materials and Methods).
Figure 2
Figure 2
Effect of PKC inhibition on ERK activation via the LPA receptor and the m5-muscarinic receptor (A + B). Effect of βARKct on LPA-mediated ERK activation in L cells (C). Quiescent L cells were pretreated with DMSO or with 100 ng/ml TPA for 16 h (A and B) or not pretreated at all (C). The cells were then stimulated as indicated with agonists [1% BSA (control, CO), 25 μM LPA, or 100 nM carbachol (Cch), 100 ng/ml TPA], lysed and subjected to an ERK mobility shift assay (see Materials and Methods). (A and B) After a 5-min incubation of cells with 1% BSA (control, CO), 25 μM LPA, or 100 ng/ml TPA, ERK activation was compared in L cells stably transfected with vector alone or with the βARKct plasmid (C).
Figure 3
Figure 3
Shc activation in response to LPA in L cells. Confluent L cells were serum-starved in phosphate-deficient medium for 16 h, pulsed with 500 μCi/ml 32P-orthophosphate for 3 h, and stimulated with 1% BSA (15 min, CO), 25 μM LPA (indicated time points), or 10 ng/ml PDGF-BB (15 min). Incorporation of 32P into Shc was measured by autoradiography and densitometric scanning. Shc isoforms of different molecular masses are indicated (p66, p52). The filter was reprobed with anti-Shc (αShc).
Figure 4
Figure 4
Effect of AG1296 and AG1478 on LPA-induced ERK activation in L cells. Confluent, serum-starved L cells were pretreated with DMSO, AG1296, or AG1478 at the indicated concentrations. Before lysis, the cells were incubated for 5 min with 1% BSA (control, CO), 25 μM LPA, or 10 ng/ml PDGF-BB. ERK activation was detected with an ERK mobility shift assay (see Materials and Methods).
Figure 5
Figure 5
LPA signaling to ERK in L cells involves PDGFR. Confluent 15-cm dishes were serum-starved for 16 h and stimulated with 1% BSA (control, CO) (10 min), 3 ng/ml PDGF-BB (10 min), or 25 μM LPA (indicated time points). Lysates were immunoprecipitated with anti-PDGF-β-R (αPDGF-β-R) followed by SDS/PAGE and immunoblotting with anti-phosphotyrosine (αPY) (Left). In a similar experiment, the filter was reprobed with anti-PDGF-β-R (αPDGF-β-R) (Right) (A). Quiescent L cells were preincubated for 10 min with DMSO or 10 μM AG1296. Tyrosine phosphorylation of the precipitated PDGF-β-R was determined by immunoblotting with anti-phosphotyrosine (αPY) (B). L cells were transfected transiently with HA-ERK2 and either vector alone or a dominant negative PDGF-β-R. The cells were serum-starved for 16 h and stimulated (5 min) before lysis with 1% BSA (control, CO), 10 ng/ml PDGF-BB, or 25 μM LPA. Lysates were immunoprecipitated with anti-HA (αHA) and subjected to SDS/PAGE. ERK activation was detected by immunoblotting with a phopho-specific anti-ERK (αPY-ERK) that only detects tyrosine-phosphorylated activated ERKs (C).
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