Differential signalling of NH2-terminal flag-labelled thrombopoietin receptor activated by TPO or anti-FLAG antibodies

doi:10.1016/j.cellsig.2003.08.010

Cellular Signalling

Volume 16, Issue 3, March 2004, Pages 355-363

https://doi.org/10.1016/j.cellsig.2003.08.010 Get rights and content

Abstract

In this report, we compared activation of NH2-terminal FLAG-labelled thrombopoietin receptor (Mpl) by anti-FLAG antibodies and by thrombopoietin (TPO). We found that anti-FLAG monoclonal antibodies M1 dimerize FLAG-labelled receptor and trigger proliferation of BaF3/FLAG-Mpl cells. In UT7/FLAG-Mpl cells, activation of the FLAG-Mpl receptor by low TPO concentrations triggered proliferation, while high concentrations triggered differentiation. Activation of FLAG-Mpl receptors in these cells by all tested concentrations of M1 antibodies induced proliferation but not differentiation. Low TPO concentrations induced similar to M1 antibodies level of Jak2, Stat3, Stat5 and Akt phosphorylation. In contrast, only TPO and not M1 antibodies activated Erks phosphorylation. Since the anti-FLAG antibodies do not react with the TPO binding site of the receptor, we hypothesize that they can trigger a distinct signal by dimerizing Mpl in a manner different from that induced by TPO.

Introduction

The protooncogene c-mpl is a receptor for thrombopoietin, a cytokine which has been shown to be the major regulator of megakaryopoiesis and platelet formation [1], [2], [3]. This receptor was first isolated as an oncogenic truncated form transduced by the myeloproliferative leukemia virus (MPLV). The Mpl receptor is a member of the hematopoietic receptor superfamily lacking intrinsic kinase activity. Other members of this family include growth hormone, granulocyte colony-stimulating factor (G-CSF) and erythropoietin (EPO) receptors. The structurally related features of the cytokine receptor superfamily consist of a single transmembrane-spanning protein with an extracellular ligand binding domain and an intracellular domain. These receptors are non-covalently associated with members of the JAK family of cytosolic tyrosine kinases. By analogy with the receptor tyrosine kinases, ligand-stimulated dimerization with cross-phosphorylation has been put forth as a model for transmembrane signalling by the hematopoietic receptors [4], [5]. Cross-phosphorylation of specific residues on the intracellular domain of the receptors and the associated JAKs is thought to be responsible for the recruitment and activation of intracellular signalling molecules [6].

Recent studies on the erythropoietin receptor show that dimerization alone is insufficient for EpoR activation and that activation of this receptor involves domain reorientation within a preformed dimer complex [7]. In accordance with this model of conformation-dependent activation of receptors, one can imagine the following scenario: different agonists of the same receptor could induce different receptor conformations, which, in turn, could change the pattern of the receptor phosphorylation and activate different signal transduction pathways. Recently, Sweeney and coworkers have uncovered evidence for ligand discrimination by individual ErbB receptor dimers. The receptors respond to the different ligands binding through differential autophosphorylation site usage [8]. These observations indicate that ligand stimulation of receptors is not generic and point to another layer in the receptor signal diversification mechanisms.

In the present study, we have examined the effects of anti-FLAG antibodies (FLAG is the eight amino acids sequence tag) [9] on cellular growth and differentiation signalling in cell lines expressing NH2-terminal FLAG-labelled G-CSFR, EpoR and Mpl. We observed that TPO and anti-FLAG antibodies induced differential signalling of FLAG-Mpl. Our observations point to the possibility of designing molecules capable of introducing different biochemical responses within the context of a single receptor dimer.

Section snippets

Cytokines, antibodies and reagents

WEHI-3B conditioned medium was used as a source of murine IL-3. Human recombinant TPO was a generous gift from Kirin (Tokyo, Japan), human glycosylated G-CSF was purchased from RPR Bellon (Neuilly-sur-Seine, France) and GM-CSF was generously provided by NOVARTIS (Rueil-Malmaison, France). Mouse anti-FLAG M1 and M2 monoclonal antibodies (MoAbs) were purchased from Sigma (Saint-Quentin Fallavier, France) and directly conjugated MoAbs PE-anti-human CD41a from Becton Dickinson (Mountain View, CA).

Anti-FLAG antibodies induced proliferation of FLAG-Mpl and FLAG-G-CSFR expressing cells

The FLAG sequence [9] has often been used for receptor targeting [14], [15], [16]. Commercially available anti-FLAG monoclonal antibodies M1 (IgG2b isotype) and M2 (IgG1 isotype) are bivalent molecules that bind to two antigen molecules at the same time. They should therefore be able to dimerize NH2-terminal FLAG-labelled receptors and trigger receptor activation. In order to test this hypothesis, we assayed proliferation of BaF3 cells expressing different NH2-terminal FLAG tagged receptors in

Discussion

Recent studies show that mouse monoclonal antibodies (IgG) to the extracellular domains of the human c-Mpl [24], or EpoR [22], [23], can trigger activation of the receptors. These antibodies activated cellular proliferation and differentiation in the same way as the natural ligands of the c-Mpl and EpoR. In the present study, we attempted to activate Mpl, G-CSFR and EpoR in a way different from those induced by their natural ligands. We used receptors labelled at their NH2-termini with the FLAG

Acknowledgments

We thank Dr. Yann Lecluse for technical assistance; Dr. Gregory Buzard for helpful comments; the Kirin Brewery (Tokyo, Japan) for the gift of PEG-rhuMGDF.

This work was supported by INSERM and the Association pour la Recherche contre le Cancer (ARC) and la Lique (Equipe labellisé Lique 2000). G.A.M. was supported by the Société Française d'Hématologie (SFH) and F.S. by the Faculte de Medicine Paris-Sud.

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Differential signalling of NH2-terminal flag-labelled thrombopoietin receptor activated by TPO or anti-FLAG antibodies

Abstract

Introduction

Section snippets

Cytokines, antibodies and reagents

Anti-FLAG antibodies induced proliferation of FLAG-Mpl and FLAG-G-CSFR expressing cells

Discussion

Acknowledgments

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