Constitutive Activation of the Prolactin Receptor Results in the Induction of Growth Factor-independent Proliferation and Constitutive Activation of Signaling Molecules

Richard C.H Lee,Jay A Walters,Mary E Reyland,Steven M Anderson

doi:10.1074/jbc.274.15.10024

Richard C.H Lee, Jay A Walters + Show 2 more

Open Access

https://doi.org/10.1074/jbc.274.15.10024

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Abstract

The ability to induce the oncogenic activation of the human prolactin receptor (PRLR) was examined by deleting 178 amino acids of the extracellular ligand-binding domain. Expression of this deletion mutant in the interleukin-3 (IL-3)-dependent murine myeloid cell line 32Dcl3 resulted in the induction of growth factor-independent proliferation. Parental 32Dcl3 cells proliferated only in the presence of exogenous murine IL-3 (mIL-3), while 32Dcl3 cells transfected with the long form of the human PRLR were able to proliferate in response to mIL-3, ovine prolactin, or human PRL. Cells expressing the Delta178 deletion mutant contained numerous phosphotyrosine-containing proteins in the absence of stimulation with either mIL-3 or ovine prolactin. Growth factor stimulation increased the number of proteins phosphorylated and the intensity of phosphorylation. These proteins included constitutively phosphorylated Janus kinase 2, signal transducer and activator of transcription 5, and SHC. Activated extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2) were observed in unstimulated 32Dcl3 cells expressing the Delta178 mutant. Likewise, transfection of Nb2 cells with the Delta178 deletion mutant induced growth factor-independent proliferation and constitutive activation of Janus kinase 2, ERK1, and ERK2. In addition to the induction of a growth factor-independent state, the expression of the Delta178 deletion mutant also suppressed the apoptosis that occurs when 32Dcl3 cells are cultured in the absence of growth factors such as IL-3. These data suggest that the constitutive activation of the PRLR can be achieved by deletion of the ligand binding domain and that this mutation leads to the oncogenic activation of the receptor as determined by the ability of the receptor to induce growth factor-independent proliferation of factor-dependent hematopoietic cells.

Highlights

Ligand-induced oligomerization of growth factor receptors is critical in regulating the proliferation and differentiation of cells
We demonstrate that the deletion of a 178-amino acid region of the extracellular domain of the prolactin receptor (PRLR) resulted in its constitutive activation and the induction of growth factor-independent proliferation
Construction of the ⌬178 Deletion Mutant—Mutation or deletion of the ligand binding domain of numerous cytokine receptor family members has been shown to result in their constitutive activation [7]

Summary

Introduction

Ligand-induced oligomerization of growth factor receptors is critical in regulating the proliferation and differentiation of cells. At least four constitutively activated transmembrane receptor tyrosine kinases have been transduced by oncogenic retroviruses: v-Erb B, v-Fms, v-Kit, and v-Ros. Erb-B, c-Fms, and c-Kit represent the receptors for epidermal growth factor, colony-stimulating factor-1, and stem cell factor, respectively [2,3,4,5,6]. Erb-B, c-Fms, and c-Kit represent the receptors for epidermal growth factor, colony-stimulating factor-1, and stem cell factor, respectively [2,3,4,5,6] These receptors can be activated by point mutations in the ligand binding domain, the transmembrane domain, and/or the intracellular domain and by deletion of the majority of the extracellular domain [2, 7,8,9,10,11,12]. The current study was undertaken to determine whether deletion of the extracellular domain of the prolactin receptor (PRLR) would lead to its constitutive activation and the ability to induce growth factor-independent proliferation of factor-de-

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