Identification of Anaplastic Lymphoma Kinase as a Receptor for the Growth Factor Pleiotrophin

Abstract

Pleiotrophin (PTN) is a secreted growth factor that induces neurite outgrowth and is mitogenic for fibroblasts, epithelial, and endothelial cells. During tumor growth PTN can serve as an angiogenic factor and drive tumor invasion and metastasis. To identify a receptor for PTN, we panned a phage display human cDNA library against immobilized PTN protein as a bait. From this we isolated a phage insert that was homologous to an amino acid sequence stretch in the extracellular domain (ECD) of the orphan receptor tyrosine kinase anaplastic lymphoma kinase (ALK). In parallel with PTN, ALK is highly expressed during perinatal development of the nervous system and down-modulated in the adult. Animal studies demonstrated that PTN can serve as a ratelimiting angiogenic factor during tumor growth, invasion, and metastasis (8 -12). Clinical studies showed elevated serum levels and tumor expression of PTN in samples from patients with colon, stomach, pancreatic, and breast cancer (5, 13). Furthermore, PTN has been implicated in neonatal brain development as well as in neurodegenerative disorders (reviewed in Ref. 14).Obviously, understanding of PTN-mediated signal transduction as well as identification of a receptor for PTN would enhance studies on the biology and pathology of this growth factor family. Our previous studies have shown that the activation of mitogen-activated protein kinase and PI 3-kinase pathways is required for mitogenic activity of PTN, and we had found that the adaptor molecule Shc participated in signal transduction (15). Based on the work of different laboratories in various cell types, it was hypothesized that proteins of 170 -220 kDa that are tyrosine-phosphorylated in response to PTN could be part of the receptor complex (15-17). More recently, several cell membrane-located proteins were shown to bind PTN at low affinity and serve as potential coreceptors or modulators of signal transduction (18 -21), but none of these molecules carried the hallmarks of a signal transducing receptor predicted from the earlier work.To identify a receptor for PTN, we rationalized that panning of a phage display cDNA library against immobilized PTN as a bait would allow us to isolate phage containing a ligand binding fragment of the receptor on their surface. Because of the high levels of expression of PTN during the perinatal development of the nervous system, we hypothesized that fetal brain would most likely also express a PTN receptor. We therefore panned a human fetal brain cDNA phage display library over several rounds against purified PTN that had been tested for biological activity (15). From this we isolated a phage insert homologous to an amino acid sequence stretch in the ECD of the receptor tyrosine kinase anaplastic lymphoma kinase (ALK), a recently described orphan receptor with an apparent molecular mass of

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“…The phage display had revealed a fragment of the ALK extracellular domain (ECD) as the ligand-binding domain (LBD) and we found that PTN binds the recombinant ALK ECD protein as well as the receptor expressed in intact cells with dissociation constants (K d ) of approximately 30 pM ( ¼ 0.5 ng/ml). Also, antibodies raised to the ALK LBD as well as to PTN inhibited receptor binding of the ligand and signal transduction via ALK as well as biologic effects were observed at EC 50 values close to the K d of PTN binding to the ALK receptor (Stoica et al, 2001;Bowden et al, 2002;Powers et al, 2002). In support of this, recent work from the laboratory of Paul Mischel (Lu et al, 2005) using different glioblastoma cell line models also reported that ALK phosphorylation and signaling are induced by PTN.…”

“…In the present paper, we analyze the contribution of IRS-1 to ALK signaling using the interleukin-3 (IL-3)-dependent, 32D murine hematopoietic cells that have no detectable ALK and IRS protein expression (Wang et al, 1993;Stoica et al, 2001;White, 2002). However, surprisingly, we found that 32D cells constitutively express the ALK ligand MK and upon transfection of ALK plus IRS-1, these cells grow and survive independent of IL-3 supplementation.…”

“…Full-length ALK was initially described as an orphan receptor tyrosine kinase that shows restricted tissue distribution and is regulated during organ development (Iwahara et al, 1997;Morris et al, 1997). Several studies show expression of full-length ALK protein in cultured fibroblasts and endothelial cells as well as cell lines derived from epithelial cancers such as pancreatic and breast carcinoma (Stoica et al, 2001 and the neuroectoderm, that is, melanoma (Dirks et al, 2002), neuroblastoma Stoica et al, 2002) glioblastoma and non-Hodgkin's lymphoma (Delsol et al, 1997), reviewed by Pulford et al (2004).…”

“…We identified ALK as a receptor for the growth factor pleiotrophin (PTN) using phage display of a human brain cDNA library with immobilized PTN as a bait and characterized the ligand-receptor interaction in cell-free and cell-based assays (Stoica et al, 2001). The phage display had revealed a fragment of the ALK extracellular domain (ECD) as the ligand-binding domain (LBD) and we found that PTN binds the recombinant ALK ECD protein as well as the receptor expressed in intact cells with dissociation constants (K d ) of approximately 30 pM ( ¼ 0.5 ng/ml).…”

Anaplastic lymphoma kinase is expressed in different subtypes of human breast cancer

“…Recently, ALK was proposed to be the physiological receptor of the 136 amino acid cytokine pleiotrophin (PTN, Ptn) [14]. However, these data were not reconciled with earlier studies that demonstrated that the Receptor Protein Tyrosine Phosphatase (RPTP) β/ζ is the functional receptor of PTN [15]; in those earlier studies, PTN was shown to inactivate RPTPβ/ζ and to increase tyrosine phosphorylation of the substrates of RPTPβ/ζ, which results from phosphorylation of these substrates by kinases that phosphorylate the same sites dephosphorylated by RPTPβ/ζ when cells are not stimulated by PTN.…”