Deorphanization of the human leukocyte tyrosine kinase (LTK) receptor by a signaling screen of the extracellular proteome

Abstract

There are many transmembrane receptor-like proteins whose ligands have not been identified. A strategy for finding ligands when little is known about their tissue source is to screen each extracellular protein individually expressed in an array format by using a sensitive functional readout. Taking this approach, we have screened a large collection (3,191 proteins) of extracellular proteins for their ability to activate signaling of an orphan receptor, leukocyte tyrosine kinase (LTK). Only two related secreted factors, FAM150A and FAM150B (family with sequence similarity 150 member A and member B), stimulated LTK phosphorylation. FAM150A binds LTK extracellular domain with high affinity (K D = 28 pM). FAM150A stimulates LTK phosphorylation in a ligand-dependent manner. This strategy provides an efficient approach for identifying functional ligands for other orphan receptors.leukocyte tyrosine kinase | extracellular protein | library screening | FAM150A | orphan receptor M any biological processes and pathogenic conditions involve ligand/receptor signaling. There are numerous transmembrane receptor-like proteins whose ligands have not been identified. Most known ligand-receptor interactions were discovered by painstaking protein purification, genetic approaches, or sequence homology. Strategies using cDNA library expressionsuch as secretion trapping (1), mammalian expression cloning (2), yeast signaling display (3), and λ phage binding display (4)-have also identified ligands for orphan receptors. However, these approaches are limited by the unknown comprehensiveness of the cDNA library and by the fact that the libraries are biased toward high-abundance transcripts and not focused on the extracellular proteome. An alternative strategy is to screen secreted factors that are individually expressed in an array format in which there is no bias in expression of each protein based on the abundance of its cDNA. The advantages of this approach for deorphanization of receptors are that it is extremely sensitive and selective and that it covers the majority of the extracellular proteome. Although arrayed proteins have been used to identify receptor partners with low-affinity, high-avidity interactions (5-7), few studies have used a cell-signaling measurement to study "classic" high-affinity ligandreceptor interactions.Leukocyte tyrosine kinase (LTK) is a receptor tyrosine kinase that was identified in 1988 (8). The extracellular domain (ECD) of LTK has no known domain structure except a glycine-rich region, leading to a proposal that LTK is not activated by a protein ligand. LTK has a close homolog named anaplastic lymphoma kinase (ALK) (9). The LTK pathway has been implicated in autoimmunity, neuronal development, and cancer. One study showed that gain-of-function polymorphism of Ltk has been associated with systemic lupus erythematosus (SLE) pathogenesis (10). Ltk is expressed throughout the adult hippocampus, and mouse knockout studies indicated that both Ltk and Alk are involved in adult neurogenesis with some functio...

Augmentor α and β (FAM150) are ligands of the receptor tyrosine kinases ALK and LTK: Hierarchy and specificity of ligand–receptor interactions

“…AUG-α stimulates robust kinase activity of both ALK and LTK at picomolar concentrations on cells and binds the ECD of ALK and LTK with dissociation constants in the low nanomolar range. We also found that AUG-β, which was previously shown to be a ligand for LTK (25), could stimulate only weak autophosphorylation of ALK at high subnanomolar concentrations, but could strongly stimulate LTK at picomolar concentrations (a schematic model of hierarchy and specificity of ligand-receptor interactions is depicted in Fig. 4F).…”

“…We have recently demonstrated that ALK expressed in the NB1 cell line can be activated by heparin (but not by pleiotrophin or midkine), suggesting a proteoglycan may regulate ALK activity and function through binding to its heparin-binding NTR (15). Two small proteins, designated FAM150A and FAM150B, were recently identified in a screening assay for secreted proteins that bind to the recombinant extracellular domain of LTK (25). It was also demonstrated that FAM150A binding results in stimulation of LTK activation, indicating that FAM150A can function as a stimulatory ligand of LTK.…”

Crosstalk of endoplasmic reticulum exit sites and cellular signaling

“…Thus, LTK remained an enigmatic receptor tyrosine kinase. The ER localization of LTK was recently questioned by reports showing that it responds to extracellular ligands . We showed that endogenous as well as overexpressed LTK localize to the ER and we did not detect any evidence that LTK ever leaves the ER .…”

Lack of evidence for GDF 11 as a rejuvenator of aged skeletal muscle satellite cells

“…It was proposed that GDF11 promotes skeletal muscle regeneration by restoring genomic integrity of old skeletal muscle satellite cells (SCs) and thereby promoting their outgrowth in response to injury (Sinha et al ., 2014). To identify factors that may regulate the growth with aging, we screened a comprehensive expression library of extracellular proteins (Zhang et al ., 2014) on aged skeletal muscle SCs (additional screen details to be published elsewhere). Given the reported activity of GDF11, we expected to identify it in our screen.…”