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.

Keywords: FAM150A; extracellular protein; leukocyte tyrosine kinase; library screening; orphan receptor.

Fig. 1.

Extracellular proteome signaling screening to identify LTK ligand(s). (A) Screening strategy: 293 cells stably transfected with LTK–HA were treated with the extracellular proteome library, one protein at a time. The readout is LTK phosphorylation by ELISA. (B) Relative LTK receptor phosphorylation is shown as the SD from the median chemiluminescent units within each assay plate (y axis) for each secreted protein (displayed on x axis). Each point within a boxed area is a technical replicate of individual wells of expressed protein from the same cDNA clone. (C) Relative activities of FAM150A and FAM150B. (D) Dose-dependent phosphorylation of LTK induced by purified FAM150A.

Fig. 2.

Expression of FAM150 family (FAM150A and FAM150B) and LTK. (A) Homology between FAM150A and FAM150B. (B) FAM150A expression in a panel of human tissue RNA samples normalized to the housekeeping gene GUSB. Error bars indicate SD of duplicate data points. (C) FAM150B expression in the same tissue panel normalized to the housekeeping gene GUSB. Error bars indicate SD of duplicate data points. (D) LTK expression in human peripheral blood mononuclear cells (PBMCs). LTK RNA levels in each cell type were analyzed by qRT-PCR. Expression levels were normalized to 18S rRNA as indicated. Each RNA sample was assayed as a single value. Data shown are representative of at least two independent experiments with two different donors.

Fig. 3.

High-affinity binding of FAM150A to LTK–ECD-Fc and LTK stimulation by purified FAM150A. (A) Purification products of FAM150A. (B) Binding kinetics of FAM150A to human LTK–ECD-Fc and mouse Ltk–ECD-Fc fusion proteins by Biacore analysis. (C) Phosphorylation of LTK and downstream proteins after FAM150A stimulation in 293 cells overexpressing LTK–HA. (D) Phosphorylation of LTK and downstream proteins after FAM150A stimulation in SK-N-SH cells.