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

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.

Discovery of a cytokine and its receptor by functional screening of the extracellular proteome.

To understand the system of secreted proteins and receptors involved in cell-cell signaling, we produced a comprehensive set of recombinant secreted proteins and the extracellular domains of transmembrane proteins, which constitute most of the protein components of the extracellular space. Each protein was tested in a suite of assays that measured metabolic, growth, or transcriptional responses in diverse cell types. The pattern of responses across assays was analyzed for the degree of functional selectivity of each protein. One of the highly selective proteins was a previously undescribed ligand, designated interleukin-34 (IL-34), which stimulates monocyte viability but does not affect responses in a wide spectrum of other assays. In a separate functional screen, we used a collection of extracellular domains of transmembrane proteins to discover the receptor for IL-34, which was a known cytokine receptor, colony-stimulating factor 1 (also called macrophage colony-stimulating factor) receptor. This systematic approach is thus useful for discovering new ligands and receptors and assessing the functional selectivity of extracellular regulatory proteins.

A comprehensive collection of chicken cDNAs.

Birds have played a central role in many biological disciplines, particularly ecology, evolution, and behavior. The chicken, as a model vertebrate, also represents an important experimental system for developmental biologists, immunologists, cell biologists, and geneticists. However, genomic resources for the chicken have lagged behind those for other model organisms, with only 1845 nonredundant full-length chicken cDNA sequences currently deposited in the EMBL databank. We describe a large-scale expressed-sequence-tag (EST) project aimed at gene discovery in chickens (http://www.chick.umist.ac.uk). In total, 339,314 ESTs have been sequenced from 64 cDNA libraries generated from 21 different embryonic and adult tissues. These were clustered and assembled into 85,486 contiguous sequences (contigs). We find that a minimum of 38% of the contigs have orthologs in other organisms and define an upper limit of 13,000 new chicken genes. The remaining contigs may include novel avian specific or rapidly evolving genes. Comparison of the contigs with known chicken genes and orthologs indicates that 30% include cDNAs that contain the start codon and 20% of the contigs represent full-length cDNA sequences. Using this dataset, we estimate that chickens have approximately 35,000 genes in total, suggesting that this number may be a characteristic feature of vertebrates.