Design and application of a cytokine-receptor-based interaction trap.

Ligand-induced clustering of type I cytokine receptor subunits leads to trans-phosphorylation and activation of associated cytosolic janus kinases (JAKs). In turn, JAKs phosphorylate tyrosine residues in the receptor tails, leading to recruitment and activation of signalling molecules. Among these, signal transducers and activators of transcription (STATs) are important in the direct transmission of signals to the nucleus. Here, we show that incorporation of an interaction trap in a signalling-deficient receptor allows the identification of protein-protein interactions, using a STAT-dependent complementation assay. Mammalian protein-protein interaction trap (MAPPIT) adds to existing yeast two-hybrid procedures, as originally explored by Fields and Song, and permits the detection of both modification-independent and of phosphorylation-dependent interactions in intact human cells. We also demonstrate that MAPPIT can be used to screen complex complementary DNA libraries, and using this approach, we identify cytokine-inducible SH2-containing protein (CIS) and suppressor of cytokine signalling-2 (SOCS-2) as interaction partners of the phosphotyrosine 402 (Tyr 402)-binding motif in the erythropoietin receptor (EpoR). Importantly, this approach places protein-protein interactions in their normal physiological context, and is especially applicable to the in situ analysis of signal transduction pathways.

ESM-1 is a novel human endothelial cell-specific molecule expressed in lung and regulated by cytokines.

We here report the identification of a novel human endothelial cell-specific molecule (called ESM-1) cloned from a human umbilical vein endothelial cell (HUVEC) cDNA library. Constitutive ESM-1 gene expression (as demonstrated by Northern blot and reverse transcription-polymerase chain reaction analysis) was found in HUVECs but not in the other human cell lines tested. The cDNA sequence contains an open reading frame of 552 nucleotides and a 1398-nucleotide 3'-untranslated region including several domains involved in mRNA instability and five putative polyadenylation consensus sequences. The deduced 184-amino acid sequence defines a cysteine-rich protein with a functional NH2-terminal hydrophobic signal sequence. Searches in several data bases confirmed the unique identity of this sequence. A rabbit immune serum raised against the 14-kDa COOH-terminal peptide of ESM-1 immunoprecipitated a 20-kDa protein only in ESM-1-transfected COS cells. Immunoblotting and immunoprecipitation of HUVEC lysates revealed a specific 20-kDa band corresponding to ESM-1. In addition, constitutive ESM-1 gene expression was shown to be tissue-restricted to the human lung. Southern blot analysis suggests that a single gene encodes ESM-1. A time-dependent up-regulation of ESM-1 mRNA was seen after addition of tumor necrosis factor alpha (TNFalpha) or interleukin (IL)-1beta but not with IL-4 or interferon gamma (IFNgamma) alone. In addition, when IFNgamma was combined with TNFalpha, IFNgamma inhibited the TNFalpha-induced increase of ESM-1 mRNA level. These data suggest that ESM-1 may have potent implications in the areas of vascular cell biology and human lung physiology.