Tumor necrosis factor (TNF) is a multifunctional cytokine that may play an important role in the pathogenesis of airway inflammation in asthma. TNF bioactivity is regulated by soluble TNF receptors that serve as TNF binding proteins. The specific aim of this project is to identify new mechanisms by which the release of soluble TNF receptors are regulated. We hypothesized that the mechanism of TNFR1 shedding might involve interactions with regulatory ectoproteins. Utilizing a yeast two-hybrid approach, we identified ARTS-1 (Aminopeptidase Regulator of TNFR1 Shedding) as a type II integral membrane protein that associates with the TNFR1 extracellular domain. An association between membrane-associated ARTS-1 and TNFR1 was confirmed by co-immunoprecipitation experiments utilizing human pulmonary epithelial and umbilical vein endothelial cells. A direct relationship exists between membrane-associated ARTS-1 protein levels and concordant changes in TNFR1 shedding. Cells over-expressing ARTS-1 demonstrated increased TNFR1 shedding and decreased membrane-associated TNFR1, while cells expressing anti-sense ARTS-1 mRNA demonstrated decreased membrane-associated ARTS-1, decreased TNFR1 shedding and increased membrane-associated TNFR1. ARTS-1 neither bound to TNFR2 nor altered its shedding, suggesting specificity for TNFR1. Although a GST-ARTS-1 fusion protein demonstrated selective aminopeptidase activity towards non-polar amino acids, multiple lines of negative evidence suggest that ARTS-1 does not possess TNFR1 sheddase activity. These findings indicate that ARTS-1 is a multi-functional ectoprotein capable of associating with and promoting TNFR1 shedding. Since release of soluble TNFR1, IL-6 receptor-alpha (IL-6R), and the type II IL-1 decoy receptor (IL-1RII) can all be inhibited by hydroxamic acid based zinc metalloprotease inhibitors, we hypothesized that ARTS-1 might also regulate IL-6R and IL-1RII shedding. Reciprocal co-immunoprecipitation experiments identified that membrane-associated ARTS-1 associates with the soluble, cleaved forms of IL-6R and IL-1RII. Further, ARTS-1 promoted IL-6R and IL-1RII shedding, as demonstrated by a direct correlation between increased membrane-associated ARTS-1 protein, increased IL-6R and IL-1RII shedding, and decreased membrane-associated IL-6R and IL-1RII in cell lines overexpressing ARTS-1. Thus, ARTS-1 promotes the shedding of three distinct cytokine receptor superfamilies, the type I cytokine receptor superfamily (IL-6R), the interleukin 1/Toll-like receptor family (IL-1RII) and the TNF receptor superfamily (TNFR1). We have also identified that TNFR1 can be released from human vascular endothelial cells into the extracellular compartment as a full-length, 55-kDa receptor within the context of membranes of exosome-like vesicles. This identifies a novel mechanism by which soluble cytokine receptors may be generated. We propose that ARTS-1 is a multi-functional aminopeptidase that may modulate inflammatory events by promoting the release of soluble receptors from three cytokine receptor superfamilies. Therefore, ARTS-1 may play an important role in regulating inflammatory events in the airway. Ongoing investigations will be aimed at identifying the mechanisms by which ARTS-1 promotes and regulates the release of soluble cytokine receptors.