Identification of ARTS-1 as a novel TNFR1-binding protein that promotes TNFR1 ectodomain shedding.
Proteolytic cleavage of TNF receptor 1 (TNFR1) generates soluble receptors that regulate TNF bioactivity. We hypothesized that the mechanism of TNFR1 shedding might involve interactions with regulatory ectoproteins. Using a yeast two-hybrid approach, we identified ARTS-1 (aminopeptidase regulator of TNFR1 shedding) as a type II integral membrane protein that binds to the TNFR1 extracellular domain. In vivo binding of membrane-associated ARTS-1 to TNFR1 was confirmed by coimmunoprecipitation experiments using 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 overexpressing ARTS-1 demonstrated increased TNFR1 shedding and decreased membrane-associated TNFR1, while cells expressing antisense 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 recombinant ARTS-1 protein demonstrated selective aminopeptidase activity toward nonpolar amino acids, multiple lines of negative evidence suggest that ARTS-1 does not possess TNFR1 sheddase activity. These data indicate that ARTS-1 is a multifunctional ectoprotein capable of binding to and promoting TNFR1 shedding. We propose that formation of a TNFR1-ARTS-1 molecular complex represents a novel mechanism by which TNFR1 shedding is regulated.
Pulmonary-Critical Care Medicine Branch, National Heart, Lung, and Blood Institute, , National Institutes of Health, Bethesda, Maryland 20892-1590, USA.
Xinle
X
Cui
Feras
F
Hawari
Sura
S
Alsaaty
Marion
M
Lawrence
Christian A
CA
Combs
Weidong
W
Geng
Farshid N
FN
Rouhani
Dianne
D
Miskinis
Stewart J
SJ
Levine
D036002:ADP Ribose Transferases / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000378:metabolism; D015703:Antigens, CD / Q000737:chemistry* / Q000378:metabolism*; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004730:Endothelium, Vascular / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism; D058851:GPI-Linked Proteins; D006801:Humans; D008168:Lung / Q000166:cytology / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D015778:Minor Histocompatibility Antigens; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D018124:Receptors, Tumor Necrosis Factor / Q000737:chemistry* / Q000378:metabolism*; D047888:Receptors, Tumor Necrosis Factor, Type I; D014407:Tumor Cells, Cultured
D015703:Antigens, CD; D002352:Carrier Proteins; D058851:GPI-Linked Proteins; D008565:Membrane Proteins; D015778:Minor Histocompatibility Antigens; D018124:Receptors, Tumor Necrosis Factor; D047888:Receptors, Tumor Necrosis Factor, Type I; D036002:ADP Ribose Transferases; C449301:ART1 protein, human; D000626:Aminopeptidases; C464841:ERAP1 protein, human