title: Extracellular TNFR1 release requires the calcium-dependent formation of a nucleobindin 2-ARTS-1 complex.
abstract: Extracellular tumor necrosis factor (TNF) receptors function as TNF-binding proteins that modulate TNF activity. In human vascular endothelial cells (HUVEC), extracellular TNFR1 (type I TNF receptor, TNFRSF1A) is generated by two mechanisms, proteolytic cleavage of soluble TNFR1 ectodomains and the release of full-length 55-kDa TNFR1 in the membranes of exosome-like vesicles. TNFR1 release from HUVEC is known to involve the association between ARTS-1 (aminopeptidase regulator of TNFR1 shedding), an integral membrane aminopeptidase, and TNFR1. The goal of this study was to identify ARTS-1 binding partners that modulate TNFR1 release to the extracellular space. A yeast two-hybrid screen of a human placenta cDNA library showed that NUCB2 (nucleobindin 2), via its helix-loop-helix domains, binds the ARTS-1 extracellular domain. The association between endogenous ARTS-1 and NUCB2 in HUVEC was demonstrated by co-immunoprecipitation experiments, which showed the formation of a calcium-dependent NUCB2.ARTS-1 complex that associated with a subset of total cellular TNFR1. Confocal microscopy experiments demonstrated that this association involved a distinct population of NUCB2-containing intracytoplasmic vesicles. RNA interference was utilized to specifically knock down NUCB2 and ARTS-1 expression, which demonstrated that both are required for the constitutive release of a full-length 55-kDa TNFR1 within exosome-like vesicles as well as the inducible proteolytic cleavage of soluble TNFR1 ectodomains. We propose that calcium-dependent NUCB2.ARTS-1 complexes, which associate with TNFR1 prior to its commitment to pathways that result in either the constitutive release of TNFR1 exosome-like vesicles or the inducible proteolytic cleavage of TNFR1 ectodomains, play an important role in mediating TNFR1 release to the extracellular compartment.
authors.affiliation: Pulmonary-Critical Care Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-1590, USA.
authors.forename: Aminul
authors.initials: A
authors.lastname: Islam
authors.forename: Barbara
authors.initials: B
authors.lastname: Adamik
authors.forename: Feras I
authors.initials: FI
authors.lastname: Hawari
authors.forename: Ge
authors.initials: G
authors.lastname: Ma
authors.forename: Farshid N
authors.initials: FN
authors.lastname: Rouhani
authors.forename: Jing
authors.initials: J
authors.lastname: Zhang
authors.forename: Stewart J
authors.initials: SJ
authors.lastname: Levine
mesh_terms: D000626:Aminopeptidases / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D022162:Cytoplasmic Vesicles / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D015536:Down-Regulation; D042783:Endothelial Cells / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism; D005110:Extracellular Space / Q000378:metabolism*; D006801:Humans; D007375:Interleukin-1 / Q000502:physiology; D018613:Microscopy, Confocal; D015778:Minor Histocompatibility Antigens; D009419:Nerve Tissue Proteins; D000080866:Nucleobindins; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000378:metabolism*; D020545:Respiratory Mucosa / Q000378:metabolism
chemical_list: D002135:Calcium-Binding Proteins; D004268:DNA-Binding Proteins; D007375:Interleukin-1; D015778:Minor Histocompatibility Antigens; C000630650:NUCB2 protein, human; D009419:Nerve Tissue Proteins; D000080866:Nucleobindins; D047888:Receptors, Tumor Necrosis Factor, Type I; D000626:Aminopeptidases; C464841:ERAP1 protein, human; D002118:Calcium