title: The protein kinase/endoribonuclease IRE1alpha that signals the unfolded protein response has a luminal N-terminal ligand-independent dimerization domain.
abstract: In response to accumulation of unfolded proteins in the endoplasmic reticulum (ER), cells activate an intracellular signal transduction pathway called the unfolded protein response (UPR). IRE and PERK are the two type-I ER transmembrane protein kinase receptors that signal the UPR. The N-terminal luminal domains (NLDs) of IRE1 and PERK sense ER stress conditions by a common mechanism and transmit the signal to regulate the cytoplasmic domains of these receptors. To provide an experimental system amenable to detailed biochemical and structural analysis to elucidate the mechanism of ER-transmembrane signaling mechanism mediated by the NLD, we overexpressed the soluble luminal domain of human IRE1alpha in COS-1 cells by transient DNA transfection. Here we report the expression, purification, and characterization of the soluble NLD. The biological function of the NLD was confirmed by its ability to associate with itself and to interact with both the membrane-bound full-length IRE1alpha receptor and the ER chaperone BiP. Functional and spectral studies suggested that the highly conserved N-linked glycosylation site is not required for proper protein folding and self-association. Interestingly, we demonstrated that the NLD forms stable dimers linked by intermolecular disulfide bridges. Our data support that the luminal domain represents a novel ligand-independent dimerization domain.
authors.affiliation: Howard Hughes Medical Institute, Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-0650, USA.
authors.forename: Chuan Yin
authors.initials: CY
authors.lastname: Liu
authors.forename: Hetty N
authors.initials: HN
authors.lastname: Wong
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Schauerte
authors.forename: Randal J
authors.initials: RJ
authors.lastname: Kaufman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002942:Circular Dichroism; D019281:Dimerization; D004722:Endoribonucleases; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014162:Transfection
chemical_list: D005656:Fungal Proteins; D008024:Ligands; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D029701:Saccharomyces cerevisiae Proteins; C403982:ERN2 protein, human; C082863:IRE1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; D004722:Endoribonucleases