Increased sensitivity to dextran sodium sulfate colitis in IRE1beta-deficient mice.

The epithelial cells of the gastrointestinal tract are exposed to toxins and infectious agents that can adversely affect protein folding in the endoplasmic reticulum (ER) and cause ER stress. The IRE1 genes are implicated in sensing and responding to ER stress signals. We found that epithelial cells of the gastrointestinal tract express IRE1beta, a specific isoform of IRE1. BiP protein, a marker of ER stress, was elevated in the colonic mucosa of IRE1beta(-/-) mice, and, when exposed to dextran sodium sulfate (DSS) to induce inflammatory bowel disease, mutant mice developed colitis 3-5 days earlier than did wild-type or IRE1beta(+/-) mice. The inflammation marker ICAM-1 was also expressed earlier in the colonic mucosa of DSS-treated IRE1beta(-/-) mice, indicating that the mutation had its impact early in the inflammatory process, before the onset of mucosal ulceration. These findings are consistent with a model whereby perturbations in ER function, which are normally mitigated by the activity of IRE1beta, participate in the development of colitis.

An immunoreceptor tyrosine-based inhibitory motif, with serine at site Y-2, binds SH2-domain-containing phosphatases.

Clustering of the mast cell function-associated antigen by its specific monoclonal antibody (G63) inhibits the FcepsilonRI-mediated secretory response. The cytosolic tail of the mast cell function-associated antigen contains a SIYSTL stretch, a potential immunoreceptor tyrosine-based inhibition motif. To investigate the possible functional role of this sequence, as well as identify potential intracellular proteins that interact with it, peptides corresponding to residues 4-12 of the mast cell function-associated antigen's N-terminal cytoplasmic domain, containing the above motif, were synthesized and used in affinity chromatography of mast cell lysates. Both tyrosyl phosphorylated and thiophosphorylated mast cell function-associated antigen peptides bound the src homology domain 2 (SH2)-containing tyrosine phosphatases-1 (SHP-1), -2 (SHP-2) and inositol 5'-phosphatase (SHIP), though with different efficiencies. Neither the nonphosphorylated peptide nor its tyrosyl phosphorylated reversed sequence peptide bound any of these phosphatases. Point mutation analysis of mast cell function-associated antigen pITIM binding requirements demonstrated that for SHP-2 association the amino acid residue at position Y-2 is not restricted to the hydrophobic isoleucine or valine. Glycine and other amino acids with hydrophilic residues, such as serine and threonine, at this position also maintain this binding capacity, whereas alanine and acidic residues abolish it. In contrast, SHP-1 binding was maintained only when serine was substituted by valine, suggesting that the Y-2 position provides selectivity for peptide binding to SH2 domains of SHP-1 and SHP-2. These results were corroborated by surface plasmon resonance measurements of the interaction between tyrosyl phosphorylated mast cell function-associated antigen peptide and recombinant soluble SH2 domains of SHP-1, SHP-2 and SHIP, suggesting that the associations observed in the cell lysates may be direct. Taken together these results clearly indicate that the SIYSTL motif present in mast cell function-associated antigen's cytosolic tail exhibits characteristic features of an immunoreceptor tyrosine-based inhibition motif, suggesting it is a new member of the growing diverse family of immunoreceptor tyrosine-based inhibition motif-containing receptors.

Interactions of the AP-1 Golgi adaptor with the polymeric immunoglobulin receptor and their possible role in mediating brefeldin A-sensitive basolateral targeting from the trans-Golgi network.

We provide morphological, biochemical, and functional evidence suggesting that the AP-1 clathrin adaptor complex of the trans-Golgi network interacts with the polymeric immunoglobulin receptor in transfected Madin-Darby canine kidney cells. Our results indicate that immunofluorescently labeled gamma-adaptin subunit of the adaptor complex and the polymeric immunoglobulin receptor partially co-localize in polarized and semi-polarized cells. gamma-Adaptin is co-immunoisolated with membranes expressing the wild-type receptor. The entire AP-1 adaptor complex could be chemically cross-linked to the receptor in filter-grown cells. gamma-Adaptin could be co-immunoprecipitated with the wild-type receptor, with reduced efficiency with receptor mutant whose basolateral sorting motif has been deleted, and not with receptor lacking its cytoplasmic tail. Co-immunoprecipitation of gamma-adaptin was inhibited by brefeldin A. Mutation of cytoplasmic serine 726 inhibited receptor interactions with AP-1 but did not abrogate the fidelity of its basolateral targeting from the trans-Golgi network. However, the kinetics of receptor delivery to the basolateral cell surface were slowed by the mutation. Although surface delivery of the wild-type receptor was inhibited by brefeldin A, the delivery of the mutant receptor was insensitive to the drug. Our results are consistent with a working model in which phosphorylated cytoplasmic serine modulates the recruitment of the polymeric immunoglobulin receptor into AP-1/clathrin-coated areas in the trans-Golgi network. This process may regulate the efficiency of receptor targeting from the trans-Golgi network.