RESUMO
Calpain is a Ca2+-regulated cytosolic protease. Mammals have 14 calpain genes, half of which are predominantly expressed in specific organ(s); the rest are expressed ubiquitously. A defect in calpains causes lethality/pathogenicity, indicating their physiological indispensability. nCL-2/calpain-8a was identified as a stomach-specific calpain, whose physiological functions are unclear. To elucidate these, we characterized nCL-2 in detail. Unexpectedly, nCL-2 was localized strictly to the surface mucus cells in the gastric epithelium and the mucus-secreting goblet cells in the duodenum. Yeast two-hybrid screening identified several nCL-2-interacting molecules. Of these, the beta-subunit of coatomer complex (beta-COP) occurs in the stomach pit cells and is proteolyzed by nCL-2 in vitro. Furthermore, beta-COP and nCL-2 co-expressed in COS7 cells co-localized in the Golgi, and Ca2+-ionophore stimulation caused the proteolysis of beta-COP near the linker region, resulting in the dissociation of beta-COP from the Golgi. These results strongly suggest novel functions for nCL-2 that involve the membrane trafficking of mucus cells via interactions with coat protein.
Assuntos
Calpaína/fisiologia , Muco/metabolismo , Animais , Northern Blotting , Células COS , Cálcio/metabolismo , Calpaína/química , Calpaína/metabolismo , Chlorocebus aethiops , Clonagem Molecular , Primers do DNA/química , DNA Complementar/metabolismo , Eletroforese em Gel de Poliacrilamida , Epitélio/metabolismo , Mucosa Gástrica/metabolismo , Glutationa Transferase/metabolismo , Complexo de Golgi/metabolismo , Immunoblotting , Imunoprecipitação , Hibridização In Situ , Ionóforos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Fatores de Tempo , Ativação Transcricional , Técnicas do Sistema de Duplo-HíbridoRESUMO
Here, we identified and characterized a Ly49 family member, designated as Ly49Q. The Ly49q gene encodes a 273-aa protein with an immunoreceptor tyrosine-based inhibitory motif (ITIM) at the N terminus of its cytoplasmic domain. We show that the ITIM of Ly49Q can recruit SHP-2 and SHP-1 in a tyrosine phosphorylation-dependent manner. In contrast to other known members of the Ly49 family, Ly49Q was found not to be expressed on NK1.1(+) cells, but instead was detectable on virtually all Gr-1(+) cells, such as myeloid precursors in bone marrow. Monocytes/macrophages also expressed low levels of Ly49Q, and the expression was enhanced by the treatment of cells with IFN-gamma. Treatment of activated macrophages with anti-Ly49Q mAb induced rapid formation of polarized actin structures, showing filopodia-like structure on one side and lamellipodial-like structure on the other side. A panel of proteins became tyrosine-phosphorylated in myeloid cells when treated with the mAb. Induction of the phosphorylation depends on the ITIM of Ly49Q. Thus, Ly49Q has unique features different from other known Ly49 family members and appears to be involved in regulation of cytoskeletal architecture of macrophages through ITIM-mediated signaling.