The polymeric immunoglobulins, IgA and IgM (pig), bind to the pig receptor (pIgR) on the basolateral surface of many types of epithelial cells. The receptor and ligand are endocytosed and transcytosed to the apical surface, where the ligand is released into apical secretions. Transcytosis is regulated by at least three mechanisms: 1) Phosphorylation of Ser664 on the cytoplasmic domain of the plgR stimulates transcytosis. 2) Binding of the ligand, dimeric lgA (dlgA), to the plgR stimulates transcytosis. These two stimuli are relatively independent, in that either one will stimulate transcytosis in the absence of the other. 3) Activation of the heterotrimeric G protein, Gs, also stimulates transcytosis; both the Gsalpha and Beta/gamma subunits appear to be involved. The goal of this research is to analyze the molecular mechanisms of regulation of transcytosis. This is important for three reasons. First, transport of pig into secretions is a major defense against >90% of all infectious agents (including HIV and other emerging infections), which enter through mucosal surfaces. Second, failure to transport lgA may lead to lgA immune complex deposition, which occurs in several diseases. Third, transcytosis of pig is an excellent model for studying general mechanisms of regulation of protein traffic in polarized epithelial cells, which can provide insights into a broad range of biomedical problems. Several approaches will be used. 1) We will examine which step(s) in transcytosis are controlled by each of the three types of stimuli. 2) We will determine which portion(s) of the 103 amino acid cytoplasmic domain of the plgR are needed for stimulation by dlgA binding. 3) We will examine the role of dimerization of the plgR and its transmembrane segment in stimulation of transcytosis. 4) Binding of dlgA to the plgR causes release of inositol 1 ,4,5-trisphosphate and activation of protein kinase C, indicating that a phosphatidyl inositol specific phospholipase C (PI-PLC) is involved. This probably involves coupling of the plgR to a heterotrimeric Gq protein. We will examine how the plgR is coupled to a Gq protein and if this is required for regulation of transcytosis. 5) Calmodulin binds with high affinity in a Ca2+ dependent fashion to the region of the cytoplasmic domain of the plgR that controls its polarized sorting. We will investigate the role of Ca2+ and calmodulin in transcytosis of the plgR.
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