Raul Rojas

Immunoglobulin transport across polarized epithelial cells

IgA, IgG and IgM are transported across epithelial cells in a receptor-mediated process known as transcytosis. In addition to neutralizing pathogens in the lumen of the gastrointestinal, respiratory and urogenital tracts, these antibody–receptor complexes are now known to mediate intracellular neutralization of pathogens and might also be important in immune activation and tolerance. Recent studies on the intracellular transport pathways of antibody–receptor complexes and antibody-stimulated receptor-mediated transcytosis are providing new insight into the nature and regulation of endocytic pathways.

The Guanine Nucleotide Exchange Factor ARNO mediates the activation of ARF and phospholipase D by insulin

BackgroundPhospholipase D (PLD) is involved in many signaling pathways. In most systems, the activity of PLD is primarily regulated by the members of the ADP-Ribosylation Factor (ARF) family of GTPases, but the mechanism of activation of PLD and ARF by extracellular signals has not been fully established. Here we tested the hypothesis that ARF-guanine nucleotide exchange factors (ARF-GEFs) of the cytohesin/ARNO family mediate the activation of ARF and PLD by insulin.ResultsWild type ARNO transiently transfected in HIRcB cells was translocated to the plasma membrane in an insulin-dependent manner and promoted the translocation of ARF to the membranes. ARNO mutants: ΔCC-ARNO and CC-ARNO were partially translocated to the membranes while ΔPH-ARNO and PH-ARNO could not be translocated to the membranes. Sec7 domain mutants of ARNO did not facilitate the ARF translocation. Overexpression of wild type ARNO significantly increased insulin-stimulated PLD activity, and mutations in the Sec7 and PH domains, or deletion of the PH or CC domains inhibited the effects of insulin.ConclusionsSmall ARF-GEFs of the cytohesin/ARNO family mediate the activation of ARF and PLD by the insulin receptor.

RhoB-dependent modulation of postendocytic traffic in polarized Madin-Darby canine kidney cells.

The Rho family of GTPases is implicated in the control of endocytic and biosynthetic traffic of many cell types; however, the cellular distribution of RhoB remains controversial and its function is not well understood. Using confocal microscopy, we found that endogenous RhoB and green fluorescent protein‐tagged wild‐type RhoB were localized to early endosomes, and to a much lesser extent to recycling endosomes, late endosomes or Golgi complex of fixed or live polarized Madin‐Darby canine kidney cells. Consistent with RhoB localization to early endosomes, we observed that expression of dominant‐negative RhoBN19 or dominant‐active RhoBV14 altered postendocytic traffic of ligand‐receptor complexes that undergo recycling, degradation or transcytosis. In vitro assays established that RhoB modulated the basolateral‐to‐apical transcytotic pathway by regulating cargo exit from basolateral early endosomes. Our results indicate that RhoB is localized, in part, to early endosomes where it regulates receptor egress through the early endocytic system.

RhoB-dependent modulation of early endocytic traffic in Madin-Darby canine kidney cells.

This article was withdrawn by the authors before final publication on September 16, 2004.