EXPRESSION AND POLARIZED TARGETING OF A RAB3 ISOFORM IN EPITHELIAL-CELLS

Pathways of polarized membrane traffic in epithelial tissues serve a v ariety of functions, including the generation of epithelial polarity a nd the regulation of vectorial transport. We have identified a candida te regulator of polarized membrane traffic in epithelial cells (i.e., rab3B), which is a member of the rab family of membrane traffic regula tors. Rab3B is highly homologous to a brain-specific rab3 isoform (rab 3A) that targets in a polarized fashion to the presynaptic nerve termi nal, where it probably regulates exocytosis. The coding region for hum an rab3B was cloned from epithelial mRNA using a reverse-transcription polymerase chain reaction strategy. This cDNA clone hybridized to a s ingle mRNA species in Northern blots of poly(A)(+) RNA isolated from e pithelial cell lines. A rab3B-specific antibody that was raised agains t recombinant fusion protein recognized a 25-kD band in immunoblots of cell lysates prepared from cultured epithelial cells (e.g., T-84 and HT29-CL19A), but not from a variety of nonepithelial cells (e.g., PC12 neuroendocrine cells). Immunofluorescence analysis confirmed that rab 3B protein is preferentially expressed in cultured epithelial cells as well as in a number of native epithelial tissues, including liver, sm all intestine, colon, and distal nephron. Rab3B localized to the apica l pole very near the tight junctions between adjacent epithelial cells within all of these cell lines and native epithelial tissues, as dete rmined by immunofluorescence and immunoelectron microscopic analysis. Moreover, this pattern of intracellular targeting was regulated by cel l contact; namely, rab3B was reversibly retrieved from the cell periph ery as epithelial cell contact was inhibited by reducing the extracell ular Ca2+ concentration. Our results indicate that neurons and epithel ial cells express homologous rab3 isoforms that target in a polarized fashion within their respective tissues. The pattern and regulation of rab3B targeting in epithelial cells implicates this monomeric GTPase as a candidate regulator of apical and/or junctional protein traffic i n epithelial tissues.