Direct interaction between Rab3D and the polymeric immunoglobulin receptor and trafficking through regulated secretory vesicles in lacrimal gland acinar cells.
The lacrimal gland is responsible for tear production, and a major protein found in tears is secretory component (SC), the proteolytically cleaved fragment of the extracellular domain of the polymeric Ig receptor (pIgR), which is the receptor mediating the basal-to-apical transcytosis of polymeric immunoglobulins across epithelial cells. Immunofluorescent labeling of rabbit lacrimal gland acinar cells (LGACs) revealed that the small GTPase Rab3D, a regulated secretory vesicle marker, and the pIgR are colocalized in subapical membrane vesicles. In addition, the secretion of SC from primary cultures of LGACs was stimulated by the cholinergic agonist carbachol (CCH), and its release rate was very similar to that of other regulated secretory proteins in LGACs. In pull-down assays from resting LGACs, recombinant wild-type Rab3D (Rab3DWT) or the GDP-locked mutant Rab3DT36N both pulled down pIgR, but the GTP-locked mutant Rab3DQ81L did not. When the pull-down assays were performed in the presence of guanosine-5'-(gamma-thio)-triphosphate, GTP, or guanosine-5'-O-(2-thiodiphosphate), binding of Rab3DWT to pIgR was inhibited. In blot overlays, recombinant Rab3DWT bound to immunoprecipitated pIgR, suggesting that Rab3D and pIgR may interact directly. Adenovirus-mediated overexpression of mutant Rab3DT36N in LGACs inhibited CCH-stimulated SC release, and, in CCH-stimulated LGACs, pull down of pIgR with Rab3DWT and colocalization of pIgR with endogenous Rab3D were decreased relative to resting cells, suggesting that the pIgR-Rab3D interaction may be modulated by secretagogues. These data suggest that the novel localization of pIgR to the regulated secretory pathway of LGACs and its secretion therefrom may be affected by its novel interaction with Rab3D.
Evans
Eunbyul
E
Dept. of Pharmacology and Pharmaceutical Sciences, Univ. of Southern California, Los Angeles, CA 90089-9121, USA.
Zhang
Wenzheng
W
Jerdeva
Galina
G
Chen
Chiao-Yu
CY
Chen
Xuequn
X
Hamm-Alvarez
Sarah F
SF
Okamoto
Curtis T
CT
D000818:Animals; D015153:Blotting, Western; D002217:Carbachol / Q000494:pharmacology; D002478:Cells, Cultured; D018679:Cholinergic Agonists / Q000494:pharmacology; D005260:Female; D006160:Guanosine Triphosphate / Q000378:metabolism; D047468:Immunoprecipitation / Q000379:methods; D007527:Isoenzymes / Q000378:metabolism; D007765:Lacrimal Apparatus / Q000166:cytology / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D018613:Microscopy, Confocal; D009154:Mutation; D011485:Protein Binding; D021381:Protein Transport; D011817:Rabbits; D019056:Receptors, Polymeric Immunoglobulin / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012635:Secretory Component / Q000378:metabolism; D022142:Secretory Vesicles / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D013997:Time Factors; D014161:Transduction, Genetic; D020692:rab3 GTP-Binding Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*
D018679:Cholinergic Agonists; D007527:Isoenzymes; D019056:Receptors, Polymeric Immunoglobulin; D011994:Recombinant Proteins; D012635:Secretory Component; D006160:Guanosine Triphosphate; D002217:Carbachol; C116711:Rab3d protein, mouse; D020692:rab3 GTP-Binding Proteins