The Monomeric Guanosine Triphosphatase rab4 Controls an Essential Step on the Pathway of Receptor-mediated Antigen Processing in B Cells

Abstract

Each member of the rab guanosine triphosphatase protein family assists in the regulation of a specific step within the biosynthetic or endocytic pathways. We have found that the early endosome-associated rab4 protein controls a step critical for receptor-mediated antigen processing in a murine A20 B cell line. Expression of the dominant negative rab4N121I mutant dramatically inhibited the processing and presentation of ovalbumin, λ cI repressor, or rabbit immunoglobulin G internalized as antigens by B cell antigen receptors or transfected Fc receptors. This defect did not reflect a block in antigen endocytosis or degradation, and transfected cells remained completely capable of presenting exogenously added ovalbumin and λ repressor peptides. Most remarkably, rab4N121I-expressing cells were undiminished in their ability to present each of these antigens when whole proteins were internalized at high concentration by fluid-phase endocytosis. Thus, expression of the rab4N121I selectively inactivated a portion of the endocytic pathway required for the processing of receptor-bound, but not nonspecifically internalized, antigens. These results suggest that elements of the early endosome-recycling pathway play an important and selective role in physiologically relevant forms of antigen processing in B cells.

Serotonin Transamidates Rab4 and Facilitates Its Binding to the C Terminus of Serotonin Transporter

“…Immunoprecipitation of this band required co-expression of SERT and preincubation with 100 M 5HT. In addition, we did not detect the band above background in cells expressing dominant negative Rab4N121I, or GTP-hydrolysisdeficient Rab4Q67L (41 neither Rab4N121I nor Rab4Q67L mutants were modified with…”

“…Rab4 and Rab4N121I constructs have been described previously (41). A list of primers used to construct each mutation is located in supplemental Table S1.…”

“…Using Rab4Q67L and Rab4N121I mutant forms of Rab4, here, we show that intracellular 5HTfirst binds to Rab4, allowing the GTPase to interact with SERT. Rab4N121I (41), which cannot bind to nucleotide, does not bind to SERT at all. This conclusion is enhanced by our finding that Rab4Q67L, which has impaired GTP-hydrolysis ability, therefore mimics the constitutively active GTP-bound form of Rab4 (47).…”

Receptor-Facilitated Antigen Presentation Requires the Recruitment of B Cell Linker Protein to Igα

“…To enter a compartment, transport vesicles must first tether, then fuse to the limiting membranes of the targeted vesicles. Rab proteins probably mediate tethering (62)(63)(64), while fusion requires the interaction of vesicle soluble N-ethylmalemide-sensitive factor attachment protein receptors (SNAREs) on the donor compartment with target SNAREs on the target or acceptor membranes (65). Mammalian structural and functional homologues of both the Rab and SNARE yeast proteins have been characterized in several cell types, including B lymphocytes (62,56), indicating that the general mechanisms of endocytic trafficking are widely conserved.…”

Dispatched mediates Hedgehog basolateral release to form the long-range morphogenetic gradient in the Drosophila wing disk epithelium

“…S2 B and B′, arrowheads), where the recycling endosome compartment is probably located (38). Vertebrate Rab4 directs rapid recycling from early endosomes to the plasma membrane (39,40). We also observed a slight buildup of Hh protein in the apical part of the epithelium when we overexpressed a dominant-negative form of Rab4 ( Based on the above, we propose that in producing cells, the apical "secreted" Hh undergoes a subsequent endocytic internalization mediated by dynamin and Rab5 that leads to its "recycling" to the basolateral domain of the plasma membrane, where we believe the long-range Hh gradient is formed and shaped.…”