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J Cell Biol. or with a mutant glycophorin transmembrane domain name to prevent dimerization. Schizandrin A Forcing dimerization stimulated transcytosis of the chimera, whereas preventing dimerization abolished ligand-stimulated transcytosis. We conclude that binding of dimeric IgA to the pIgR induces its dimerization and that this dimerization is necessary and sufficient to stimulate pIgR transcytosis. INTRODUCTION The polymeric immunoglobulin receptor (pIgR) is usually a type I membrane protein that is responsible for the transcytosis of dimeric IgA (dIgA) and pentameric IgM across the epithelial monolayer (Mostov 1994; Mostov and Cardone, 1995). Transcytosis of dIgA by pIgR is the first immunological defense against infections entering through mucosal surfaces, which constitute >95% of all infections. The pIgR itself is usually constitutively transcytosed across the cell; however, binding of dIgA stimulates its rate of transcytosis both in vitro (Hirt 1993; Track (1993) found that when MDCK cells expressing transfected rabbit pIgR were Schizandrin A rapidly boiled in 3% SDS and then immunoprecipitated for the pIgR and analyzed by nonreducing SDS-PAGE, a high molecular weight species could be detected. Second, the pIgR contains within its transmembrane domain name (TMD) a consensus sequence proposed to mediate ligand-dependent dimerization (Sternberg and Gullick, 1990). The presence of this motif in the pIgR suggests that the binding of ligand might alter the conformation of the pIgR TMD to facilitate receptor dimerization, leading to the modulation of pIgR transcytosis. The stoichiometry of the conversation of to dIgA has been under investigation for many years with conflicting results (Kuhn and Kraehenbuhl, 1982; Kraehenbuhl and Neutra, 1992). In fact, the results of two recent investigations that examined SC and dIgA are suggestive of a 1:1 ratio of SC to dIgA rather than a 2:1 ratio (Rindisbacher role of dimerization around the intracellular trafficking of the pIgR, both in the presence and absence of the ligand. To study the effects of dimerization around the intracellular trafficking of the wild-type polymeric Ig receptor (pIgR-WT), we replaced its TMD with the TMD of human glycophorin A (pIgR-GpA). The glycophorin transmembrane domain name contains the dimerization motif LIxxG79VxxG83VxxT, which has been demonstrated to be both necessary and sufficient for the oligomerization of glycophorin (Lemmon 1992a,b). GpA or a peptide corresponding to its TMD forms dimers that are stable even in the presence of SDS in a reducing environment. Previous studies found that the transfer of the GpA TMD onto the heterologous proteins v-neu, EGFR, and nuclease resulted in their detection as a SDS stable dimer (Lemmon (Rockford, IL). NP40 was from Calbiochem (San Diego, CA). The anti-mouse IgG-horseradish peroxidase (HRP) secondary antibody was purchased from Gene Schizandrin A Pulser ((1996). A total of 107 Jurkat cells stably expressing pIgR-WT, pIgR-, and pIgRWT- were transiently transfected with 20 g of NFAT-luciferase reporter plasmid as explained above. Twenty-four hours later, 105 cells in a total volume of 90 l were stimulated with increasing concentrations of monomeric, dimeric, and tetrameric IgA. Cells were lysed 6.5 h later with 10 l of 10 lysis buffer (final concentration of 100 mM KPO4, pH 7.8, 5 mM dithiothreitol, and 1% Triton X-100). The lysate was then mixed with 100 l of assay buffer (200 mM KPO4, pH 7.8, 10 mM ATP, 20 mM MgCl2) followed by 100 l of 1 1 mM luciferin. In general, stimulation of the TCR with C305 gave a two- to threefold stronger response (Singer, unpublished data). Luciferase activity, expressed in arbitrary models, was decided in triplicate for each experimental condition. Jurkat Cell Activation A total of 2 107 Jurkat cells in 200 l were stimulated with C305 (anti-TCR at 1:500 dilution of ascites), tetrameric IgA (2 mg/ml), or left unstimulated for 2 min at 37C, as explained by Chu (1996). Cells were rapidly lysed in Schizandrin A buffer made up of 1% NP40, 125 mM NaCl, 20 mM HEPES (pH 7.4), 10 mM NaF, 2 mM Na Vanadate, and a cocktail of protease inhibitors. After the nuclei had been spun out, lysates were cleared 2 with vacant protein A Sepharose (Pharmacia, Uppsala, Sweden), immunoprecipitated with anti- monoclonal 6810.2 (a kind gift from Art Weiss) bound to protein A, resolved by 15% SDS-PAGE, and transferred to Immobilon P (Millipore, Bedford, MA) for Western blot with mouse monoclonal 4G10 (anti-phosphotyrosine, Upstate Biotechnology, Lake Placid, NY). Blots were not Sp7 stripped, but were directly reprobed with anti- monoclonal 6810.2. Antibodies were detected by secondary antibody conjugated to HRP detected by enhanced chemiluminescence. Receptor Dimerization Cells transfected with the various constructs were examined for receptor dimerization by reducing SDS-PAGE. Cells were lysed from confluent 10-cm plastic tissue culture Petri dishes in ice-cold HEPES buffered saline (HBS, 50 mM HEPES, pH 8.0, 125 mM NaCl), plus 1% NP40 and protease inhibitors. The lysate was centrifuged at.