Prewarmed pH 8.0 buffer was added, and the cells were returned to 37C for various times, after which cells were again cooled on ice. is required for transport, the ability of rFcRn to transcytose and recycle wild-type Fc homodimers (wtFc; two FcRn-binding sites) and a heterodimeric Fc (hdFc; one FcRn-binding site) was compared. We show that ligand bivalency is not required for transcytosis or recycling, but that wtFc is transported more efficiently than hdFc, particularly at lower concentrations. We also demonstrate that hdFc and wtFc have different intracellular fates, with more hdFc than wtFc being trafficked to lysosomes and degraded, suggesting a role for avidity effects in FcRn-mediated IgG transport. Keywords:apical, basolateral, FcRn, FcRn-GFP, heterodimeric Fc (hdFc), IgG, MadinDarby canine kidney (MDCK) cells, transcytosis The plasma membranes of epithelial cell barriers are segregated into two spatially and functionally distinct domains that serve as a means for complex organisms to distinguish GDC-0623 between the external environment and the underlying tissue. ACVRL1 A vital characteristic of such cellular barriers is their ability to selectively allow the passage of materials, such as ions, small molecules, peptides, lipids and proteins, either by passive or active (receptor mediated) transport mechanisms. The neonatal Fc receptor (FcRn), a class I major histocompatibility complex (MHC)-related protein that associates with the MHC light chain 2-microglobulin (2m), mediates the transfer of maternal IgG across epithelial cell barriers to the fetus or newborn (1,2). In newborn suckling rodents, FcRn is expressed in the polarized epithelium of the GDC-0623 intestine. The FcRn present at the apical surface of the intestinal cells binds to maternal IgG from ingested milk, transcytoses it across the epithelium and releases it into circulation from the basolateral cell surface (1). This process confers passive humoral immunity to the newborn during the first weeks of independent life. The difference in pH between the intestinal lumen (pH 6.0) and the bloodstream (pH 7.4) promotes the efficient unidirectional transport of IgG, as FcRn binds IgG at pH values 6.5, but not at neutral or higher pH (1,3). The FcRn-mediated transport of IgG can also occur in the absence of a pH gradient. In gestating primates, IgG in the maternal bloodstream is transferred to the fetal bloodstream in a process that consists of passive uptake of IgG by syncitiotrophoblast cells, followed by transcytotic delivery to the fetal bloodstream on the opposite surface (46). In adult mammals, FcRn plays a key role in serum IgG homeostasis by protecting IgG taken up by vascular endothelial cells from a default degradative pathway (79). For both these functions, it is believed that IgG internalized at pH 7.4 via fluid-phase endocytosis is bound by FcRn in acidic endosomes. This results in FcRnIgG complexes being transcytosed in the case of maternofetal IgG transfer, or returned to the cell surface rather than catabolized in the case of serum IgG homeostasis. We previously described a structure-based hypothesis to account for the ability of cells to distinguish endosomes containing FcRnIgG complexes destined for recycling or transcytosis from endosomes destined for a degradative pathway (10). The hypothesis suggests that an oligomeric ribbon of FcRn dimers GDC-0623 bridged by the homodimeric Fc regions of IgG molecules, as seen in the crystal structure of an rFcRn/Fc complex (11), forms inside acidic trafficking vesicles. Formation of the oligomeric ribbon between GDC-0623 the adjacent membranes of a tubular endosome could act as an intracellular trafficking signal, designating vesicles containing such complexes for entry into the transcytotic or recycling pathways. A requirement for the formation of the oligomeric ribbon is a homodimeric Fc or IgG molecule capable of bridging between FcRn proteins on opposing membrane faces. Consistent with this hypothesis, previous studies demonstrated that a heterodimeric Fc molecule (hdFc), which contains one FcRn-binding chain and one non-FcRn-binding chain, is less efficiently transcytosed across neonatal mouse intestine (12) and exhibits a shorter serum half-life (13) than a wild-type homodimeric Fc molecule (wtFc). These results indicate that two FcRn-binding sites on a ligand are.