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First published online May 28, 2005
doi: 10.1242/10.1242/jcs.02367

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Characterization of basolateral-targeting signals in the neonatal Fc receptor

Estelle E. Newton, Zhen Wu^* and Neil E. Simister

Rosenstiel Center for Basic Biomedical Sciences and Biology Department, Brandeis University, Waltham, MA 02254-9110, USA

View larger version (23K): [in a new window]   Fig. 1. Mutations in the cytoplasmic tail of FcRn. Schematic representation of the amino acid sequences of the cytoplasmic domains of wild-type and mutant rat FcRn chains used in this study. Mutated residues are in bold. The residues previously identified as components of the tryptophan-based (black) and dileucine-based (gray) basolateral-targeting signals are highlighted.

View larger version (37K): [in a new window]   Fig. 2. Expression of wild-type and mutant forms of FcRn chain in IMCD cells. Stable cell lines were selected after transfection of IMCD cells with cDNA encoding the chain of wild-type or mutant FcRn. Extracts of these cells were analyzed on western blots using polyclonal rabbit antiserum against rat FcRn chain. The two forms of FcRn detected differ in their glycosylation (see Results).

View larger version (16K): [in a new window]   Fig. 3. Steady-state distributions of wild-type and mutant FcRn in polarized IMCD cells measured by binding Fc at the apical or basolateral cell surface. Cell monolayers on Transwells were incubated at 4°C with 125I-Fc at the apical or basolateral surface with or without excess unlabeled IgG. Binding in the presence of competing IgG was considered non-specific and was subtracted from the total. Specific Fc binding at each surface is represented as a percentage of the total specific binding at both surfaces. Columns represent the mean apical and basolateral percentage±s.e.m. from triplicate samples from one experiment representative of 2-5 experiments.

View larger version (18K): [in a new window]   Fig. 4. Endocytosis of Fc by IMCD cells expressing wild-type and mutant FcRn. Non-polarized IMCD cells were incubated at 37°C with 125I-Fc with or without excess unlabeled IgG. Cell-associated CPM in the presence of competing IgG was considered non-specific and subtracted. Internal specific CPM/surface specific CPM was plotted as a function of time. Points show the means from duplicate plates from one experiment representative of three experiments. Data for wild-type FcRn are represented by open circles and mutant FcRn by filled circles in all panels, as indicated: (A) A309L/L322A/L323A, (B) L314F/L322A/L323A, (C) W311A/L322A/L323A, (D) D317A/D318A, (E) E331A/E333A.

View larger version (26K): [in a new window]   Fig. 5. Transcytosis and recycling of Fc by wild-type and mutant FcRn. Monolayers of IMCD cells were incubated at 4°C with 125I-Fc at either the apical or the basolateral surface. Unbound Fc was removed and the cells were warmed to initiate transport. Media at both surfaces were maintained at pH 8.0, and both sets of media were collected and counted at the indicated times. After the last time point, the cells were lysed and counted. At each time point, the cumulative Fc that underwent transcytosis or recycling was represented as a percentage of the total Fc associated with the cells. Points are means of triplicates; bars showing s.e.m. were smaller than the symbols and were omitted for clarity. Each graph is from one experiment representative of 3-7 experiments. Panels A-D show recycling to the apical surface (circles) and apical to basolateral transcytosis (downward-pointing triangles) after loading from the apical surface; panels E-H show recycling to the basolateral surface (circles) and basolateral to apical transcytosis (upward-pointing triangles) after loading from the basolateral surface. Data for wild-type FcRn are represented by open symbols and mutant FcRn by filled symbols: (A,E) A309L/L322A/L323A; (B,F) L314F/L322A/L323A; (C,G) D317A/D318A; (D,H) E331A/E333A.

View larger version (71K): [in a new window]   Fig. 6. (A) Surface delivery of newly synthesized wild-type and mutant FcRn chains. Cells were pulsed with 35S-labeled Met+Cys. After the chase times shown, cells were biotinylated at either the apical or the basolateral surface. FcRn was immunoprecipitated from lysates and biotinylated receptors were re-precipitated with streptavidin-agarose. Precipitated proteins were resolved on polyacrylamide gels, which were then dried and exposed to PhosphorImager screens. Arrowheads indicate the immature (i) and mature (m) forms of FcRn chain. (B) Evaluation of the biotinylation of an intracellular protein. Following pulse-labeling and biotinylation without chase, actin was immunoprecipitated from cell lysates and biotinylated actin was re-precipitated as above. The amount of total actin loaded on the gel represents 0.2% of the cell lysate, whereas the amount of biotinylated actin represents 50% of the cell lysate.

View larger version (29K): [in a new window]   Fig. 7. Basolateral-targeting signals discussed in this paper. Numbers in parentheses indicate multiple signals in the same protein; FcRn and CD1d contain overlapping signals. The numbers of amino acids between the transmembrane (TM) regions and each sequence are shown. Amino acids that are essential elements of basolateral sorting signals or that enhance those signals are in bold. Abbreviations: LAP, lysosomal acid phosphatase; LDL-R, low-density lipoprotein receptor; Ii, MHC class II-associated invariant chain; SCF, stem cell factor.

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