Interactions of the AP-1 Golgi Adaptor with the Polymeric Immunoglobulin Receptor and Their Possible Role in Mediating Brefeldin A-sensitive Basolateral Targeting from the trans-Golgi Network

Ena Orzech,Karni Schlessinger,Aryeh Weiss,Curtis T Okamoto,Benjamin Aroeti

doi:10.1074/jbc.274.4.2201

Abstract

We provide morphological, biochemical, and functional evidence suggesting that the AP-1 clathrin adaptor complex of the trans-Golgi network interacts with the polymeric immunoglobulin receptor in transfected Madin-Darby canine kidney cells. Our results indicate that immunofluorescently labeled gamma-adaptin subunit of the adaptor complex and the polymeric immunoglobulin receptor partially co-localize in polarized and semi-polarized cells. gamma-Adaptin is co-immunoisolated with membranes expressing the wild-type receptor. The entire AP-1 adaptor complex could be chemically cross-linked to the receptor in filter-grown cells. gamma-Adaptin could be co-immunoprecipitated with the wild-type receptor, with reduced efficiency with receptor mutant whose basolateral sorting motif has been deleted, and not with receptor lacking its cytoplasmic tail. Co-immunoprecipitation of gamma-adaptin was inhibited by brefeldin A. Mutation of cytoplasmic serine 726 inhibited receptor interactions with AP-1 but did not abrogate the fidelity of its basolateral targeting from the trans-Golgi network. However, the kinetics of receptor delivery to the basolateral cell surface were slowed by the mutation. Although surface delivery of the wild-type receptor was inhibited by brefeldin A, the delivery of the mutant receptor was insensitive to the drug. Our results are consistent with a working model in which phosphorylated cytoplasmic serine modulates the recruitment of the polymeric immunoglobulin receptor into AP-1/clathrin-coated areas in the trans-Golgi network. This process may regulate the efficiency of receptor targeting from the trans-Golgi network.

Highlights

Polarized epithelial cells possess two surface domains as follows: the apical plasma membrane that faces the external environment and the basolateral plasma membrane that is in contact with internal milieu
Evidence from several laboratories strongly argues that both adaptor proteins (AP)-2 and AP-1 clathrin adaptors recognize tyrosine-based signals conforming to the YXXØ type cytoplasmic signals, which mediate endocytosis from the plasma membrane and may confer intracellular sorting in the trans-Golgi network (TGN) [30]
The functional role of these interactions in polarized sorting remains speculative. Other sorting signals, such as the acidic cluster comprising the CKII site (ESEER) juxtaposed to a di-leucine motif in the CD-mannose 6-phosphate receptor (MPR) cytoplasmic tail is essential for high affinity binding of AP-1 adaptors in vitro [45] and probably acts as a dominant determinant controlling cation-dependent mannose 6-phosphate receptor (CD-MPR) sorting in the TGN

Summary

EXPERIMENTAL PROCEDURES

Characterization of MDCK Cells Stably Expressing the pIgR—MDCK cells expressing the wild-type or mutant receptors were maintained for up to 10 passages in minimal Eagle’s medium (MEM, Biological Industries Co, Beit Haemek, Israel) supplemented with 5% (v/v) fetal bovine serum (Biological Industries Co, Beit Haemek, Israel), 100 units/ml penicillin, and 0.1 mg/ml streptomycin in 5% CO2, 95% air. Detection of pIgR1⁄7AP-1 Complexes by Chemical Cross-linking— MDCK cells expressing the wild-type pIgR were cultured for 3–5 days on a 100-mm Transwell porous filter and rinsed twice with ice-cold TGH, and the basolateral surface of the cells was selectively permeabilized by placing the filter onto 1 ml of TGH buffer (50 mM Hepes-NaOH, pH 7.4, 1 mM EGTA-NaOH, 10 mM MgCl2, 150 mM NaCl, 1 mM sodium orthovanadate, 10% v/v glycerol) containing 50 ␮g/ml digitonin (Sigma) for 20 min on ice, as described [50]. Co-immunoprecipitation of 35S-Labeled pIgR with ␥-Adaptin— MDCK cells expressing the wild-type pIgR were cultured for 3 days on 24 mm Transwell filters prior to the experiment. In some experiments cells were treated with 10 ␮g/ml BFA (EpiCenter, stored as a 10 mg/ml stock solution in Me2SO at Ϫ20 °C), as described [42]

RESULTS

Findings

DISCUSSION