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

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 ␥-adaptin subunit of the adaptor complex and the polymeric immunoglobulin receptor partially co-localize in polarized and semi-polarized cells. ␥-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. ␥-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 ␥-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/clathrincoated areas in the trans-Golgi network. This process may regulate the efficiency of receptor targeting from the trans-Golgi network.

Polymeric immunoglobulin receptor traffics through two distinct apically targeted pathways in primary lacrimal gland acinar cells

“…On the other hand, non-phosphorylated pIgR or pIgR phosphorylated only on serine 726 may be trafficked into the secretory pathway via its interaction with the AP-1 clathrin adaptor at the TGN (Orzech et al, 1999). Moreover, the direct binding between pIgR and Rab3D shown previously (Evans et al, 2008) and in this study may explain the sequestration of a subset of newly synthesized pIgR by Rab3D into SVs or the recruitment of Rab3D onto nascent SVs by pIgR.…”

The Epidermal Growth Factor Receptor Juxtamembrane Domain Has Multiple Basolateral Plasma Membrane Localization Determinants, Including a Dominant Signal with a Polyproline Core

“…First, what is the nature of the protein interactions mediated by these basolateral localization signals? AP-1-regulated sequestration of membrane cargo with tyrosine-dependent sorting signals into clathrin-coated transport vesicles is thus far the only mechanism implicated in regulated sorting of basolateral proteins (23,24). However, the nature of the EGF receptor sorting signals, as well as the accuracy of EGF receptor sorting in LLCPK 1 cells lacking the AP-1 isoform necessary for polarized sorting (37), suggests that these motifs underlie a novel mechanism.…”

“…The three major classes of mammalian APs are AP-1 found predominantly at the trans-Golgi network (TGN), AP-2 at the plasma membrane, and AP-3 at the TGN and endosomes (19,21,22). Although a role for AP-facilitated transport in clathrin-dependent pathways is well-established (19), it is only recently that these molecules have been implicated in basolateral transport at the TGN (23,24). Other basolateral sorting signals have been identified, however, that bear no relation to known clathrin-coated membrane localization motifs.…”

The Carboxyl Terminus of the Cystic Fibrosis Transmembrane Conductance Regulator Binds to AP-2 Clathrin Adaptors

“…Co-immunoprecipitation and Immunoblot-Complexes between CFTR and adaptors were detected by chemical cross-linking according to previously published methods (26). Briefly, Calu-3 cells were cultured for 3-5 days on 100-mm plates.…”