A Region from the Medium Chain Adaptor Subunit (μ) Recognizes Leucine- and Tyrosine-based Sorting Signals

Abstract

Tyrosine-based sorting signals in the cytosolic tails of membrane proteins have been found to bind directly to the medium chain subunit () of the adaptor complexes AP-1 and AP-2. For the leucine-based signals, an interaction with AP-1 and AP-2 has been reported, but no specific interacting subunit has been demonstrated. After searching for molecules interacting with the leucinebased sorting signals within the cytosolic tail of the major histocompatibility complex class II-associated invariant chain using a phage display approach, we identified phage clones with homology to a conserved region of the AP-1 and AP-2 chains. To investigate the relevance of these findings, we have expressed regions of mouse 1 and 2 chains on phage gene product III and investigated the binding to tail sequences from various transmembrane proteins with known endosomal targeting signals. Enzyme-linked immunosorbent binding assays showed that these phages specifically recognized peptides containing functional leucine-and tyrosinebased sorting signals, suggesting that these regions of the 1 and 2 chains interact with both types of sorting signals.The cytosolic tails of membrane proteins contain the information for targeting to various intracellular destinations. Two main classes of endosomal sorting signals have been identified: one class characterized by an essential tyrosine and one by a double leucine (LL) motif or variants thereof (1, 2). There is increasing evidence that both classes of sorting signals interact directly with the adaptor complexes AP-1 and AP-2 for clathrindependent sorting from the trans-Golgi network (TGN) 1 or the plasma membrane, respectively (3-11). Studies on adaptor complexes binding to tail columns indicate a multivalent attachment of aggregated complexes with a binding constant in the micromolar range (3, 4,7). This is confirmed by recent data obtained by surface plasmon resonance (9), suggesting a model where binding to the signals is associated with clustering of the adaptors. In fact, adaptor complexes tend to aggregate and drive the formation of coats in vitro (12), and a requirement for AP clustering is in agreement with the current model where adaptors are first recruited to the membrane by a docking protein and then associate to form a coat to where the membrane proteins diffuse laterally.A specific interacting subunit of the AP complexes was recently demonstrated for the tyrosine-based signals as they were shown to interact with both 1 and 2 , the medium chain subunits of AP-1 and AP-2, respectively (10, 13). No such interaction was demonstrated for the leucine class signals in those assays, although there are data indicating that these also mediate binding to the AP complexes (9).The latest information about how signals may be recognized by the sorting machinery was obtained using the yeast twohybrid system (13), a library-based method for studying protein-protein interactions. We have used phage display technology as an alternative library-based approach to obtain more information about protein seque...

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

“…3 Thus, a distinct subunit of the AP-1 complex may interact with the cytoplasmic tail. Although 2 and 1 chains recognize Tyr-based signals (60) and some leucine-based signals (61,62), the adaptor subunit that interacts with Ser-based determinants has not yet been identified. Recent studies have even suggested the N-terminal trunk domain of the ␥-subunit of membrane bound AP-1 adaptors to mediate the interactions with the bovine papillomavirus E6 protein (63), indicating that adaptin subunits other than medium chains may mediate interactions with signal motifs.…”

μ2 Binding Directs the Cystic Fibrosis Transmembrane Conductance Regulator to the Clathrin-mediated Endocytic Pathway

“…AP-2 complexes containing mutant 2 failed to bind to CFTR in pulldown experiments, confirming that the tyrosine-based sorting motif of CFTR solely interacts with the 2 subunit of AP-2. Another tyrosine-based sorting motif binding site has been proposed at the amino-terminal domain of 2 (residues 102-125); however, as the D176A/W421A mutations blocked the interaction between 2 and CFTR, it does not appear that these residues contribute significantly to the interaction with the tyrosine sorting motif of CFTR (51).…”

“…For example, AP complexes have been shown to interact with dileucine signals in vitro much the same as tyrosine-based signals. However, co-immunoprecipitation studies have yet to show the interaction between AP-2 complexes and dileucine-containing proteins in vivo, prompting some groups to conclude that dileucine signals constitute a low affinity site for AP-2 interactions (51,52). Although acknowledging the potential for multiple signals in CFTR that could bind to AP-2, the results presented here clearly demonstrate that the YXX⌽ sorting motif identified in the carboxyl terminus of CFTR follows the paradigm of other well described YXX⌽ motifs, such that it interacts with the defined YXX⌽ binding site on 2, and is important in the efficient endocytosis of CFTR.…”

Glycosylation Signals That Separate the Trimerization from the MHC Class II-binding Domain Control Intracellular Degradation of Invariant Chain

“…A dissection of certain segments revealed that functional domains of Ii could be grouped to sequences encoded by relatively short exons of the Ii gene (13). Exon 1 encodes the sorting sequence of Ii that is based on a Leu-Ile motif responsible for interaction with cytoplasmic components (14). The stretch of Ii that spans the cell membrane is encoded by exon 2.…”