Hiroshi Ohno

AP-3: an adaptor-like protein complex with ubiquitous expression

We have identified two closely related human proteins (σ3A and σ3B) that are homologous to the small chains, σ1 and σ2, of clathrin‐associated adaptor complexes. Northern and Western blot analyses demonstrate that the products of both the σ3A and σ3B genes are expressed in a wide variety of tissues and cell lines. σ3A and σ3B are components of a large complex, named AP‐3, that also contains proteins of apparent molecular masses of 47, 140 and 160 kDa. In non‐neuronal cells, the 47 kDa protein most likely corresponds to the medium chain homolog p47A, and the 140 kDa protein is a homolog of the neuron‐specific protein β‐NAP. Like other members of the medium‐chain family, the p47A chain is capable of interacting with the tyrosine‐based sorting signal YQRL from TGN38. Immunofluorescence microscopy analyses show that the σ3‐containing complex is present both in the area of the TGN and in peripheral structures, some of which contain the transferrin receptor. These results suggest that the σ3 chains are components of a novel, ubiquitous adaptor‐like complex involved in the recognition of tyrosine‐based sorting signals.

Tyrosine Phosphorylation Controls Internalization of CTLA-4 by Regulating Its Interaction with Clathrin-Associated Adaptor Complex AP-2

CTLA-4 is a costimulation receptor that binds to the same ligands, CD80 and CD86, as CD28 with high affinity and is transiently expressed on the cell surface of activated T cells. CTLA-4 delivers an inhibitory signal through association of a phosphotyrosine-containing motif in the cytoplasmic domain with Syp tyrosine phosphatase. We now demonstrate that CTLA-4 interacts with the mu2 subunit of the plasma membrane-associated adaptor complex, AP-2, through the same motif involved in the interaction with Syp, except that the interaction with mu2 requires unphosphorylated tyrosine. The interaction with mu2 likely induces rapid internalization of CTLA-4 from the cell surface. Our results suggest that the phosphorylation state of a single tyrosine residue determines whether CTLA-4 delivers a negative signal or is internalized.

Structural Determinants of Interaction of Tyrosine-based Sorting Signals with the Adaptor Medium Chains

Many integral membrane proteins contain tyrosine-based signals within their cytoplasmic domains that mediate internalization from the cell surface and targeting to lysosomal compartments. Internalization depends on an interaction of the tyrosine-based signals with the clathrin-associated adaptor complex AP-2 at the plasma membrane, whereas lysosomal targeting involves interaction of the signals with an analogous complex, AP-1, at the trans-Golgi network. Recent studies have identified the medium chains μ2 of AP-2 and μ1 of AP-1 as the recognition molecules for tyrosine-based signals. We have now investigated the structural determinants for interaction of the signals with μ2 and μ1. The position of the signals was found to be an important determinant of interactions with μ2 and μ1; signals were most effective when present at the carboxyl terminus of a polypeptide sequence. Another important determinant of interactions was the identity of residues surrounding the critical tyrosine residue. Mutation of some residues affected interactions with μ2 and μ1 similarly, whereas other mutations had differential effects. These observations suggest that both the position and the exact sequence of tyrosine-based sorting signals are major determinants of selectivity in their interaction with clathrin-associated adaptor complexes.

The medium subunits of adaptor complexes recognize distinct but overlapping sets of tyrosine-based sorting signals.

Tyrosine-based sorting signals conforming to the motif YXXØ (Y is tyrosine, X is any amino acid, and Ø is an amino acid with a bulky hydrophobic side chain (leucine, isoleucine, phenylalanine, methionine, valine)) interact with the medium (μ) subunits of clathrin adaptor (AP) complexes. We have analyzed the selectivity of interaction between YXXØ signals and the μ1, μ2, and μ3 (A or B) subunits of the AP-1, AP-2, and AP-3 complexes, respectively, by screening a combinatorial XXXYXXØ library using the yeast two-hybrid system. All the medium subunits were found to prefer proline at position Y+2, suggesting that YXXØ signals are stabilized by a bend in the polypeptide backbone. Other than for this common preference, each medium subunit favored specific sets of residues at the X and Ø positions; these preferences were consistent with the proposed roles of the different adaptor complexes in rapid endocytosis and lysosomal targeting. A considerable specificity overlap was also revealed by these analyses, suggesting that additional factors, such as the context of the signals, must be important determinants of recognition.

Functional Domain Mapping of the Clathrin-associated Adaptor Medium Chains μ1 and μ2

The clathrin-associated adaptors AP-1 and AP-2 are heterotetrameric complexes involved in the recognition of sorting signals present within the cytosolic domain of integral membrane proteins. The medium chains of these complexes, μ1 and μ2, have been implicated in two types of interaction: assembly with the β1 and β2 chains of the corresponding complexes and recognition of tyrosine-based sorting signals. In this study, we report the results of a structure-function analysis of the μ1 and μ2 chains aimed at identifying regions of the molecules that are responsible for each of the two interactions. Analyses using the yeast two-hybrid system and proteolytic digestion experiments suggest that μ1 and μ2 have a bipartite structure, with the amino-terminal one-third (residues 1–145 of μ1 and μ2) being involved in assembly with the β chains and the carboxyl-terminal two-thirds (residues 147–423 of μ1 and 164–435 of μ2) binding tyrosine-based sorting signals. These observations support a model in which the amino-terminal one-third of μ2 is embedded within the core of the AP-2 complex, while the carboxyl-terminal two-thirds of the protein are exposed to the medium, placing this region in a position to interact with tyrosine-based sorting signals.

Cytokine-independent Jak3 Activation upon T Cell Receptor (TCR) Stimulation through Direct Association of Jak3 and the TCR Complex

Jak3 is responsible for growth signals by various cytokines such as interleukin (IL)-2, IL-4, and IL-7 through association with the common γ chain (γc) in lymphocytes. We found that T cells from Jak3-deficient mice exhibit impairment of not only cytokine signaling but also early activation signals and that Jak3 is phosphorylated upon T cell receptor (TCR) stimulation. TCR-mediated phosphorylation of Jak3 is independent of IL-2 receptor/γc but is dependent on Lck and ZAP-70. Jak3 was found to be assembled with the TCR complex, particularly through direct association with CD3ζ via its JH4 region, which is a different region from that for γc association. These results suggest that Jak3 plays a role not only in cell growth but also in T cell activation and represents cross-talk of a signaling molecule between TCR and growth signals.

Cloning of the gene encoding the murine clathrin-associated adaptor medium chain μ2: gene organization, alternative splicing and chromosomal assignment

The mu 2 chain of the clathrin-associated adaptor complex AP-2 is a member of the adaptor medium chain family, a group of proteins involved in the sorting of integral membrane proteins in endocytic/exocytic pathways. Here, we report the cloning of the (MMU)CLAPM1 gene encoding the murine mu 2 chain, the first member of the family for which this information has become available. The mu 2 gene is approximately 8.5 kb long and is organized into 12 exons and 11 introns. Two transcripts are generated by alternative splicing of exon 5, a mini-exon of only six nucleotides. Proteins encoded by both transcripts are capable of interacting with tyrosine-based sorting signals, suggesting that they are functionally equivalent. The mu 2 gene is localized to the proximal region of mouse chromosome 16, which is syntenic to the proximal region of human chromosome 3. The isolation and characterization of the mu 2 gene should be instrumental for future studies of the genetics and physiological role of the adaptor medium chains in mammals.