The EMBO Journal vol. 10 no. 12 pp.3735 - 3742, 1991

In vitro binding of the asialoglycoprotein receptor to the adaptin of plasma membrane coated vesicles

James P.Beltzer and Martin Spiess Department of Biochemistry, Biocenter, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland Communicated by G.Schatz

The asialoglycoprotein (ASGP) receptor was used to probe total clathrin-coated vesicle proteins and purified adaptor proteins (APs) which had been fractionated by gel electrophoresis and transferred to nitroceliulose. The receptor was found to interact with proteins of -100 kDa. The cytoplasmic domain of the ASGP receptor subunit Hl fused to dihydrofolate reductase competed for receptor binding to the 100 kDa polypeptide in the plasma membrane-type AP complexes (AP-2). A fusion protein containing the cytoplasmic domain of the endocytic mutant haemagglutinin HA-Y543 also competed, but a protein with the wild-type haemagglutinin sequence did not. This indicates that the observed interaction is specific for the cytoplasmic domain of the receptor and involves the tyrosine signal for endocytosis. When fractionated by gel electrophoresis in the presence of urea, the ASGP receptor binding polypeptide displayed a characteristic shift in electrophoretic mobility identifying it as the (3 adaptin. Partial proteolysis of the AP-2 preparation followed by the receptor binding assay revealed that the aminoterminal domain of the (3 adaptin contains the binding site for receptors. Key words: adaptins/asialoglycoprotein receptor/assembly proteins/clathrin-coated vesicles/endocytosis

Introduction

The main component of the coat is clathrin. Soluble triskelions composed of a trimer of clathrin heavy chains and three molecules of clathrin light chain aggregate on the cytoplasmic face of the membrane to form polyhedral cages. However, no direct interaction of clathrin with the passenger proteins in coated pits (or buds) and vesicles has been demonstrated. Other coat-associated proteins, termed adaptor or assembly proteins (APs), appear to mediate the interaction between receptors and clathrin. Two classes of AP have been described using monoclonal antibodies and fluorescence immunocytochemistry, AP-1 in clathrin-coated membranes budding off the trans-Golgi, and AP-2 associated with coated pits and vesicles of the plasma membrane (Ahle et al., 1988; Robinson, 1987). APs were first characterized for their ability to promote clathrin assembly into cage-like lattices (Keen et al., 1979). AP-2 complexes have also been shown to be necessary for the in vitro formation of clathrin-coated pits on purified plasma membranes (Moore et al., 1987;

Mahaffey

et al.,

1990).

The AP-2 plasma membrane complex is a tetramer composed of two 100 kDa proteins, ax and 3, and two smaller polypeptides of 50 kDa and 17 kDa. There are at least four subtypes of a polypeptides, aal, %a2, act and (c2 (Ahle et al., 1988). The Golgi AP-1 complex is similarly a tetramer composed of two 100 kDa molecules, (' and -y, and two smaller proteins of 47 kDa and 20 kDa. The 100 kDa proteins of these complexes were called adaptins (Pearse, 1988) as they were postulated to interact with clathrin and with the cytoplasmic domains of receptors. Recently, both the ca and (3 subunits of AP-2 complexes were shown to be required for correct clathrin polymerization (Prasad and Keen, 1991). AP complexes exhibit a characteristic tripartite structure consisting of a central core (or 'brick') containing the amino-terminal portions of the two 100 kDa adaptins and the two smaller polypeptides, and two appendages (or 'ears') made of the carboxy-terminal portions of the two adaptins (Heuser and Keen, 1988; Keen and Beck, 1989; Kirchhausen et al., 1989). By limited proteolysis the ear-like projections of the AP-2 complexes are released and the assembly promoting activity but not clathrin binding is eliminated (Keen and Beck, 1989; Matsui and Kirchhausen, 1990). The (3 adaptin of the AP-2 complex has been purified and has been shown to bind clathrin (Ahle and Ungewickell, 1989) with efficient binding requiring both domains of the molecule (Schroder and Ungewickell, 1991). That AP complexes interact not only with clathrin but also with receptors was first demonstrated for the mannose-6-phosphate (M6P) receptor which could be coassembled into clathrin-AP cages (Pearse, 1985). Furthermore, Pearse (1988) also showed specific binding of the AP-2 complex to the immobilized cytoplasmic domain of the low density lipoprotein (LDL) receptor. In a similar manner, both AP- 1 and AP-2 complexes bound to the immobilized cytoplasmic domain of the M6P receptor -

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The first step in the process of receptor-mediated endocytosis is the specific clustering of receptors into clathrin-coated pits on the plasma membrane. The determinants for clustering were shown for several receptors to reside within their cytoplasmic domains (Lehrman et al., 1985; Mostov et al., 1986; Prywes et al., 1986; Rothenberger et al., 1987). By analysis of natural receptor mutants (Davis et al., 1986) and by in vitro mutagenesis experiments (Davis et al., 1987; Lazarovits and Roth, 1988; Lobel et al., 1989; Alvarez et al., 1990; Chen et al., 1990; Jing et al., 1990; Ktistakis et al., 1990), a degenerate recognition signal has been identified within the diverse sequences of these cytoplasmic domains. It consists of a short sequence containing at least one aromatic amino acid, typically a tyrosine, within a context that most likely assumes a turn conformation (Collawn et al., 1990; Ktistakis et al., 1990). Binding of coat proteins to these recognition signals is presumed to be important for the clustering of receptors and for the formation of coated pits (Iacopetta et al., 1988). (D) Oxford University Press

3735

J.P.Beltzer and M.Spiess

(Glickman et al., 1989). Mutation of the only two tyrosines within this domain to non-aromatic residues selectively abolished its ability to bind to the AP-2 fraction, suggesting separate binding sites for each class of AP complex. In this study we have investigated the interaction between APs and the human asialoglycoprotein (ASGP) receptor (reviewed by Spiess, 1990), a transport receptor of hepatocytes which is responsible for the removal of desialylated, galactose-terminal serum glycoproteins from the circulation. The distribution of the ASGP receptor in intracellular and surface membranes, its clustering in clathrin-coated pits and the kinetics of its internalization and recycling have been well characterized (Breitfeld et al., 1985). The ASGP receptor is composed of two homologous subunits, HI and H2 (Spiess and Lodish, 1985), which are assembled in a complex with an HI:H2 ratio of 3:1 (Henis et al., 1990). Formation of the hetero-oligomeric complex is necessary for high-affinity ligand binding (Shia and Lodish, 1989); however, the major subunit HI contains all the signals for constitutive endocytosis and recycling (Geffen et al., 1989). HI contains an amino-terminal cytoplasmic domain of 40 residues with a single tyrosine residue at position 5. Mutation of this tyrosine to an alanine significantly reduced the rate of endocytosis (Fuhrer et al., 1991), suggesting that a tyrosine-containing signal for endocytosis similar to those identified in other receptors, but without obvious sequence similarity, is also operative in the ASGP receptor. We have developed an in vitro assay to examine the interaction between the ASGP receptor and individual APs derived from clathrin-coated vesicles. The receptor binds with its cytoplasmic domain to a 100 kDa protein of AP-2 complexes which was identified as the j3 adaptin based on a characteristic shift in electrophoretic mobility in an SDS-urea gel system. Upon partial proteolysis of AP-2 complexes, the amino-terminal portion of the adaptin was shown to retain receptor-binding activity. -

Results A blot assay for receptor -AP interaction To identify individual proteins that recognize plasma membrane receptors, we have developed a blot assay similar to those successfully used to detect receptor - ligand interactions (Gershoni, 1988; e.g. LDL binding to its receptor, Daniel et al., 1983). Coated vesicles or purified adaptor complexes were fractionated by SDS -PAGE, transferred to nitrocellulose and probed with purified asialoglycoprotein (ASGP) receptor. We took advantage of the fact that the ASGP receptor can be purified in milligram amounts from human liver by affinity chromatography (Baenziger and Maynard, 1980). Receptor bound by at least partially renatured proteins on the nitrocellulose blot was visualized with a rabbit antiserum directed against the carboxy-terminal ten residues of the major subunit HI of the ASGP receptor in combination with 125I-iodinated protein A and autoradiography. A silver-stained SDS gel of coated vesicle proteins as prepared from human liver is shown in Figure 1 (lane 1). It displays a complex mixture of polypeptides (coat components and passenger proteins). After transfer to nitrocellulose, only the ASGP receptor protein naturally present in liver coated vesicles was recognized by the antiHI antiserum (lane 2). If the blot was first incubated with purified ASGP receptor, an additional band corresponding to a protein of 100 kDa was decorated (lane 3). Since this corresponds to the molecular weight range of coat-associated adaptins, isolated adaptor complexes were analyzed with this assay. Two fractions of AP complexes, AP-1, specific for the Golgi, and AP-2, specific for the plasma membrane, were purified from cow brain coated vesicles by a combination of gel filtration and hydroxylapatite chromatography (see Materials and methods). These two AP preparations, as visualized by silver-staining (Figure 1, lanes 4 and 5), were strongly enriched in 100-115 kDa adaptins, -

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