Correspondence to: James H. Keen, Kimmel Cancer Institute and the Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, PA 19107. Tel:(215) 503-4624 Fax:(215) 503-0622 E-mail:jim.keen{at}mail.tju.edu.
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Abstract |
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The clathrin-associated AP-2 adaptor protein is a major polyphosphoinositide-binding protein in mammalian cells. A high affinity binding site has previously been localized to the NH2-terminal region of the AP-2 subunit (
residues 2180 comprise a discrete folding and inositide-binding domain. Further, positively charged residues located within this region are involved in binding, with a lysine triad at positions 5557 particularly critical. Mutant peptides and protein in which these residues were changed to glutamine retained wild-type structural and functional characteristics by several criteria including circular dichroism spectra, resistance to limited proteolysis, and clathrin binding activity. When expressed in intact cells, mutated
subunit showed defective localization to clathrin-coated pits; at high expression levels, the appearance of endogenous AP-2 in coated pits was also blocked consistent with a dominant-negative phenotype. These results, together with recent work indicating that phosphoinositides are also critical to ligand-dependent recruitment of arrestin-receptor complexes to coated pits (
Key Words: clathrin, adaptor, phosphatidylinositols, endocytosis, adaptins
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Introduction |
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RECEPTOR-MEDIATED endocytosis is a multistep process by which certain cell surface proteins are specifically and efficiently internalized into cells through plasma membrane coated pits. Clathrin, the major structural component of the cell surface coated pit, is a triskelion-shaped protein that forms the regular polygonal surface lattice of the coat and provides its structural integrity. Another stoichiometric component of the coat is the multimeric protein complex termed adaptor or AP1, for assembly or associated proteins. Most of the APs are heterotetrameric proteins and multiple forms have been identified (reviewed in and ß' in AP-1 and
and ß in AP-2), a medium subunit (µ1 in AP-1 and µ2 in AP-2), and a small subunit (
sigma}">1 in AP-1 and
sigma}">2 in AP-2). The
subunits have some homology to the
subunit and both are very distantly related to the ß/ß' subunits (reviewed in
AP-2 is critical for two of the key functions of the early steps of the endocytosis pathway: the formation of the clathrin lattice and selection of specific cargo proteins for internalization. AP-2 interacts with clathrin through the and ß subunits (
The function of AP-2 in endocytosis is probably modulated by multiple factors. Proteinprotein interactions of AP-2 with other macromolecules implicated in the endocytosis pathway such as dynamin (
In previous reports we identified and characterized high affinity, stoichiometric binding of various PPIs to AP-2 ( 0.1 µM), which is the focus of this report, appears not be involved. We were subsequently able to localize the PPI binding site to the NH2-terminal amino acids 5 to 80 of the
subunit of AP-2 (
A and
C genes (
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Materials and Methods |
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Materials
Clathrin was purified from bovine braincoated vesicles as described (
Construction of Deletion and Site Mutants in Maltose-binding Protein 5-80
Deletion and site-directed mutations in the 5-80 insert in the plasmid pMAL
A5-80 (
Preparation of Wild-type and Mutant 5-80 Peptides
Purified maltose-binding protein chimera were dialyzed into 20 mM Tris-HCl, pH 8.0, 100 mM NaCl, and 2 mM CaCl2, and digested with the Factor Xa (1 mg enzyme/200 mg fusion protein) for 2436 h at 4°C. The digestion mixture was then incubated with amylose beads to remove MBP and undigested fusion protein. The supernatant was then applied to an S-Sepharose column (Pharmacia) and the column was washed with 20 mM sodium phosphate, pH 7.3, 200 mM NaF. The bound peptide was eluted with 20 mM sodium phosphate and 1 M NaF. The eluate was dialyzed into 10 mM sodium phosphate, pH 7.3, and 100 mM NaF, and used for circular dichroism spectroscopy. Purity of the peptide was checked by SDS-electrophoresis on an 825% gradient gel using a PhastSystem (Pharmacia).
Inositol Polyphosphatebinding Assay
The binding of [3H]IP6 to recombinant fusion proteins was determined by a polyethylene glycol precipitation procedure as described previously (
Circular Dichroism Spectroscopy
Circular dichroism (CD) spectroscopy was performed on an Aviv-62DS instrument at ambient temperature under nitrogen atmosphere with peptides at 0.10.4 mg/ml in 10 mM sodium phosphate, 100 mM NaF. The CD spectra were analyzed using secondary structure prediction software based on the method described by
In Vitro Transcription-Translation of Wild-type and Mutant AP-2A
Wild-type and mutant A subunits were expressed in vitro using a TnT rabbit reticulocyte lysate transcription-translation system. First, plasmid pSP65
A (
A cDNA. Analysis showed that the resulting plasmid pSP65
A2 gave 510-fold higher expression than pSP65
A. Mutant
A cDNA fragments were cloned into the pSP65
A2 by a series of subcloning procedures, the details of which are available on request. The resulting plasmid pSP65
AKKK-Q and the plasmid pSP65
A2 were used for in vitro expression of mutant and wild-type
A.
Transcription-translation reactions were performed according to the manufacturer's recommendations in the presence of 35S-Translabel. After incubation for 2 h at 30°C, translation reactions were centrifuged at 100,000 rpm for 20 min at 4°C in a TLA100 rotor (Beckman).
Proteolysis and Clathrin Cage Binding Assays of In Vitro Translated Polypeptides
Limited tryptic proteolysis and clathrin cage binding experiments with in vitrotranslated wild-type and mutant AP-2 polypeptides were performed essentially as described previously (
Expression of Wild-type and Mutant Adaptin Constructs in Mammalian Cells
Plasmid containing the construct (derived from bovine AP-2
C) in pBluescript SK- (
. This plasmid was cut with SacI and SalI, and the excised fragment was ligated together with the linker connecting NdeI and SacI sites into the plasmids pSP65
A2 and pSP65
AKKK-Q from which NdeI-SalI fragments were excised. The resultant plasmids pSP65-WT
and pSP65-M18
contained wild-type and mutant
chimeric inserts. These plasmids were consecutively treated with SalI, blunted with Klenow fragment and dNTP, and then digested with EcoRI. The excised inserts were ligated into eukaryotic expression vector pcDNA3 that had been cut with EcoRI and EcoRV, resulting in plasmid constructs pcDNA3
WT and pcDNA3
KKK-Q.
For transient expression, BALB/c-3T3 cells were grown in T-75 flasks in a humidified atmosphere with 5% CO2 in DME supplemented with 5% fetal bovine serum, 5% calf serum, 100 U/ml penicillin, and 100 mg/ml streptomycin. The cells were grown to 6070% confluence and transfected with expression constructs using Lipofectamine reagent. In brief, 15 µg of pcDNA3WT or pcDNA3
KKK-Q were incubated with 80 µl of Lipofectamine in 5 ml of DMEM for 30 min at room temperature. After the incubation 5 ml of DME was added and the mixture was transferred to a flask with DMEM-rinsed BALB/c-3T3 cells. After 4 h of incubation the mixture was substituted with complete media and the cells were incubated for 1518 h. Cells were then trypsinized and plated on 12-mm round glass coverslips in a 24-well plate. Immunofluorescence analysis was performed 48 h after transfection.
Coimmunoprecipitation of ß-Adaptin with Polypeptides
MOP8 cells transiently transfected for 48 h with the wild-type or KKK/Q mutant
constructs (3 T-75 flasks) were washed with PBS and scraped into 5 mM Tris-HCl, pH 7.0, supplemented with protease inhibitors. After homogenization, 1 M Tris-HCl, pH 7.0, was added to the broken cells to a final concentration of 0.5 M, the suspension was incubated for 30 min on ice and centrifuged at 100,000 rpm for 30 min in TLA100 rotor (Beckman). The supernatant was diluted to yield a concentration of 125 mM Tris-HCl and supplemented with protease inhibitors and Triton X-100 (0.05%). The AP-2
protein was immunoprecipitated with the antibody 100/3 (
-adaptins, respectively, as well as affinity-purified antibodies against µ2 and
sigma}">2 (
Immunofluorescence Microscopy Analysis
Immunofluorescence analysis was performed as described previously ( polypeptide and rabbit Ab31 (1:150) for detection of endogenous AP-2 (
C, reacts only with
hinge and not with the ear domain (data not shown). Treatment of the
subunit with trypsin is known to generate two major fragments corresponding to the core and the intact hinge + ear domains, while elastase cleaves the ear fragment leaving intact core + hinge (
ear, recognized both the hinge + ear fragment generated by trypsin and the ear domain generated by elastase. This allowed us to use Ab 31 to detect endogenous
but not
, which lacks the hinge region of
.
Clathrin was detected using rabbit polyclonal antibody 27004 (1:150 dilution) (
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Results |
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Dissection of the AP-2 PPI Binding Site by Truncation Mutagenesis
In our previous study, using photoaffinity labeling and bacterially expressed fusion proteins, we localized the high affinity PPI binding site on the clathrin adaptor protein AP-2 to the region between residues 5 and 80 at the NH2-terminal of the subunit. To determine whether PPI binding could be localized to a shorter sequence within this region, we produced several maltose-binding protein (MBP) fusion proteins containing smaller fragments (Figure 1). Among fusion proteins containing either residues 521, 2180, 549, or 5080, only those containing residues 2180 retained specific IP6 binding, with affinity similar to that of the full fragment 580 (Figure 1); the other fusion proteins did not display any detectable binding. This suggested that the PPI binding site in the AP-2
subunit may not be represented by a short stretch of residues, but that a relatively large portion of the sequence between amino acids 2180 may be required to form a discrete domain with proper tertiary structure.
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Site-directed Mutagenesis of the AP-2 PPI Binding Domain
The AP-2 sequence between residues 5 and 80 is a fairly basic region with several clusters of cationic residues. As it is likely that positive charges are involved in the interaction with the negatively charged phosphate groups of PPIs, we investigated more closely the role of these basic residues in IP6 binding. Accordingly, we produced a series of fusion proteins of MBP with the AP-2
5-80 fragment (denoted MBP-
5-80) in which each basic amino acid (10 lysines and 4 arginines) was changed to a glutaminyl residue. Glutamine was chosen because it contains a substantial side chain, similar to lysyl and arginyl residues, but is uncharged. Each of these fusion proteins was purified by affinity chromatography and tested for IP6 binding.
We found that residues scattered throughout the 580 region affected IP6 binding, though to differing extents (Figure 2). The mutations could be divided into several groups in terms of their effects on IP6 binding: no reduction in IP6 binding activity (R21); slight (~20%) reduction (K24, K26, R41, K43, K48); substantial (~40%) reduction (K31, R32, K35, K45, K61); and large (>60%) inhibition (K55, K56, K57). To investigate further the role of lysyl residues 5557 whose alteration to glutamines had the most pronounced effect on IP6 binding, we generated an additional mutant in which residue K56 was changed to glutamic acid with reversal of charge. The IP6 binding ability of this mutant was even more greatly diminished, to ~30% of the wild-type protein, compared with substitution with glutamine. When all three lysyl residues were changed to glutamines in a single mutant, denoted KKK/Q, the IP6 binding ability was decreased to <10% of the wild-type protein. This mutant, essentially devoid of IP6 binding, was characterized further using biophysical methods and functional assays described below to determine whether these residues are directly involved in PPI binding, or whether the decrease in PPI binding is the result of gross conformational change in the structure of the protein.
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Structural Analysis of the Wild-type and Mutant AP-2 PPI Binding Domains by Circular Dichroism Spectroscopy
CD spectroscopy is a very useful method for the rapid determination of secondary structures of peptides and proteins. We employed CD spectroscopy to characterize the secondary structure of the AP-2 PPI binding domain and to monitor any changes resulting from the mutations introduced in its sequence. The AP-2 5-80 fragments of the wild-type and KKK/Q mutant fusion proteins were cleaved with Factor Xa and purified from MBP by consecutive affinity and ion-exchange chromatography steps as described in Materials and Methods. The purification procedure resulted in peptide preparations that were uniformly >95% homogeneous (data not shown). A CD spectrum of the isolated wild-type AP-2
5-80 fragment is shown in Figure 3 a. The positive absorption peak at 195 nm and two negative peaks at 207 and 222 nm indicate that the conformation of the
5-80 peptide has substantial
-helical content. Secondary structure calculated by the method of
-helix and 26% ß sheet. Also shown in Figure 3 a is the CD spectra of the wild-type AP-2
5-80 fragment in 50% trifluoroethanol, known to induce an
-helical conformation in oligopeptides (
-helicity.
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The CD spectrum of the mutant AP-2 5-80-KKK/Q fragment is presented in Figure 3 b, along with the spectrum of the wild-type peptide. The mutant
5-80-KKK/Q has secondary structure content practically identical to that of the wild-type protein. The CD spectrum of the purified peptide derived from the charge inversion mutant AP-2
5-80-K56E was also indistinguishable from that of the wild-type (data not shown). These results argue that the decrease in PPI binding observed with these mutants is not the result of gross conformational changes induced by the amino acid substitutions, but rather results from disruption of direct interactions between the ligand and basic residues on the protein.
We tried to determine whether ligand binding induces any conformational change in the PPI binding domain 5-80. Unfortunately, in the presence of IP6 at concentrations as low as 1 µM the peptide aggregated. This problem could not be overcome by the addition of salt and/or nonionic detergents.
Structural and Functional Characterization of the KKK/Q Mutant in the Context of the Full-length In Vitro Translated AP-2 Subunit
To further evaluate the effects of the KKK/Q mutation on the overall properties of the AP-2 subunit, we performed structural and functional assays on full-length wild-type and mutant AP-2
polypeptides generated in a rabbit reticulocyte in vitro translation system. We have previously shown that the in vitro translated AP-2
polypeptide is folded similarly to that in the native AP-2 complex isolated from bovine brain (
generates fragments of ~5566 kD and 40 kD, corresponding to the NH2-terminal core and COOH-terminal appendage domain generated on similar treatment of the native bovine brain AP-2 (
A polypeptide produced a pattern with the characteristic core and appendage domains, virtually identical to that of the wild-type polypeptide (Figure 4 a). This result demonstrates that alteration of the K(55-57) residues in the NH2-terminal region of the AP
polypeptide does not cause gross misfolding of the entire subunit on synthesis.
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The isolated AP-2 subunit generated by in vitro translation has also been shown to bind specifically to clathrin (
polypeptide required for this interaction. We found that binding of the in vitrotranslated mutant KKK/Q
polypeptide to clathrin cages was indistinguishable from that of the wild-type polypeptide (Figure 4 b). Collectively, these results indicate that the mutant KKK/Q AP-2
polypeptide retained the native tertiary structure and function of the wild-type protein, but that it is essentially devoid of PPI binding ability.
Functional Characterization of Mutant KKK/Q AP-2 in Intact Cells
To investigate the functional role of the AP-2 PPI binding site in intact cells, we employed an construct described previously (
construct encodes mouse
C polypeptide in which the hinge region between the core and appendage domains, corresponding to
C residues 620700, has been substituted with the hinge region of the bovine Golgi-specific AP-1
subunit. This enabled us to specifically localize the expressed
polypeptides in transiently transfected mouse fibroblasts using a
-specific monoclonal antibody (mAb 100/3) which does not recognize the endogenous (mouse) protein (
polypeptide could also be uniquely localized using Ab31, a rabbit polyclonal anti-
antibody kindly provided by Dr. A. Sorkin (University of Colorado). We found that though Ab31 was produced by inoculation with a fragment consisting of the hinge and appendage domains of the rat brain
C subunit (
hinge region and not with the
appendage (see Materials and Methods). Thus, the endogenous and the transiently expressed exogenous
polypeptides could be detected independently, providing important tools for the study of mutant
subunits.
First, we asked whether wild-type and KKK/Q mutant AP-2 polypeptides expressed after transfection are incorporated into AP-2 adaptor complexes in intact cells. Lysates of mock, wild-type
, and KKK/Q mutant
transfected MOP8 mouse fibroblasts were challenged with monoclonal antibody 100/3, and the resultant immunoprecipitates were fractionated by SDS-PAGE and analyzed by immunoblotting with antibodies to the other AP-2 subunits. As shown in Figure 5, reactivity with the anti-
100/3 antibody was detected only in immunoprecipitates from cells transfected with the wild-type or mutant
constructs, consistent with the inability of this antibody to recognize the endogenous mouse AP-2
polypeptide. On immunoblotting with antibodies to the ß, µ2, or
sigma}">2 subunits of AP-2, no signal was detected in the immunoprecipitates from mock-transfected cell lysates demonstrating that recovery of the endogenous AP-2 subunits were dependent on their incorporation into complexes containing exogenous
polypeptide. However, anti-
immunoprecipitates of cells transfected with either the wild-type or the KKK/Q
constructs contained the endogenous ß2, µ2, and
sigma}">2 subunits in similar amounts. These findings confirm the results of
polypeptide becomes incorporated into AP-2 complexes, which we denote AP-2WT. Furthermore, the results presented here demonstrate that the mutant KKK/Q
polypeptide behaves indistinguishably from the wild-type, associating with the other AP-2 subunits and forming complexes (which we denote AP-2PPI-) in the transiently transfected cells.
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To investigate the cellular phenotype resulting from knockout of the PPI binding site of AP-2, we analyzed transfected BALB/c-3T3 cells by confocal fluorescence microscopy. Though the transfection efficiency of BALB/c-3T3 cells was lower than that of MOP-8 cells in our hands, the former were chosen for this experiment because their morphology after fixation is much more amenable to immunofluorescence analysis of plasma membrane coated pits. Cells transfected with wild-type (AP-2WT) or mutant KKK/Q (AP-2PPI-) constructs were double-labeled with mouse monoclonal antibody 100/3 to reveal the localization of the exogenous
product, and with either rabbit Ab31 or 27004 to localize endogenous
-adaptin or clathrin, respectively.
Figure 6 a shows the localization of the AP-2WT product at several different expression levels in transiently transfected cells. The vast majority of the expressed AP-2WT protein (upper panels) had a punctate distribution in the plane of the plasma membrane, with very little diffuse signal detectable. Comparison with the distribution of endogenous AP-2
(lower panels) indicated almost complete colocalization (Figure 6 a). The images also show that the presence of the
hinge did not misdirect the protein to the Golgi region. Consistent with this finding, the AP-2WT distribution was also largely coincident with the localization of plasma membrane coated pits stained with anti-clathrin antibody, but did not colocalize with anti-clathrin staining in the trans-Golgi network (data not shown). Similar observations were made by
construct for expression in Rat1 cells.
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At low expression levels, AP-2WT had no detectable effect on the distribution of the endogenous adaptin (Figure 6 a, left panels). Interestingly, in cells with higher levels of expression there is an apparent dominant-negative effect in that the level of endogenous
adaptin in clathrin-coated pits is decreased compared with untransfected cells in the same field (Figure 6 a, center and right panels). Only at unphysiologically elevated levels of expression is there any evidence for significant accumulation of soluble AP-2WT protein (data not shown), and there is no detectable effect on the normal distribution of clathrin at the plasma membrane or in the Golgi region.
The localization of the mutant AP-2PPI- protein at several different levels of expression are shown in Figure 6 b and 7. The distribution of the mutant protein differed radically from that of the wild-type protein. Generally, most of the AP-2PPI- localization was diffuse and at any level of expression, no significant amount of the mutant polypeptide could be detected in clathrin-coated pits at the plasma membrane. In some cells a small amount of finely punctate signal was detectable, most of which was intracellular. With few exceptions this signal did not coincide with that of endogenous AP-2 (Figure 6 b), nor did it colocalize with either early or recycling endosomes (labeled with endocytosed fluorescent transferrin), or with the late endosome/lysosomal compartment (labeled with endocytosed fluorescent dextran) (data not shown). Interestingly, with increasing expression levels of the mutant AP-2PPI- protein, the proper localization of AP-2 to discrete plasma membrane sites was diminished (Figure 6 b, right panels).
Similarly, at low levels of mutant AP-2PPI- expression, the localization of clathrin to plasma membrane was not noticeably affected (Figure 7, left and middle panels). However, clathrin localization was clearly abnormal at higher levels of mutant expression with a reduced number of plasma membrane coated pits present in comparison to adjacent, nonexpressing cells (Figure 7, right panels). Interestingly, the clathrin signal in the Golgi region also seemed to be affected by elevated levels of AP-2PPI- expression, consistent with continuity between the plasma membrane and Golgi pools of clathrin.
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Finally, we evaluated the internalization of the fluorescently tagged transferrin by cells expressing constructs. In cells expressing low levels of either the AP-2WT or AP-2PPI- mutant proteins, internalization of transferrin was indistinguishable from that in neighboring cells that were not expressing either product (data not shown), consistent with the absence of an effect on coated pit distribution in these cells. Interestingly, transferrin internalization was greatly diminished in cells expressing moderate levels of the mutant AP-2PPI- protein, consistent with the disruption of clathrin-coated pits in that population. In contrast, AP-2WT did not detectably affect transferrin uptake until very high levels of expression were attained.
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Discussion |
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In this study, we have sought to determine the importance of the high affinity PPI binding site located in the NH2-terminal region of the AP-2 subunit in the process of receptor-mediated endocytosis. In previous reports we identified the polypeptide region involved in binding (
In some PPI-binding proteins short peptides (820 residues) have been found to be sufficient for high affinity binding of inositol phosphates or phosphoinositides. Examples include certain actin-associated proteins such as gelsolin (
The mutagenesis analysis reported here suggests that the latter characterization is more applicable to the PPI binding site in AP-2 . It has a highly organized secondary structure and seems to require a
60 residue region for full binding activity, from which we infer that this portion of the
structure comprises a distinct structural and functional domain. Positively charged amino acids throughout the region contribute to the binding interaction (Figure 2), with two clusters of basic residues toward each end (a lysine triad at 5557 and K31/R32/K35) appearing to be most important. In parallel with the PH domains whose tertiary structure in complex with ligands has been determined (e.g., that in ß-spectrin;
This region of the AP-2 sequence, and the basic residues in particular, are virtually entirely conserved in both Drosophila and C. elegans homologues of mammalian
A. Furthermore, although the overall identity of two recently identified yeast
homologues with the mammalian protein in this region is 3040%, most of the basic residues required for inositide binding in the mammalian protein, in particular the lysine triad, are also conserved (Figure 8). This extends the inference of a functional PPI binding domain to these lower eukaryotes. Interestingly, the mammalian AP-1
, AP-3
, and recently identified
subunit of a novel AP-4 complex show distinct but considerably less conservation of several of these basic residues (Figure 8): to the best of our knowledge the PPI binding properties of these proteins have not been reported. Finally, the COPI coatomer (
. Collectively, these observations suggest that PPI binding by coat subunits involved in membrane transport is a ubiquitous phenomenon, and that the nature of specific residues in this binding domain may impart inositide binding specificity.
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There is increasing evidence for an essential role of phosphoinositides in transport vesicle function at different locations in mammalian cells. Phosphoinositides, particularly PIP2, formed secondarily to ARF activation of phospholipase D, have been implicated in the recruitment of COPI coat proteins onto the membranes of the Golgi stacks (, had an inhibitory effect on AP-2 recruitment to the plasma membrane, indirectly implicating phosphoinositides in AP-2 targeting (
Our in vitro binding data indicated that the (assembled) coat form of AP-2 shows the highest affinity for phosphoinositides, as compared with inositol phosphates, and that the converse is true for the soluble (disassembled) AP-2 protein (
The results reported here provide direct support for the notion that PPIs play a physiologically important role in membrane recruitment of AP-2. The mutant AP-2PPI- assayed in vitro is almost totally defective in PPI binding, but otherwise indistinguishable from the wild-type protein by multiple structural and functional criteria. However, in intact cells AP-2PPI- is almost completely defective in incorporation into plasma membrane clathrin-coated pits. Unlike the wild-type protein, it tends to have a diffuse distribution throughout the cell (Figure 6 a and 7). At high expression levels, a small amount of punctate signal is also detectable which may reflect the inability of AP-2PPI- to bind PPIs and resist self-association (/
chimeras, their results indicated that the plasma membrane/Golgi targeting signals are localized primarily between residues 130 and 330350 in the
and
sequences, respectively. Interestingly, chimeric proteins in which 132, or even 36, residues from the NH2-terminal region of the AP-2
subunit had been replaced by corresponding
sequences gave substantial diffuse signal and considerably reduced, though still detectable, recruitment to plasma membrane coated pits (for example see
sequence, with the action of a hypothetical PPI binding domain in the NH2-terminal region of the
sequence (see above and Figure 8). According to this reasoning, the AP-2PPI- is not detectably recruited to coated pits despite presence of a plasma membrane targeting signal because it lacks PPI binding.
Interestingly, AP-2PPI- also acts as a dominant-negative inhibitor of coated pit formation. This suggests that excess inactive AP-2PPI- complexes effectively sequester the other AP-2 subunits and/or occupy the limited sites that must be available for coat formation (
This general model is supported by our recent demonstration that PPIs are also involved in the ligand-dependent internalization of another class of receptors, the G-proteincoupled receptors. Nonvisual arrestins, which have been shown to act as adaptors in the internalization of ß2-adrenergic receptors (
Together, these findings point to common themes of phosphoinositide action in membrane trafficking events: they may serve either as recruitment signals for coat components and/or to modulate the interaction of coat components with receptor complexes. The presence in clathrin-coated pits and vesicles of synaptojanin (Haffne et al., 1997), a phosphoinositide 5-phosphatase, suggests that adaptor functions may be regulated by a complex interplay of different enzymes involved in site-specific phosphoinositide metabolism. Additional enzymes involved in adaptor/coat regulation, and the factors, which modulate their activity, are yet to be discovered.
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Acknowledgements |
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We are grateful to Dr. Esteban Dell'Angelica (NIH) for sharing unpublished information, to Dr. Oscar Goodman for help with cage binding experiments, Dr. J. Johannson (University Pennsylvania) for help with CD spectroscopy, Dr. M. Robinson (Cambridge, UK) for cDNA constructs and antibodies to AP-2 subunits, and to Dr. A. Sorkin (University of Colorado) for antibodies against -adaptin.
Supported by National Institutes of Health grant GM-49217 to J.H. Keen.
1.used in this paper: AP, assembly or associated protein; CD, circular dichroism; MBP, maltose-binding protein; PH, pleckstrin homology; PPI, polyphosphoinositide or inositol polyphosphate
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References |
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