©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Inhibition of Clathrin Assembly by High Affinity Binding of Specific Inositol Polyphosphates to the Synapse-specific Clathrin Assembly Protein AP-3 (*)

(Received for publication, October 7, 1994; and in revised form, November 11, 1994)

Weilan Ye (2) Nawab Ali (3) Michael E. Bembenek (4) Stephen B. Shears (3) Eileen M. Lafer (1)(§)

From the  (1)From theCenter for Molecular Medicine, Institute of Biotechnology, University of Texas Health Science Center, San Antonio, Texas 78245, the (2)Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, the (3)Inositol Lipid Section, Laboratory of Cellular and Molecular Pharmacology, NIEHS, Research Triangle Park, North Carolina 27709, and (4)New England Nuclear, Medical Products Department, E. I. Du Pont Nemours & Co. (Inc.), Boston, Massachusetts 02118

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Bacterially expressed synapse-specific clathrin assembly protein, AP-3 (F1-20/AP180/NP185/pp155), bound with high affinity both inositol hexakisphosphate (InsP(6)) (K = 239 nM) and diphosphoinositol pentakisphosphate (PP-InsP(5)) (K = 22 nM). The specificity of this ligand binding was demonstrated by competitive displacement of bound [^3H]InsP(6). IC values were as follows: PP-InsP(5) = 50 nM, InsP(6) = 240 nM, inositol-1,2,4,5,6-pentakisphosphate (Ins(1,2,4,5,6)P(5)) = 2.2 µM, inositol-1,3,4,5,6-pentakisphosphate (Ins(1,3,4,5,6)P(5)) = 5 µM, inositol-1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P(4)) > 10 µM, inositol-1,4,5-trisphosphate (Ins(1,4,5)P(3)) > 10 µM. Moreover, 10 µM inositol hexasulfate (InsS(6)) displaced only 15% of [^3H]InsP(6). The physiological significance of this binding is the ligand-specific inhibition of clathrin assembly (PP-InsP(5) > InsP(6) > Ins(1,2,4,5,6)P(5)); Ins(1,3,4,5,6)P(5) and InsS(6) did not inhibit clathrin assembly. We also observed high affinity binding of InsP(6) to purified bovine brain AP-3. We separately expressed the 33-kDa amino terminus and the 58-kDa carboxyl terminus, and it was the former that contained the high affinity inositol polyphosphate binding site. These studies suggest that specific inositol polyphosphates may play a role in the regulation of synaptic function by interacting with the synapse-specific clathrin assembly protein AP-3.


INTRODUCTION

InsP(6), (^1)a near-ubiquitous constituent of mammalian cells, is a particularly enigmatic polyphosphate. Even the routes of its synthesis and metabolism remain incompletely resolved(1) . Indeed, it was only recently that InsP(6) was found not to be the metabolically lethargic compound that most laboratories had assumed; now it is known that InsP(6) and a diphosphoinositol derivative (PP-InsP(5)) participate in a rapid, ongoing cycle of phosphorylation and dephosphorylation(2) . It is unclear how the cell is rewarded by the considerable investment of ATP in this cycle. More uncertainty surrounds the intracellular concentration and distribution of InsP(6) in mammalian cells; total cellular levels are generally around 15 µM(3) , although there are examples of cells with around 50 µM InsP(6)(4) . Early suggestions that all of this InsP(6) was unlikely to be free in the cytosol arose from the consideration that this polyphosphate would have a very limited solubility in the cytosolic ionic environment (5) . Indeed, there is now evidence that much of the cell's InsP(6) could be nonspecifically bound to cellular membranes (6) . Against this puzzling background, the physiological significance of InsP(6) has also not been determined. However, one promising line of enquiry follows from the demonstration of tight binding of InsP(6) to AP-2, an adaptor protein that promotes the formation of clathrin-coated vesicles involved in receptor-mediated endocytosis(7, 8, 9) . Binding of InsP(6) inhibits AP-2-mediated clathrin assembly(7) . The idea that this observation is of some fundamental importance has been reinforced by the demonstration that InsP(6) also binds with high affinity to coatomer, a protein complex associated with vesicle traffic between Golgi cisternae(10) . It is now an exciting possibility that there may be a family of InsP(6)-binding proteins that are important to the process of vesicle trafficking. This consideration has prompted us to pursue the observation that the synapse-specific clathrin assembly protein AP-3 has some weak homology with the polyphosphate-binding alpha-adaptin domain of AP-2(11) .

AP-3 was independently discovered in a variety of contexts and has been known as pp155(12) , AP180(13) , NP185(14) , and F1-20(15, 16) . pp155, AP180, and NP185 were shown to be the same protein and renamed AP-3(17) . F1-20 and AP-3 were then shown to be identical(11, 18) . Characterization of the biochemical properties of AP-3 revealed that it is an unusually acidic (12, 13, 16) phosphoprotein (12, 16, 19, 20) and glycoprotein(20) , which migrates anomalously on SDS-PAGE(13, 16, 17, 21) . AP-3 has the functional property that it binds to clathrin triskelia and promotes their assembly into a homogeneous population of clathrin cages(13, 21) . AP-3 was first cloned and sequenced in 1992 (16) . AP-3 was expressed in bacteria (11) and shown to have full clathrin binding and assembly properties(22) , establishing this as an ideal system in which to pursue structure-function studies.

AP-3 expression is neuronal specific(14, 18, 23) . Within both the peripheral and central nervous systems, AP-3 localizes to nerve terminals(23, 24, 25) . The developmental expression of AP-3 is coincident with synaptic maturation(23, 24) . AP-3 is the only clathrin assembly protein shown to be specific for synapses, which led to the suggestion that it is involved in synaptic vesicle biogenesis and recycling(11) . We considered that it would be of particular significance if AP-3 bound InsP(6), because it is in neuronal cell types where there is the strongest evidence that levels of InsP(6) are acutely regulated by extracellular stimuli. For example, [^3H]InsP(6) levels in [^3H]inositol-labeled N1E-115 neuroblastoma cells were increased by up to 50% by either carbachol, elevated extracellular [K], or prostaglandin E1(26) . [^3H]InsP(6) levels in cultured rat cerebellar granule cells respond in a similar manner to increases in extracellular [K](27) .

We now report that AP-3 is indeed another member of this growing family of vesicle trafficking proteins that bind InsP(6). We also describe the impact of ligand binding on the clathrin assembly functions of AP-3. Furthermore, we report on the specificity of this association, with particular reference to PP-InsP(5), since coatomer was found to bind PP-InsP(5) with even higher affinity than that for InsP(6)(10) .


EXPERIMENTAL PROCEDURES

Materials

Proteins

The proteins GST-AP-3 (AS15AS108)(11) , GST-NH(2)-33kDa (22) , and GST, were all expressed exactly as described previously (22) and purified on glutathione-Sepharose, as described previously(22) . Bovine brain clathrin and AP-3 were purified from bovine brain clathrin-coated vesicles as described previously(22) , based on subtle modifications of (13) . SDS-PAGE analysis of the purified clathrin revealed three silver-stained bands, corresponding in apparent molecular weight to clathrin heavy chain, and to the two clathrin light chains. Clathrin was determined to be free of detectable contaminating AP-3 by Western blot analysis with the F1-20 monoclonal antibody utilizing the ECL detection system as described previously(11) . One cycle of assembly-disassembly was carried out as described previously (22) . Protein concentrations were determined spectrophotometrically using extinction coefficients which were calculated from the reported amino acid sequences of clathrin (29, 30, 31) and AP-3(16) , according to the relation = number of tryptophan residues(5690) + number of tyrosine residues(1280)(32) , and found to be as follows: AP-3, 39,970, GST-AP-3, 80,650; GST-AP-3-58kDa-COOH, 63,440; GST-AP-3-33kDa-NH(2), 57,890; GST, 40,680. Bovine AP-3 was shown to be free of detectable contaminating clathrin by Western blot analysis with anti-clathrin heavy chain monoclonal antibody F21-5 utilizing the ECL detection system. Monoclonal antibody F21-5 was generated by fusion of spleens from mice immunized with clathrin which had been purified as described (22) and checked to be free of contaminating AP-3 by Western blot analysis. The monoclonal antibody was found to be very specific for clathrin heavy chain and did not display detectable cross-reactivity with bacterially expressed or bovine AP-3, clathrin light chains, AP-1 or AP-2. (^2)Monoclonal antibody F1-20, has been shown to be specific for AP-3(11, 15, 16, 23) . A construct expressing the carboxyl-terminal 58 kDa of AP-3 was constructed by polymerase chain reaction using two primers: 58k5`, 5`-AGCAGGATCCCCGGGTCTTCTCCAGCCACAACTGTTACA-3` and SZ405, 5`-CTCAGGTTAGTTTTTTCCTATTCAGTCACA-3` and plasmid pGEX3X-F1-20 (AS15) as the template. The polymerase chain reaction product was digested with XmaI and StuI and the 1.3-kilobase fragment was gel-purified. The XmaI-StuI fragment (2.2 kilobase) in plasmid pGEX3X-F1-20 (AS15) was replaced by the 1.3-kilobase fragment. The resulting construct expresses the carboxyl 58-kDa portion of AP-3 (from amino acid 304 to the stop codon) fused with the 26-kDa GST fragment in the amino terminus. This new plasmid is called pGEX3X-F1-20-COOH-58kDa and was introduced into Escherichia coli BL21. GST-58-kDa COOH terminus of AP-3 was expressed and purified under the same conditions as described previously for GST-AP-3 (22) .

Inositol Polyphosphates

Defined mass amounts of PP-InsP(5) were prepared by phosphorylation of InsP(6) (purchased from Calbiochem and repurified by us on HPLC(2) ) using kinase activity present in homogenates of the AR4-2J pancreatoma(2) : 12-ml incubations contained 25 mM HEPES (pH 7.2), 20 mM phosphocreatine, 7 mM MgSO(4), 5 mM Na(2)ATP, 1 mM Na(2)EDTA, 1 mM dithiothreitol, 0.2 mg/ml phosphocreatine kinase, 5 µM [^3H]InsP(6) (5000 dpm/nmol) plus 0.3 mg of AR4-2J homogenate protein/ml. After 90 min, the reaction was quenched with 4 volumes of ice-cold buffer containing 2 mM EDTA, 10 mM triethylamine, 0.1 M Tris-HCl (pH 7.7 at 25 °C). Next, the quenched reaction was divided into 10-ml aliquots, each of which was applied to an NENSORB Preparative deproteinizing cartridge. The flow-throughs were saved. Each column was washed with 2 times 4-ml aliquots of the quenching buffer, and all the flow-throughs were combined and loaded onto a Partisphere SAX HPLC column. The HPLC gradient was as described previously(28) , except that buffers A and B were each supplemented with 1 mM Na(2)EDTA. The PP-InsP(5) peak was saved and desalted(2) .

Ins(1,2,4,5,6)P(5) was prepared by dephosphorylation of InsP(6) using Aspergillus ficuum phytase (Sigma) which we further purified as described previously(33) : 0.001 unit (as defined in (33) ) of phytase were incubated for 25 min at 37 °C in a 3-ml incubation containing 50 mM BisTris (pH 6), 1 mM EDTA, 0.5 mM EGTA, 0.5% (w/v) bovine serum albumin, 2 mM [^3H]InsP(6) (30 dpm/nmol). Reactions were quenched with perchloric acid, neutralized, and chromatographed on an Adsorbosphere SAX HPLC column(2) . Fractions containing Ins(1,2,4,5,6)P(5) were saved and desalted and then rechromatographed on a Partisphere SAX HPLC column(28) . The Ins(1,2,4,5,6)P(5) was again saved and desalted.

Ins(1,4,5)P(3) was obtained from LC Services, Woburn, MA. Ins(1,3,4,5)P(4) was obtained from the University of Rhode Island Foundation (Kingston, RI). Ins(1,3,4,5,6)P(5) was purchased from Boehringer Mannheim. [^3H]InsP(6) (NET 1023) and PP-[^3H]InsP(5) (NET 1093) were obtained from DuPont NEN.

Methods

Inositol Polyphosphate Binding Assay

The binding of inositol polyphosphates to AP-3 was determined by a slight modification of a polyethylene glycol precipitation procedure(10) . AP-3 (preparation indicated in the figure legend) was incubated at 4 °C in 50 µl of a solution containing 25 mM Tris-HCl (pH 7.5 at 25 °C), 5 mg/ml bovine -globulin, 100 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, [^3H]InsP(6) (approximately 20,000 dpm) or PP-[^3H]InsP(5) (approximately 3000 dpm). Other additions are given in the figure legends. After 20 min, protein was precipitated by addition of 35 µl of ice-cold 30% (w/v) polyethylene glycol, followed by immediate vortexing. After a further 10 min on ice, samples were centrifuged for 10 min at 10,000 times g at 4 °C. The supernatants were carefully aspirated and the pellets were quickly washed with 1 ml of the incubation buffer without immunoglobulin. The final pellet was dissolved in 1 ml of 1% SDS, followed by 8 ml of Monoflow scintillation fluid, and was then counted for ^3H. Immunoblots with a monoclonal antibody against AP-3 (F1-20(15) ) indicated that the majority of the AP-3 protein was precipitated by polyethylene glycol.

Any background binding of [^3H]inositol polyphosphates to -globulin itself (<5% of total) was subtracted from the total binding observed in the presence of AP-3. We also subtracted nonspecific binding which was measured in the presence of excess (10 µM) InsP(6); this was less than 5% of the total binding. As a control for the AP-3 preparations that were fusions with GST, we carried out binding studies with GST, and found that GST by itself did not bind any of the inositol polyphosphates. The parameters for the Scatchard plots were calculated using the ``LIGAND'' program developed by the Analytical and Biostatistical Section, Division of Computer Research and Technology, National Institutes of Health, Bethesda, MD. In all experiments, data could only be fitted to a single binding site.

Clathrin Assembly Assays

GST-AP-3, and bovine brain clathrin triskelia, were dialyzed into isolation buffer (pH 6.7). 1.4 mg/ml clathrin triskelia were incubated with either 1.2 mg/ml GST-AP-3 or 1.2 mg/ml GST (negative control) in ice-cold isolation buffer for 4 h. Clathrin cage assembly was evaluated by the sedimentation method (18, 34) . The pellet and supernatant fractions were analyzed by 10-15% gradient SDS-PAGE. Clathrin heavy chain was visualized by ECL-Western blotting using a clathrin heavy chain-specific monoclonal antibody F21-5. The distribution of clathrin heavy chain between the pellet and supernatant fractions was quantitated using the Millipore BioImage system with 3cx scanner. Assembly percentage was calculated as [pellet/(pellet + supernatant)] times 100. Background sedimentation was subtracted from the negative control (clathrin triskelia incubated with GST protein). Under these conditions, 55% of the clathrin triskelia were assembled into cages by GST-AP-3.


RESULTS

AP-3 Binds to Inositol Polyphosphates with High Affinity and Specificity

Even in highly purified preparations of AP-3 from brain, it is difficult to exclude the possibility of contaminants being present, which even in small quantities might still contribute significantly to the overall ligand binding parameters. Therefore, we assessed the binding of bacterially expressed GST-AP-3 to InsP This protein preparation has been characterized previously and was shown to bind and assemble clathrin as well as AP-3 purified from bovine brain(22) . We found the average K(d) for the binding of InsP(6) to GST-AP-3 to be 239 nM, with an average B(max) of 0.16 mol/mol of protein. A representative Scatchard plot is shown (Fig. 1A). The finding that the B(max) value is less than one may reflect some proteolysis. Because the protein under analysis was expressed as a fusion with GST, we also examined the binding of GST to InsP(6). We found no significant binding, at concentrations of InsP(6) up to 10 µM. We purified AP-3 from bovine brain cerebral cortex and found that it bound to InsP(6) with a similarly high affinity as the bacterially expressed protein (data not shown). InsP(6) was not the ligand with the highest affinity for AP-3. Rather, PP-InsP(5), a recently discovered metabolite of InsP(6)(2) , binds to bacterially expressed GST-AP-3 with an average K(d) of 22 nM, and an average B(max) of 0.20 mol/mol of protein (Fig. 1B).


Figure 1: Scatchard analyses of [^3H]InsP(6) and PP-[^3H]InsP(5) binding to AP-3. GST-AP-3 (0.5-2 µg) was incubated with the indicated concentrations of InsP(6) or PP-InsP(5), and the proportions of bound and free ligand were estimated as described under ``Experimental Procedures'' (after subtraction of nonspecific binding, which was determined with 10M ligand, see insets). For InsP(6) (A), the calculated K is 190 nM, and the B(max) is 0.17 mol/mol of protein. Three further experiments gave K values of 170, 400, and 195 nM with corresponding B(max) values of 0.074, 0.18, and 0.2 mol/mol of protein. For PP-InsP(5) (B), the calculated K is 22 nM, and the B(max) is 0.21 mol/mol of protein. Two further experiments gave K values of 26 and 17 nM with corresponding B(max) values of 0.21 and 0.17 mol/mol of protein.



We measured the displacement of [^3H]InsP(6) from GST-AP-3 by the following: PP-InsP(5), InsP(6), Ins(1,2,4,5,6)P(5), Ins(1,3,4,5,6)P(5), Ins(1,3,4,5)P(4), and Ins(1,4,5)P(3). As a control for specificity, we also examined the displacement of [^3H]InsP(6) from GST-AP-3 by InsS(6). Displacement curves (Fig. 2), and IC values (Table 1) indicate that the relative affinities were PP-InsP(5) > InsP(6) > Ins(1,2,4,5,6)P(5) > Ins(1,3,4,5,6)P(5) > Ins(1,3,4,5)P(4) > Ins(1,4,5)P(3) >> InsS(6). We conclude that AP-3 has a high affinity binding site for specific inositol polyphosphates.


Figure 2: Displacement of [^3H]InsP(6) from AP-3 by competing ligands. GST-AP-3 (1.3-2.0 µg) was incubated with 5 nM [^3H]InsP(6) and indicated concentrations of one of the following competing ligands (from left to right): PP-InsP(5) (inverted triangles), InsP(6) (closed circles), Ins(1, 2, 4, 5, 6) P(5) (open circles), Ins(1, 3, 4, 5, 6) P(5) (triangles), Ins(1,3,4,5)P(4) (closed squares), and InsS(6) (open squares). [^3H]InsP(6) binding, as a percentage of total [^3H]InsP(6), was determined as described under ``Experimental Procedures.'' All data points are means of duplicate determinations. Each curve is representative of two or more experiments.





The Binding of Inositol Polyphosphates to AP-3 Inhibits Clathrin Assembly

In order to asses the functional consequences of inositol polyphosphates binding to AP-3, we examined the effects of inositol polyphosphates on AP-3-mediated clathrin assembly, using a modification of the quantitative clathrin assembly assay (18, 34) (Fig. 3). The modification was to avoid dialysis, since we found that if we carried out the quantitative clathrin assembly assay exactly as described(18, 34) , the concentration of inositol polyphosphates changed as a function of time due to movement through the dialysis membrane. We found strong inhibition of clathrin assembly by PP-InsP(5) > InsP(6) > Ins(1,2,4,5,6)P(5). Note that the rank order of potency for inhibition parallels the relative binding affinities of these ligands. For each polyphosphate, the values for the K(d) were lower than the concentrations that inhibited clathrin assembly, but this is not surprising, since the two assays were, out of necessity, performed under different experimental conditions. There may be marginal inhibition of assembly by Ins(1,3,4,5,6)P(5), and there was no measurable inhibition by InsS(6) at concentrations up to 150 µM (Fig. 3). Thus, we conclude that the binding of specific inositol polyphosphates to AP-3 inhibits clathrin assembly.


Figure 3: The binding of specific inositol polyphosphates to AP-3 inhibits clathrin assembly. Clathrin cages were assembled by GST-AP-3 as described under ``Experimental Procedures,'' in the presence of the indicated concentrations of ligands and the % inhibition was calculated. Each data point was derived from three independent experiments, and the error bars represent the standard deviations. Because of the different scales, inhibition of GST-AP-3-mediated clathrin assembly by InsP(6) (open triangle), InsS(6) (closed triangle), Ins(1, 2, 4, 5, 6) P(5) (open circle), and Ins(1,3,4,5,6)P(5) (closed circle) is shown in A, and inhibition by PP-InsP(5) is shown in B.



Mapping of the High Affinity Inositol Polyphosphate Binding Site to the 33-kDa Amino Terminus of AP3

The amino-terminal one-third of AP-3 is relatively neutral, with an amino acid composition typical of a globular structure(11, 16) . The carboxyl-terminal two-thirds of AP-3 is extraordinarily acidic, with an unusually high amount of proline, serine, threonine, and alanine, and a self-repeating structure(11, 16) . It has been shown that while the 33-kDa amino terminus of AP-3 has the ability to bind to clathrin triskelia(17, 22) , it cannot assemble clathrin triskelia into clathrin cages(17, 22) , or bind to preassembled clathrin cages(22) . Here we have examined the ability of the bacterially expressed 33-kDa amino terminus of AP-3(11) , and 58-kDa carboxyl terminus of AP-3, to bind to inositol polyphosphates. There was no measurable binding of GST-58kDa-COOH-AP-3 to InsP(6) in the range 5 nM to 10 µM. In contrast, Scatchard analyses revealed that GST-33kDa-NH(2)-AP-3 binds to InsP(6) with an average K(d) of 173 nM and an average B(max) of 0.77 mol/mol of protein (Fig. 4A) and to PP-InsP(5) with an average K(d) of 76 nM and an average B(max) of 0.60 mol/mol of protein (Fig. 4B). We conclude that the high affinity inositol polyphosphate binding site is located in the 33-kDa amino terminus of AP-3.


Figure 4: Scatchard analyses of [^3H]InsP(6) and PP-[^3H]InsP(5) binding to the 33-kDa amino terminus of AP-3. GST-33-kDa NH(2) terminus of AP-3 (0.3 µg) was incubated with the indicated concentrations of InsP(6) or PP-InsP(5), and the proportions of bound and free ligand were estimated as described under ``Experimental Procedures'' (after subtraction of nonspecific binding, which was determined with 10M ligand, see insets). For InsP(6) (A), the calculated K is 165 nM, and the B(max) is 0.86 mol/mol of protein. One further experiment gave a K value of 180 nM with a corresponding B(max) value of 0.68 mol/mol of protein. For PP-InsP(5) (B), the calculated K is 75 nM, and the B(max) is 0.65 mol/mol of protein. One further experiment gave a K value of 77 nM with a corresponding B(max) value of 0.55 mol/mol of protein.




DISCUSSION

We have found that specific inositol polyphosphates bind to AP-3 with high affinity and inhibit AP-3-mediated clathrin assembly. The crucial finding that inositol hexasulfate has at least a 50-fold lower affinity for AP-3 than InsP(6) testifies to the protein being specific for certain polyphosphates of inositol, rather than simply negative charge density. Furthermore, our observation that PP-InsP(5), InsP(6), and Ins(1,2,4,5,6)P(5) were all more potent ligands than Ins(1,3,4,5,6)P(5) confirms that AP-3 has a distinct preference for a specific configuration of phosphate groups. Although these findings have provided useful information on the specificity for Ins(1,2,4,5,6)P(5), which had only an 8-fold lower affinity for AP-3 than InsP(6), this observation has a limited physiological bearing, since there is approximately 50-fold less Ins(1,2,4,5,6)P(5) in cells than InsP(6). The finding that the strength of inhibition of clathrin assembly by the various ligands paralleled their binding affinity for AP-3 provides a functional as well as structural measure of the specificity. In addition to investigating the consequences of changes in ligand concentration upon AP-3 in vivo, another important line of enquiry will be to determine if alterations in the degree of glycosylation of the protein(20) , its phosphorylation state(12, 16, 19, 20) , or alternative RNA splicing(11, 16, 18) , will prove to be regulatory processes that act by altering the affinity of the ligands to modulate clathrin assembly.

A key consequence of our study with AP-3 is that inhibition of adaptin-mediated clathrin assembly by specific inositol polyphosphates was previously only known to be a characteristic of AP-2(7) . Our experiments have now demonstrated that this is a more widespread phenomenon. It is of further importance to understand the molecular basis for these effects. For this task, our results highlight that AP-3 provides the simpler model, since it is a single 91-kDa polypeptide (16) , whereas AP-2 is a heterotetramer of 270 K(d)(35) . Indeed, our mapping of the high affinity polyphosphate binding site to the 33-kDa amino terminus of this 91-kDa polypeptide further narrows the search for the amino acids that comprise a polyphosphate binding domain. The latter may even provide a general motif for polyphosphate-dependent influences upon vesicle traffic, which may be mediated not only by AP-2 and AP-3, but also by coatomer (10) and perhaps other proteins. It is of particular interest that the 33-kDa amino terminus of AP-3 has been well conserved in evolution. Cloning of the Xenopus homologue of AP-3 revealed that while the overall identity between mouse and Xenopus AP-3 was 77%, the identity in the 33-kDa amino terminus was 97%. (^3)

We also think that it is particularly significant that AP-3, as a new member of this family of inositol polyphosphate binding proteins, is synaptically localized(23, 24, 25) . It is also fascinating to consider that an interaction of InsP(6) with AP-3 may be at the heart of the provocative finding that injection of this polyphosphate into the nucleus tractus solitarius of rat brain resulted in a decrease in both arterial blood pressure and heart rate(36, 37) . An acceptable molecular basis for these putative ``neurotransmitter-like'' effects has never been developed previously. Our data now raise the novel possibility that extracellularly applied InsP(6) might gain access to synaptic adaptor proteins such as AP-3, likely through endocytosis, and thereby perturb synaptic signaling. In this respect, it is notable that InsP(6) was more potent than Ins(1,3,4,5,6)P(5) at inducing hypotension and bradycardia (36) ; this is the same rank order of affinity of these ligands for AP-3.

PP-InsP(5) had a 5-10 fold higher affinity for AP-3 compared with InsP(6). Therefore our experiments should provide further impetus to the goal of defining the intracellular distribution of both of these inositol polyphosphates. Total cellular InsP(6) is around 15 µM(3) , but it has often been considered likely that much of this material does not have immediate access to the cytosol, but instead is sequestered inside an organelle, or bound to cellular membranes(5, 6) . Indeed, the only enzyme known to dephosphorylate InsP(6) inside mammalian cells is itself restricted to the interior of endoplasmic reticulum (33) . However, levels of InsP(6) in N1E-115 neuroblastoma cells and cerebellar granule cells have been reported to change rapidly in response to extracellular stimuli(26) . Total cellular levels of PP-InsP(5) are about 5% those of InsP(6)(2, 28) , but it is also not known where in the cell this particular compound may be located. Nevertheless, levels of PP-InsP(5) are regulated by changes in intracellular Ca(28) , which opens up another potential molecular basis for the regulation of AP-3 function and synaptic vesicular traffic by extracellular stimuli.


FOOTNOTES

*
This work was supported by NINDS Grant NS29051 (to E. M. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Institute of Biotechnology, University of Texas Health Science Center, 15355 Lambda Dr., San Antonio, TX 78245. Tel.: 210-567-7220; Fax: 210-567-7277.

(^1)
The abbreviations used are: InsP(6), inositol hexakisphosphate; PP-InsP(5), diphosphoinositol pentakisphosphate; Ins(1,2,4,5,6)P(5), inositol-1,2,4,5,6-pentakisphosphate; Ins(1,3,4,5,6)P(5), inositol-1,3,4,5,6-pentakisphosphate; Ins(1,3,4,5)P(4), inositol-1,3,4,5-tetrakisphosphate; Ins(1,4,5)P(3), inositol-1,4,5-trisphosphate; InsS(6), inositol hexasulfate; GST, glutathione S-transferase; BSA, bovine serum albumin.

(^2)
E. M. Lafer, W. Ye, and N. Hrinya-Tannery, manuscript in preparation.

(^3)
S. Zhou, W. Ye, and E. M. Lafer, manuscript in preparation.


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