Vol. 749, No. 2, 1987 December 16, 1987

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Pages 680-685

SPECIFIC BINDING SITES FOR [3H]INOSITOL(l ,3,4,5)TETRAKISPHOSPHATE

ON MEMBRANES OF HL-60 CELLS

Peter G. Bradford* and Robin F. Irvine’

*Department of Pharmacology, Hahnemann University, Broad & Vine, Philadelphia, PA 19102-l 192

+Department of Biochemistry, AFRC Institute of Animal Physiology, Babraham. Cambridge CB2 4AT, UK.

Received November 3, 1987

Membranes of HL-60 cells were shown to possess saturable binding sites for [3H]inositol(l,3,4,5) tetrakisphosphate, with nanomolar affinity (K = 90 nM) and a density of 250 fmollmg protein. The specificity of the binding sites for Ins(l,?,4,5)P was assessed by competition studies utilising a variety of inositol polyphosphates; results indicate cl that both the presence and the correct grouping of the phosphates were important for high afftnity recognition. The apparent affinity of the binding sites for Ins(l,3,4,5)P, was over 200-fold greater than for Ins(l,4,5)P,. The possibility is discussed that this binding site represents the receptor which mediates the action of Ins(l,3,4,5)P, as a putative intracellular second messenger. Q 1987 Academic Press, Inc.

Ins(l,4,5)Ps is now thought to be the intracellular messenger responsible for Ca*+-mobilization

(1,2), and its biological activity has been correlated directly with specific and saturable binding of

radiolabelled Ins(l,4,5)Ps to cell membranes (3). Recently, another inositol phosphate which is also

of potential physiological importance has been discovered, Ins(1 ,3,4,5)P4 (ref. 4). This compound

shows specific biological activity at micromolar concentrations in eggs of Lytechirtus variegutus (5,6)

and in mouse lacrimal glands (7). These data are consistent with the proposal that Ins(l,3,4,5)P,

regulates Ca 2+ . -Influx into an Ins(l,4,5)P,-sensitive store (6,8), i.e. its proposed biological function

is to regulate the amount of Ca*+ to which Ins(l,4,5)Ps has access.

One of the criteria necessary to define further this putative physiological role of

Ins(l,3,4,5)P, is the demonstration of an intracellular receptor for this compound , As a first step in

this process we have here documented the evidence for a specific and saturable

r3H]Ins(l ,3,4,5)P4-binding site on membranes of HL-60 cells.

MATERIALS AND METHODS

[‘H]Ins(l,3,4$)P, was either purchased from New England Nuclear (specific activity 3-4 Ci/mmol) or was made from [s$I]Ins(l,4,5)P, of the same specific activity by methods similar to those described in refs. 10 and 20. [ P]Ins(l,4,5)Ps was a generous gift from Amersham (U.K.).

Preparation of inositol phosphates. Ins(l,3,4,5,6)Ps and Ins(l,4,5,)Ps were purchased from Calbiochem (San Diego, CA) and InsP from

Sigma (St. Louis, MO) and were not purified further. InsP from avian erythrocytes was purchase B from

Abbreviations: InsP phosphates 1’

InsP4, In@, and InsPs: Inositol tris-, tetrakis-, pentakis- and hexakis- respective y. with tsomeric numbering as appropriate.

assumed. Unless stated otherwise, D-numbering is

0006-291X/87 $1.50 Copyright 0 1987 by Academic Press, Inc. AN rights of reproduction in any form reserved. 680

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Boehringer Mannheim (FRG); note that the recent work P rom Stephens er al. (9) has established this InsP,, advertised as Ins(l,4,5,6)P,, to be actually D-Ins(3,4,5,6)P,. Ins(l,3,4)P, (HPLC-pure) and Ins(1,3,4,5)P, were prepared as in Irvine er ul. (lo), and Ins(w,x,y,z)P4 - a random mixture of InsP, isomers produced by acid hydrolysis of InsPs - was prepared as in Irvine & Moor (5).

Ins(3,4,5)Ps was prepared by partial dephosphorylation of Ins( 1,3,4,5)P, by alkaline phosphatase as in Batty er al. (4). followed by HPLC as in Irvine et al. (11). Ins(3,4,5)Ps chromatographs just after Ins(1,4,5)Ps apzd fractions were taken to ensure less than 1% contamination with Ins(1,4,5)Ps as determined using [ P]Ins(l,4,5)Pz as an internal standard. They were desalted as in ref. 10 and checked for absence of inorganic phosphate by ionophoresis (12).

HL-60 cell culture and preparation of membranes. HL-60 cell cultures were maintained in RPM1 1640 medium (Grand Island Biological Co., Grand

Island, N.Y.) supplemented with 10% heat-inactivated fetal bovine serum, 5 mM glutamine, penicillin (100 units/ml) and streptomycin (100 units/ml) in a 7% CO, atmosphere at 37‘C. Cells were harvested by centrifugation (200x g, 10 min), and then homogenized (10 passes, Wheaton teflon-to-glass) and sonicated (Branson Cell Disrupter 185, setting 4 for 30 set) in ice-cold isotonic buffer (250 mM sucrose, 20 mM Tris-maleate pH 6.8, 1 mM dithiothreitol, 0.2 mM benzamidine, 0.1 mM phenylmethylsulfonyl fluoride and 1 mg soybean trypsin inhibitor/l00 ml). Unbroken cells and nuclei were removed by centrifugation (800x g, 10 min), and the resultant supernatant was recentrifuged (100,000x g, 40 min) to yield a crude membrane pellet. The membranes were resuspended at 10 mg protein/ml of 25 mM Tris pH 7.2, 1 mM EDTA, and either used fresh or frozen at -20’C until use (less than 4 weeks maximum).

Radioligand binding assay. [‘H]Ins(l,3,4,5)P,-bind& to HL-60 membranes was ayessed by a filtration assay in a medium

similar to that described for [ P]Ins(l,4,5)P binding (3). [ H]Ins(1,3,4,5)P,, ca. 40 nM in a total volume of 0.25 ml, was incubated with 0.4 - 0.6 mg membrane protein for 20 min at 0°C in a cytosolic- type medium (13) of the following composition: 100 mM KCI, 20 mM NaCI. 25 mM NaHCO , 5 mM Hepes (pH 7.2), 1 mM MgSO , 0.96 mM NaHzPO 1.5 mM ATP, 5 mM creatine phosphate, lC?U/ml creatine phosphokinase, 1 mM E&TA and 0.4 mM CaC?l yielding a buffered free Ca*+ concentration of approximately 120 nM. For association kinetic studies, in&bation times were from 1 to 30 min. For dissociation kinetic studies, the radioligand was allowed to incubate with membranes for 20 min. then nonradiolablled Ins(1,3,4,5)P4 was added (final concentration of 10 PM) and samples were taken at specified times up to 10 min. Reactions were stopped by dilution with 3 ml of ice-cold 25 mM Tris (pH 7.2) 1 mM EDTA followed by immediate vacuum filtration through Whatman GF/C filters and washing with an additional 3 ml of Tris/EDTA buffer. This stop-procedure takes approximately 5 sec. Dried filters were assayed for radioactivity by scintillation-counting with 10 ml Ecolume (ICN Radiochemicals, Irvine, CA). Nonspecific binding was determined by including 10 PM Ins(1,3,4,5)P in the incubation medium; increasing the Ins(1,3,4,5)P4 concentration to 30 PM did not further d re uce binding. Specific binding generally exceeded 600 dpm. Approximately 90% of the radioligand remained at the end of the 20 min incubation period as InsP,, as assessed by anion-exchange chromatography of the assay filtrates.

RESULTS

Binding of 40 nM [3H]Ins(1,3,4,5)P, to membranes of HL-60 cells attained a steady state within

20-30 min at 4’C, and could be readily reversed by the addition of a 250-fold excess of unlabelled

Ins(1 ,3,4,5)P4 (Fig. 1). Experiments performed with increasing concentrations of radioligand in either

the presence or absence of 10 PM Ins(1,3,4,5)P,, demonstrated that the binding was saturable, with

nonspecific binding representing about 10% of total binding. Scatchard analysis of specific

[3H]Ins(l,3,4,5)P,-binding data indicated that the ligand recognised apparently a single binding site,

present at a density of 250 f 19 fmohmg protein, and having an apparent dissociation constant (K,) of

90 f 12 nM (Fig. 2); this estimated K, agreed well with that calculated from the ratio of the

dissociation and association rate constants (100 nM).

Competition experiments, performed using 40 nM [3H]Ins(1,3,4,5)P, and an extensive range of

inositol polyphosphates, demonstrated a clear specificity of the binding site for Ins(l,3,4,5)P,, which was over 200-fold more potent than its precursor, Ins(1 ,4,5)P3 (Fig. 3).

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-

1 I

0 IO 20 30

TIME (minutes)

80

60

40

20

0

I. ?gure Time course of binding of [‘H]Ins(l,3,4,.5)P, to (a). and dissociation of bound [ H]Ins(1,3,4,5)Pq from (0) HL-60 cell membranes. For dissociation experiments, 10 $M Ins(I ,3,4,5)f’, was added to the reachon mixture (arrow) after a 20 min incubation in the presence of [ H]Ins(l .3,4,5)P4 and the time course of the decrease in specific binding was determined. Specific binding for the assoctation reaction is expressed as a percentage of total specific binding at 30 min. Values are means of two experiments each done in duplicate. One hundred percent specific binding averaged 550 dpm.

The data presented in Figure 3 also provided some information on the structural requirements of

this binding site. A phosphate in position 3 of the myo-inositol ring was clearly essential for

recognition [comparing Ins(1,3,4,5)P, and Ins(l,4,5)PJ. Removal from Ins(1,3,4,5)P, of the

50 100 150 200 250

I BOUND (fmollmg)

50 100 150 200 250 300

TOTAL lns(1,3,4,5)P4 (pmoi)

2. Figure Specific binding of [3H]Ins(l ,3,4,5)P4 to HL-60 membranes as a function of increasing concentration of ligand. Incubations were performed as described in “Materials and Methods”. The maximum density of binding sites was 250 f 19 fmollmg. The calculated dissociation constant (KJ was 90 f 12 nM (n = 3). Results from a typical experiment are shown. (INSET) Scatchard plot of the binding data.

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100

80 / lrlS(1,4,5iP3

a ++l ,lrlS(1,3,4P ‘0 f sfs ‘?s,, \ 3 bls(w.x,y,z)P4

60

\

40

20

0 03 8 7 6 5 4

-log[COMPETITORl (M)

Figure 3. Cfmpetition by inositol polyphosphates for [‘H]Ins(l,3,4,5)P, binding sites in HL-60 membranes. [ H]Ins(l,3,4.5)P4-binding was determined in the presence and absence of the indicated concentrations of the various inositol polyphosphates: o, Ins(1.3.4,5)P; 0, Ins(1,3.4,5.6)P,; 0 Ins(3.4.5)Ps; n , Ins(w.x,y,z)Pd; x, InsP,; ‘1. Ins(3.4.5.6)P ; A, Ins(l,3,4)Ps: v, Ins(l,4,5)f,.

, Values

are either means f standard errors (n = 3 or 4) or means of cl uplicate experiments.

5-phosphate [to give Ins(1,3,4)P,] also resulted in a marked decrease in the apparent affinity. while

removal of the l-phosphate [Ins(3,4,5)P,] caused only a 4-fold decrease in the apparent affinity. From

these observations we can speculate that the 3.4.5grouping of the phosphates on the ring is essential

for high-affinity binding. Transfer of the phosphate from the l- to the 6-position [Ins(3,4,5,6)P,]

resulted in a marked decrease in apparent affinity, and yet having phosphates in both these positions

[Ins(l,3,4,5,6)P,] resulted in only a 3-fold decrease in apparent affinity: addition to

Ins(1,3,4,5,6)Ps of a phosphate (InsPJ resulted in a further decrease in apparent affinity. These

results indicate that, as for Ins(l,4,5)Ps-binding to the receptor associated with Ca*+-mobilization

(refs. 14,15), the presence of a phosphate on the l-position may be necessary to orientate the “active”

grouping. The results also suggest that the 6-position has little interaction with the recognition

site.

We have at present no other myo-inositol polyphosphates to test whether any configuration

consisting of three adjacent phosphates plus a single phosphate, will suffice to be recognised with

high affinity by this binding site. The assumed random mixture of InsP, isomers [Ins(w,x,y,z)PJ

should have about 40% of its components with this configuration and would therefore be expected to have

a potency about 2.5~times less than that for Ins(l ,3,4,5)P4. In fact, the binding site had an affinity

for Ins(w,x,y,z)P, which was about lo-times less than that for Ins(1,3,4,5)P,, consistent with the

suggestion that the binding site has an absolute requirement for the 3, 4 and 5 phosphates.

DISCUSSION

Although the data presented here demonstrate a specific and saturable site for

r3H]Ins(l ,3,4,5)P4-binding, they do not establish that this is the intracellular receptor for the

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physiological action of Ins(l .3,4,5)Pd. However, we suggest that it may be, because (a) the binding

characteristics are not consistent with other known inositol polyphosphate-binding proteins which might

interact with Ins(1,3,4,5)Pd and (b) the binding characteristics are consistent with the presently

known specificity of the biological activity of Ins( I ,3,4.5)/6.

With respect to (a) there are six proteins which might conceivably bind Ins(1,3,4,5)P4. The one

most likely is Ins(l,3,4,5)P,-5-phosphatase (16,17). However, the available evidence, both with the

purified enzyme from platelets (16) and in permeabilized hepatocytes (17), suggests that Ins( 1,4,5)Ps

also binds to that enzyme (and is a substrate for it). As substrates for this phosphatase,

Ins(l,4,5)Ps and Ins(1,3,4,5)P, compete with each other, the former having a Km about ten times higher

than the latter (16); the inability of Ins(1,4,5)Ps to compete out Ins(1,3,4,5)P, in the present

binding assays therefore argues against the idea that this binding is to the 5-phosphatase. The

selectivity of Ins(1,3,4,5)P, over Ins(l,4,5)1; (Fig. 3) also rules out the possibility that the

binding site represents the Ins(1,4,5)Ps-“receptor”, which is connected with Ca*+ mobilization (3), for

which Ins(1,3,4,5)P, does not compete at micromolar levels (e.g. ref. 10). One possibility that we

cannot eliminate for lack of experimental information, is that Ins(1,3,4,5)P, could bind to

Ins(l,4,5)P3-3-kinase (which is partly membrane-bound, at least in turkey erythrocytes, ref. 19).

Ins(1 ,3,4,5)P4 is the product of the reaction catalysed by this kinase, so that, depending on the

mechanism of the enzyme, Ins(1,3,4,5)P, could bind reversibly to it. However, the affinity of

Ins(l,4,5)P,-kinase for Ins(l,4,5)P, is around 0.6 PM (e.g. ref. 20) and simplistically one would

therefore expect Ins(l,4,5)Ps to compete with about that affinity [i.e. in these experiments, an

affinity roughly equal to that of Ins(1,3,4,5)PJ; the possible modulation of inositol

phosphate-binding by the other components of the reaction (ATP and ADP) however, makes such a

prediction speculative. Nevertheless, we believe that in summary, the inability of Ins(1,4,5)Ps to

compete effectively with Ins(1,3,4,5)P, (Fig. 3) does argue against this binding being to any of the

three known Ins(l,4,5)Ps-binding proteins.

The much weaker binding of Ins(1,3,4)Ps (Fig. 3) probably also rules out the

Ins(l,3,4)Ps-1-phosphatase and Ins(l,3,4)Ps-4-phosphatase isolated by Inhorn et al. (21) and Bansal er

al. (22), neither of which hydrolyses Ins(1,3,4,5)P,. Finally, the low apparent afftnity of

Ins(3,4,5,6)P, (Fig. 3) makes it unlikely that an InsP,-kinase is being studied, as the data of

Stephens ef al (23) show that in brain homogenates Ins(3,4,5,6)P, is a much better substrate than

Ins(1,3,4,5)P, for conversion of InsP, to InsPs.

While some of the characteristics of this binding are therefore not consistent with known

proteins which bind inositol polyphosphates, a number of them support the suggestion that the binding

site is associated with the physiological “receptor” for Ins(l,3,4,5)P,. For example, the low apparent

affinity of Ins(l,4,5)Ps is consistent with the properties of the site of Ins(l,3,4.5)P4’s action in

mouse lacrimal glands (7). because in that tissue whereas 100 PM Ins(1,4,5)PJ produces no effect on

Ca*+-controlled K+-efflux, 10 PM Ins(1,3,4,5)P, (with 10 ,EM Ins(l,4,5)P, present) causes a marked

stimulation. The rank order of potency in Fig. 3 also correlates well with the action of

Ins(l,3,4,5)P, in eggs of Lyrechinus vuriegarus (5.6). in which Ins(1,3,4)P, is without effect (ref.

5), Ins(w,x,y,z)P4 has a IO-fold lower potency (ref. 5). while Ins(1,3,4,5,6)Ps has a potency similar

to Ins(1,3,4,5)P, (ref. 6).

In conclusion, while these data do not prove that this binding is to the site of biological

action of Ins(1 ,3,4,5)P4, they are consistent with it; this identification of a saturable and specific

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binding site for Ins(1,3,4,5)P, is thus a further step in the demonstration of the physiological role

of this compound.

ACKNOWLEDGYENTS

We are very grateful to A.3. Letcher and D.J. Lander for help in preparing the inositol phosphates, and to K.A. Wreggett and L.R. Stephens for helpful discussions. We thank Mrs S.L. Saunders for typing the manuscript. This work was supported in part by NIH grant R29GM39588 to PGB.

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Irvine, R.F.. Brown, K.D. & Berridge, M.J. (1984). Biochem. J. 222, 269-272. Burgess, G.M., Irvine, R.F., Berridge, M.J., McKinney, J.S. & Putney, J.W. Jnr. (1984). Biochem. J. 224. 741-746. Connolly, T.. Bansal, VS., Bross, T.E.. Irvine, R.F. & Majerus, P.W. (1987). J. Biol. Chem. 262, 2146-2149.

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Joseph, S.K., Hansen, C.A. & Williamson, J.R. (1987). FEBS Letters 274, 125-129. Willocks, A.L., Crooke, A.M., Potter, B.V.L. & Nahorski, S.R. (1987). Biochem. Biophys. Res. Commun. 146, 1071-1078.

19. Morris, A.J., Harden, T.K., Downes. C.P. & Michell. R.H. (1987). Biochem. J. (in press). 20. Irvine, R.F., Letcher, A.J., Heslop, J.P. & Berridge, M.J. (1986). Nature 320, 631-634. 21. Inhorn, R.C., Bansal, V.S. & Majerus, P.W. (1987). Proc. Natn. Acad. Sci. USA 84, 2170-2174. 22. Bansal, V.S., Inhorn. R.C. & Majerus, P.W. (1987). J. Biol. Chem. 262, 9444-9447. 23. Stephens, L.R., Hawkins, P.T., Morris, A.J. & Downes. C.P. (1987). Biochem. J. (in press).

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