Abstract
The specificity of the binding site of p42 IP4 , a high-affinity 42 kDa Ins (1,3,4,5)P 4 receptor protein identified by photoaffinity labelling (Reiser et al., Biochem. J. 280, 533, 1991) was analyzed by determining the affinities for all possible inositoltris-, -tetrakis-, and -pentakisphosphate regioisomers. We tested the purified receptor protein displaying a K d of 2.2 nM for Ins (1,3,4,5)P 4 which was unequalled by any of the other inositoltetrakis-and -trisphosphate regioisomers. The affinities of inositoltetrakisphosphates were 25 to 150 times lower, with a substitution at C-2 having the largest effect in reducing the affinity. The inositoltrisphosphate isomers were three orders of magnitude less potent than Ins (1,3,4,5)P 4 , apart from D/L-Ins (3,4,5)P 3 . The pentakisphosphate Ins(1,3,4,5,6)P 5 had an affinity for the solubilized and purified receptor comparable to that of D-Ins(1,3,4,5)P 4 . This lack of discrimination was unique for the solubilized receptor, since it was not observed with the membrane-associated receptor protein. D-Ins(1,3,4,5)P 4 has been suggested to be a second messenger regulating the intracellular Ca 2/ concentration but its precise function is still largely unknown. Specific high-affinity binding sites for D-Ins(1,3,4,5)P 4 have been found in a wide range of tissues [4] . The subcellular distribution of Ins(1,3,4,5)P 4 binding sites has been studied in human platelets [5] and rat liver [6] .
Ins(1,3,4,5)P 4 binding proteins have been purified from different species and tissues. A 104 kDa InsP 4 receptor purified from pig platelets [7] identified by cDNA sequencing codes for a member of the GTPase-activating protein (GAP) family and was therefore named GAP1 IP4BP [8] . Another 104 kDa InsP 4 binding protein probably identical to GAP1 IP4BP was recently 1 Please send correspondence and reprint request to Professor G. Reiser, Otto-von-Guericke-Universität Magdeburg,

D-myo-inositol 1,4,5-trisphosphate (D-Ins(1,4,5)P 3 ), a product of receptor -activated hydrolysis of phosphatidylinositol 4,5-bisphosphate, is a well established second messenger in Ca 2/ -signalling by releasing Ca 2/ from intracellular stores via Ins(1,4,5)P 3 -gated channels [1, 2] . D-Ins(1,4,5)P 3 is metabolized partially by dephosphorylation catalysed by InsP 3 5-phosphatase, yielding D-Ins(1,4)P 2 which is further metabolized to free inositol. Alternatively, phosphorylation leads to D-inositol 1,3,4,5-tetrakisphosphate (D-Ins(1,3,4,5)P 4 ) through the action of a InsP 3 3-kinase (reviewed in [3] ).
D-Ins(1,3,4,5)P 4 has been suggested to be a second messenger regulating the intracellular Ca 2/ concentration but its precise function is still largely unknown. Specific high-affinity binding sites for D-Ins(1,3,4,5)P 4 have been found in a wide range of tissues [4] . The subcellular distribution of Ins(1,3,4,5)P 4 binding sites has been studied in human platelets [5] and rat liver [6] .
Ins(1,3,4,5)P 4 binding proteins have been purified from different species and tissues. A 104 kDa InsP 4 receptor purified from pig platelets [7] identified by cDNA sequencing codes for a member of the GTPase-activating protein (GAP) family and was therefore named GAP1 IP4BP [8] . Another 104 kDa InsP 4 binding protein probably identical to GAP1 IP4BP was recently purified from human platelets and separated from an InsP 6 binding protein which was found to be vinculin [9] . An InsP 4 binding protein from mouse cerebellum which showed higher affinity for Ins (1, 3, 4, 5, 6 )P 5 than Ins(1,3,4,5)P 4 was identified as synaptotagmin II [10] . From rat liver nuclei a 74 kDa high-affinity Ins(1,3,4,5)P 4 receptor was purified [11] . Besides an InsP 6 binding protein which was identified as clathrin assembly protein AP2 [12] two distinct Ins(1,3,4,5)P 4 binding protein complexes were purified fom rat cerebellum [13] . Recently a phosphatidylinositol 3,4,5-trisphosphate (PtdPIP 3 ) binding protein from rat brain was purified and molecularly cloned [14] . This 46 kDa protein was designated as centaurin-a and was shown to bind phosphatidylinositol 3,4,5-trisphosphate (PtdPIP 3 ) with higher affinity than Ins(1,3,4,5)P 4 . We have previously purified and characterized p42 IP4 an Ins(1,3,4,5)P 4 receptor from pig cerebellum which binds D-Ins(1,3,4,5)P 4 with high affinity [15] . The 42 kDa protein was identified by photoaffinity-labelling [16] . Immunological studies using an antipeptide antiserum suggested that this protein exists in a membrane-associtated and in a cytosolic form [17] . Here we investigate the binding specificity of this Ins(1,3,4,5)P 4 receptor protein using all InsP 3 , InsP 4 and InsP 5 regioisomers (in the case of asymmetric regioisomers as racemic pairs) and some other inositolphosphates. The aim of this study was to identify the stereochemistry of the phosphate groups needed for high affinity binding to the purified receptor protein. Synthesis of inositolphosphates. All regioisomers of InsP 4 , InsP 3 and InsP 5 were synthesized by S.-K. Chung and coworkers, as previously described [18, 19, 20] .
MATERIALS AND METHODS

Materials
Receptor purification. The InsP 4 receptor protein was purified from pig cerebellum as described previously [15] . The last step of the purification protocol was modified by using the detergent LDAO (N,N-dimethyl-dodecylamine-N-oxide) instead of Brij 58. The purity of the fraction containing the receptor protein was checked by SDS-PAGE. Western blot using an antipeptide antiserum directed against a 19 amino acid peptide of the receptor protein allows to identify the 42 kDa protein [17] . In some experiments the monoclonal antibody Mab Cl 41.1 directed against the cytosolic fragment of synaptotagmin I [21] or Mab 46-1 directed against the myelin protein CNP (2,3-Cyclic Nucleotide 3-phosphodiesterase; [22, 23] ) was employed.
Binding assays. Binding of Ins(1,3,4,5)P 4 to the purified receptor protein was determined as described previously [15] . The InsP 4 receptor protein (50 to 250 ng) was assayed in 280 ml of buffer (25 mM sodium acetate/25 mM potassium phosphate, pH 5.0, 0.05 % Brij 58, 1 mM EDTA) supplemented with 0.8 to 1.1 nM [ After incubation for 20 min at 4ЊC, an aliquot of the reaction mixture was centrifuged through columns, containing 1.2 ml packed resin of BioGel P-4 (grade fine 65 mm; Bio-Rad) equilibrated with binding buffer, to separate bound from free ligand. 3 ml of scintillation fluid (Ultima Gold, Packard) were added for measuring the bound radioactivity. All determinations made in duplicate or triplicate showed less than 15 % deviations.
Data analysis. K d values and the number of binding sites were estimated by using the RADLIG data-analysis computer program (Version 4; BIOSOFT, Cambridge, UK) as described previously [24] . In competition assays the data obtained with the displacing inositolphosphate were fitted to the data obtained for the displacement of [ protein which has been identified functionally by photoaffinity labeling [16] and in some cases traces of other proteins with molecular mass of 46, 37 and rarely another one with 32 kDa, as shown previously by SDS-PAGE ( [16] and Fig. 1 lane 1) . We identified two of these proteins by isolation and peptide sequencing. The 46 kDa protein was demonstrated to be the myelin protein CNP [22] . The 37 kDa protein turned out to be the cytosolic fragment of synaptotagmin I as demonstrated by the N-terminal sequence of the protein (Stricker and Reiser, unpublished data). The identity of the 37 kDa protein being the cytosolic fragment of synaptotagmin I was confirmed by Western blotting using the monoclonal antibody MAb Cl 41.1 [21] . In Fig. 1 , lane 1 the SDS-PAGE of a fraction typically used for the binding assays described in the following section of this paper is shown. The identity of each, the InsP 4 receptor protein (lane 4), the CNP (lane 2) and the cytosolic part of synaptotagmin I (lane 3) is demonstrated by Western blotting. Fig. 1 , moreover, shows that the 42 kDa protein is the major component in this fraction. In most cases the minor components could hardly be detected in the protein gel analysis. For immunological detection of the minor proteins in Western blot analysis a total amount of protein had to be applied which was at least 10 times higher than that needed to identify the InsP 4 receptor protein.
Binding assays were carried out with fractions similiar to that shown in Fig. 1 . We employed all the trisphosphate, tetrakisphosphate and pentakisphosphate regioisomers of myo-inositol, the mesoforms and in the case of asymmetric regioisomers the racemic pairs. For some compounds the D-enantiomers were available. We also tested the water-soluble phosphatidylinositol (3,4,5)trisphosphate-analogue D-GroPIP 3 .
The incubations were carried out in an acetate/phosphate buffer, pH 5.0 which was shown to yield highest binding of [ Among the inositoltrisphosphates only D/L-Ins(1,5,6)P 3 displayed unique behaviour (see below). All other inositoltrisphosphates tested were more than three orders of magnitude less effective in displacing [ higher affinity than all the other InsP 3 , regioisomers tested. As indicated in Table 2 [31] . This inositoltrisphosphate analogue showed no affinity for the InsP 4 receptor at concentrations of up to 10 mM, which is comparable to the data obtained with D-Ins(1,4,5)P 3 ( Table 2) .
For further characterization of the InsP 4 binding site all inositolpentakisphosphates (two mesoforms and two racemic pairs) were synthesized and used in displacement experiments. The results of these experiments are also summarized in Table 2 . Ins(1,2,3,4,6)P 5 is the weakest ligand in displacing We previously reported that the affinity of the InsP 4 receptor for Ins(1,3,4,5,6)P 5 in mem-branes was more than ten times lower than the value found for D-Ins(1,3,4,5)P 4 . After solubilization of the receptor protein by detergent from the membrane fraction the affinity for Ins (1,3,4,5,6)P 5 was increased 10-fold [15] . Similarly the affinity of the receptor for D/LIns(1,2,4,5,6)P 5 was increased by a factor of 5 to 10 when the protein was solubilized with detergent from membranes, whereas there was no effect of solubilization and purification on the K i for D/L-Ins (1,2 (Table 2) . However, these compounds are only poor displacers of [ 3 H]Ins(1,3,4,5)P 4 when tested with membranes from pig cerebellum displaying K i -values higher than 1 mM (data not shown). InsP 6 which has already been tested previously [15, 16] inhibited binding of DIns(1,3,4,5)P 4 with a K i value of at least 10 mM.
DISCUSSION
We show here the ligand specificity of a previously identified InsP 4 receptor by testing all possible InsP 3 , InsP 4 and InsP 5 regioisomers (asymmetric compounds as racemic mixtures) synthesized as described [18, 19, 20] . From some of the asymmetric regioisomers also the stereochemically pure D-isomers, were used as well as D-GroPIP 3 and the diphosphoinositol polyphosphates PP-InsP 5 and PP-InsP 4 -PP. Among all the compounds tested we found three isomers which displaced nearly equipotently [ In membranes from pig cerebellum D-Ins(1,3,4,5)P 4 and D-GroPIP 3 also show the same high affinity (not shown). However, the affinity of Ins(1,3,4,5,6)P 5 is at least tenfold lower with the receptor in membranes than with the detergent solubilized receptor protein [15] . The reason for the difference in selectivity for Ins(1,3,4,5,6)P 5 versus Ins(1,3,4,5)P 4 may due to the presence of detergent rather than to the removal of membrane lipids, since we recently found that the receptor protein exists also in a cytosolic, soluble form. This cytosolic protein has the same ligand affinity as the membrane associated protein for Ins(1,3,4,5,6)P 5 (not shown).
The binding data given in Tables 1 and 2 . This is also highlighted by the fact that phosphorylation at C-1, C-3 and C-4 or at C-1, C-3 and C-5 does not yield a high-affinity ligand but that phosphorylation at C-3, C-4 and C-5 or the additional phosphorylation of C-4 or C-5 in Ins(1,3,5)P 3 or D/L-Ins(1,3,4)P 3 , respectively, enhances the ligand affinity.
The phosphorylation of the hydroxyl at C-1 increases the affinity of p42 IP4 for inositolphosphates which are phosphorylated at C-3, C-4 and C-5 (compare D/L-Ins(1,3,4,5)P 3 and D/LIns(3,4,5)P 3 or D/L-Ins(3,4,5,6)P 4 and Ins(1,3,4,5,6)P 4 ). Further substitution at C-1, i.e. esterification of the phosphate group with the glycerol moiety in D-GroPIP 3 does not diminish but seems to increase the affinity of the ligand for the binding site (Table 1) . Previously we had reported the synthesis and usage of a photoaffinity analogue of D-Ins(1,3,4,5)P 4 in which the phosphate group at C-1 of the inositol ring was coupled to an N-(4-azidosalicyl)aminoethanol moiety. This modification reduced the ability of this compound to displace D- [ 3 H]Ins(1,3,4,5)P 4 from receptor binding by a factor of three [16] . Investigations with phosphatidylinositol 3,4,5-trisphosphate derivatives which have additional fatty acids esterified to the glycerin moiety may give more insight whether these compounds are possible endogenous ligands for the InsP 4 -receptor.
For the 104 kDa Ins(1,3,4,5)P 4 binding protein purified from porcine platelets a specificity different from that reported here was described [33] . The binding site on the 104 kDa protein was shown to be highly selective for D-Ins(1,3,4,5)P 4 and to recognize primarily the phosphates at C-1, C-3 and C-5, whereas a phosphate group at C-4 and C-6 had only little influence on binding. A phosphate group at C-2 was not tolerated [33] as with the 42 kDa InsP 4 receptor from pig brain.
The relatively high affinities of the inositolpentakisphosphates and of the diphosphoinositol polyphosphates seen here could be due to the high negative charge of these molecules interacting with the basic InsP 4 receptor molecule at the binding assay conditions [15, 30] .
The receptor preparations used throughout the experiments described here were shown to contain, beside the 42 kDa InsP 4 receptor protein (Fig. 1 lane 1 and 4) traces of some contaminating proteins [15] . Two of these proteins could be identified, the 46 kDa protein as the myelin protein CNP [22] and the 37 kDa protein as the cytosolic fragment of synaptotagmin I ( Fig. 1 lane 2 and 3) . The cytosolic fragment of synaptotagmin I contains two C2-domains from which the C2B-domain was reported to bind Ins(1,3,4,5)P 4 and other inositolpolyphosphates [34] . Interestingly this domain was also found to bind Ins(1,3,4,5)P 4 in the GAP1 IP4BP from pig platelet [8] . C2B-domains are present in different proteins, for example synaptotagmin I-IV, rabphilin 3A, protein kinase Ca, GTPase activating proteins (GAP), but inositolphosphate binding is only found in those C2B-domain containing proteins which possess a cluster of consecutive lysin residues [8, 34] . A sequence homologous to this inositolphosphate binding motive from C2B-domains was recently described for centaurin-a, a possible PIP 3 receptor protein from rat brain [14] .
The following findings exclude that the inositolphosphate binding measured in the experiments described in this paper could be partly due to the contaminating synaptotagmin I fragment. The B max values found for the receptor preparations (approx. 10 nmol/mg protein) are much too high to correlate with the trace amount of the synaptotagmin I fragment in this fraction (compare Fig. 1, lane 1, lane 3 and lane 4 ). Photoaffinity labelling with N-(4-azidosalicyl)aminoethanol(1)-1-phospho-D-myo-inositol 3,4,5-trisphosphate using a receptor fraction comparable to that in Fig. 1 resulted in the specific labelling of the 42 kDa protein [16] but not the 37 kDa protein. Another striking point is the difference in specificity and affinity for Ins(1,3,4,5)P 4 and other inositolphosphates between synaptotagmin C2B-domains and the InsP 4 -receptor described here (compare [34] , and Table 1 and 2) .
Furthermore, using our purification scheme [15] the synaptotagmin I fragment was highly enriched during the purification of the InsP 4 -receptor from pig cerebellum in a specific fraction (the eluate obtained with buffer containing 500 mM NaCl from a heparin agarose column). Binding assays using this eluate yielded no significant specific Ins(1,3,4,5)P 4 binding. Therefore, we can conclude that the binding measured in the experiments described here is due to Ins(1,3,4,5)P 4 binding to the 42 kDa InsP 4 receptor protein.
The property of the 42 kDa InsP 4 receptor p42 IP4 to bind GroPIP 3 with an affinity comparable to that for Ins(1,3,4,5)P 4 is also found with centaurin-a [14] . Centaurin-a, a protein of 46 kDa, binds both PtdIns(3,4,5)P 3 and Ins(1,3,4,5)P 4 with, however, moderate affinities (IC 50 values of 120 and 620 nM for PIP 3 and Ins(1,3,4,5)P 4 , respectively). The absolute values of the affinities reported for these two proteins differ by more than two orders of magnitude. One possible reason for this discrepancy is the fact that Hammonds-Odie and coworkers made their determinations under non-equilibrium conditions i.e. by photoaffinity labelling. Surprisingly with [ 3 H]Ins(1,3,4,5)P 4 they could not detect reversible binding. In contrast to this, D-GroPIP 3 is only a weak ligand when tested with the GAP1 IP4BP displaying a ten times lower affinity than D-Ins(1,3,4,5)P 4 [7] . Molecular cloning of centaurin-a [14] showed that the peptide sequence identically contains peptide-3 from the 42 kDa InsP 4 receptor from pig cerebellum which we used previously for the generation of the antipeptide antiserum [17] . Using this antiserum we found in Western blots of membranes from different parts of the rat brain a protein comigrating in SDS-PAGE with the 42 kDa protein from pig cerebellum [17] , but not a protein from rat brain at 46 kDa (cf. Fig. 4 in [17] (Table 1) . This compound was also the strongest displacer when tested with the GAP1 IP4BP from pig platelets and further analysis showed that the GAP1 IP4BP had slightly higher affinity for D-Ins(1,4,5,6)P 4 /LIns(3,4,5,6)P 4 than for D-Ins(3,4,5,6)P 4 /L-Ins(1,4,5,6)P 4 [33] . In contrast to these results the D-Ins(3,4,5,6)P 4 isomer displaced [ 3 H]Ins(1,3,4,5)P 4 from binding to the purified Ins(1,3,4,5)P 4 receptor from rat liver nuclei with an IC 50 of 90 nM [11] . From our data presented above, i.e. the requirement of phosphate groups at C-3, C-4 and C-5 for high affinity binding we conclude that at the 42 kDa InsP 4 receptor from pig cerebellum, D-Ins (3,4,5,6)P 4 /L-Ins(1,4,5,6)P 4 is the displacing compound. Both compounds, D-Ins(3,4,5,6)P 4 and D-Ins(1,4,5,6)P 4 , were postulated to be possible second messengers in physiological processes ( [35, 36] and the references therein).
Here we made an in-depth analysis of the specificity of the binding site of the 42 kDa InsP 4 receptor. This is important for further experiments delineating the properties of the binding pocket of the protein p42 IP4 . Furthermore, our study using all regioisomers underpins the possibly unique role of the 42 kDa protein in the cell physiology, since all the inositolpolyphosphates possibly formed upon hormonal stimulation besides Ins(1,4,5)P 3 , e.g. Ins(3,4,5,6)P 4 have a significantly lower affinity for this protein. Physiological effects of Ins(3,4,5,6)P 4 reported [36, 37] are mediated via completely different ligand recognition sites which can be deduced from the affinity profiles [36, 37] which are not overlapping with the one described here.

{'paper_id': '18960959', 'title': 'Determination of Specificity of a High-Affinity Inositol 1,3,4,5-Tetrakisphosphate Binding Site at a 42 kDa Receptor Protein, p42IP4: Comparison of Affinities of All Inositoltris-, -Tetrakis-, and -Pentakisphosphate Regioisomers', 's2_url': 'https://api.semanticscholar.org/CorpusID:18960959', 'source': 'S2ORC'}