Mutational Analysis of the Ligand Binding Site of the Inositol 1,4,5-Trisphosphate Receptor
1996; Elsevier BV; Volume: 271; Issue: 30 Linguagem: Inglês
10.1074/jbc.271.30.18277
ISSN1083-351X
AutoresFumio Yoshikawa, Mitsuhiro Morita, Toshiaki Monkawa, Takayuki Michikawa, Teiichi Furuichi, Katsuhiko Mikoshiba,
Tópico(s)Drug Transport and Resistance Mechanisms
ResumoTo define the structural determinants for inositol 1,4,5-trisphosphate (IP3) binding of the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1), we developed a means of expressing the N-terminal 734 amino acids of IP3R1 (T734), which contain the IP3 binding region, in Escherichia coli. The T734 protein expressed in E. coli exhibited a similar binding specificity and affinity for IP3 as the native IP3R from mouse cerebellum. Deletion mutagenesis, in which T734 was serially deleted from the N terminus up to residue 215, markedly reduced IP3 binding activity. However, when deleted a little more toward the C terminus (to residues 220, 223, and 225), the binding activity was retrieved. Further N-terminal deletions over the first 228 amino acids completely abolished it again. C-terminal deletions up to residue 579 did not affect the binding activity, whereas those up to residue 568 completely abolished it. In addition, the expressed 356-amino acid polypeptide (residues 224-579) exhibited specific binding activity. Taken together, residues 226-578 were sufficient and close enough to the minimum region for the specific IP3 binding, and thus formed an IP3 binding "core." Site-directed mutagenesis was performed on 41 basic Arg and Lys residues within the N-terminal 650 amino acids of T734. We showed that single amino acid substitutions for 10 residues, which were widely distributed within the binding core and conserved among all members of the IP3R family, significantly reduced the binding activity. Among them, three (Arg-265, Lys-508, and Arg-511) were critical for the specific binding, and Arg-568 was implicated in the binding specificity for various inositol phosphates. We suggest that some of these 10 residues form a basic pocket that interacts with the negatively charged phosphate groups of IP3. To define the structural determinants for inositol 1,4,5-trisphosphate (IP3) binding of the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1), we developed a means of expressing the N-terminal 734 amino acids of IP3R1 (T734), which contain the IP3 binding region, in Escherichia coli. The T734 protein expressed in E. coli exhibited a similar binding specificity and affinity for IP3 as the native IP3R from mouse cerebellum. Deletion mutagenesis, in which T734 was serially deleted from the N terminus up to residue 215, markedly reduced IP3 binding activity. However, when deleted a little more toward the C terminus (to residues 220, 223, and 225), the binding activity was retrieved. Further N-terminal deletions over the first 228 amino acids completely abolished it again. C-terminal deletions up to residue 579 did not affect the binding activity, whereas those up to residue 568 completely abolished it. In addition, the expressed 356-amino acid polypeptide (residues 224-579) exhibited specific binding activity. Taken together, residues 226-578 were sufficient and close enough to the minimum region for the specific IP3 binding, and thus formed an IP3 binding "core." Site-directed mutagenesis was performed on 41 basic Arg and Lys residues within the N-terminal 650 amino acids of T734. We showed that single amino acid substitutions for 10 residues, which were widely distributed within the binding core and conserved among all members of the IP3R family, significantly reduced the binding activity. Among them, three (Arg-265, Lys-508, and Arg-511) were critical for the specific binding, and Arg-568 was implicated in the binding specificity for various inositol phosphates. We suggest that some of these 10 residues form a basic pocket that interacts with the negatively charged phosphate groups of IP3. INTRODUCTIONMany cellular responses to hormones, neurotransmitters, growth factors, etc. are mediated by the intracellular second messenger inositol 1,4,5-trisphosphate (IP3 or (1,4,5)IP3) 1The abbreviations used are: IP3 and (1,4,5)IP3inositol 1,4,5-trisphosphateIP3Rinositol 1,4,5-trisphosphate receptorIP3R1type 1 inositol 1,4,5-trisphosphate receptormAbmonoclonal antibody (1Berridge M.J. Nature. 1993; 361: 315-325Google Scholar)IP1inositol 1-phosphate(1,4)IP2inositol 1,4-bisphosphate(4,5)IP2inositol 4,5-bisphosphate(2,4,5)IP3inositol 2,4,5-trisphosphate(1,3,4,5)IP4inositol 1,3,4,5-tetrakisphosphateRyRryanodine receptor. (1Berridge M.J. Nature. 1993; 361: 315-325Google Scholar). IP3 releases Ca2+ from intracellular stores by binding to the IP3 receptor (IP3R) (2Supattapone S. Worley P.F. Baraban J.M. Snyder S.H. J. Biol. Chem. 1988; 263: 1530-1534Google Scholar), which is a tetrameric IP3-gated Ca2+ release channel (3Ferris C.D. Huganir R.L. Supattapone S. Snyder S.H. Nature. 1989; 342: 87-89Google Scholar, 4Miyawaki A. Furuichi T. Maeda N. Mikoshiba K. Neuron. 1990; 5: 11-18Google Scholar, 5Maeda N. Kawasaki T. Nakade S. Yokota N. Taguchi T. Kasai M. Mikoshiba K. J. Biol. Chem. 1991; 266: 1109-1116Google Scholar). There are at least three types of IP3R derived from distinct genes in mammals (6Furuichi T. Yoshikawa S. Miyawaki A. Wada K. Maeda N. Mikoshiba K. Nature. 1989; 342: 32-38Google Scholar, 7Mignery G.A. Newton C.L. Archer B.T. Sudhof T.C. J. Biol. Chem. 1990; 265: 12679-12685Google Scholar, 8Sudhof T.C. Newton C.L. Archer III, B.T. Ushkaryov Y.A. Mignery G.A. EMBO J. 1991; 10: 3199-3206Google Scholar, 9Blondel O. Takeda J. Janssen H. Seino S. Bell G.I. J. Biol. Chem. 1993; 268: 11356-11363Google Scholar, 10Maranto A.R. J. Biol. Chem. 1994; 269: 1222-1230Google Scholar, 11Yamada N. Makino Y. Clark R.A. Pearson D.W. Mattei M.G. Guenet J.L. Ohama E. Fujino I. Miyawaki A. Furuichi T. Mikoshiba K. Biochem. J. 1994; 302: 781-790Google Scholar, 12Yamamoto-Hino M. Sugiyama T. Hikichi K. Mattei M.G. Hasegawa K. Sekine S. Sakurada K. Miyawaki A. Furuichi T. Hasegawa M. Mikoshiba K. Recept. and Channels. 1994; 2: 9-22Google Scholar). Structural and functional studies on type I IP3R (IP3R1) (2749 amino acids, 313 kDa) have revealed that it is structurally divided into three parts: a large N-terminal cytoplasmic arm (83% of the receptor molecule); a putative six membrane-spanning domains clustered near the C terminus, which are thought to constitute an ion channel by forming a tetramer; and a short C-terminal cytoplasmic tail (13Furuichi T. Kohda K. Miyawaki A. Mikoshiba K. Curr. Opin. Neurobiol. 1994; 4: 294-303Google Scholar, 14Michikawa T. Hamanaka H. Otsu H. Yamamoto A. Miyawaki A. Furuichi T. Tashiro Y. Mikoshiba K. J. Biol. Chem. 1994; 269: 9184-9189Google Scholar).The binding of IP3 to this receptor purified from mouse cerebella is stoichiometric (Kd = ∼100 nM, Hill coefficient = ∼1.0) (2Supattapone S. Worley P.F. Baraban J.M. Snyder S.H. J. Biol. Chem. 1988; 263: 1530-1534Google Scholar, 15Maeda N. Niinobe M. Mikoshiba K. EMBO J. 1990; 9: 61-67Google Scholar). To localize the IP3 binding site, deletion mutagenesis studies showed that IP3R1 binds IP3 within the N-terminal 650 amino acids independently of the tetramer formation (16Mignery G.A. Sudhof T.C. EMBO J. 1990; 9: 3893-3898Google Scholar, 17Miyawaki A. Furuichi T. Ryou Y. Yoshikawa S. Nakagawa T. Saitoh T. Mikoshiba K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4911-4915Google Scholar). Newton et al. (18Newton C.L. Mignery G.A. Sudhof T.C. J. Biol. Chem. 1994; 269: 28613-28619Google Scholar) have reported that the N-terminal 576 amino acids fused to glutathione S-transferase specifically bound IP3 with high affinity, whereas further N- or C-terminal deletions of this region completely abolished the specific binding. Furthermore, Mourey et al. (19Mourey R.J. Estevez V.A. Marecek J.F. Barrow R.K. Prestwich G.D. Snyder S.H. Biochemistry. 1993; 32: 1719-1726Google Scholar) have reported that residues 471-501 in this region were labeled with a photoaffinity ligand. These results indicated that the IP3 binding site is localized within the N-terminal 576 amino acids and consists of some distantly separated motifs.IP3 is characterized by three negatively charged phosphate groups at equatorial positions 1, 4, and 5 of an inositol ring. Ca2+ release experiments using various synthetic inositol phosphate analogues showed that the IP3 recognition site is markedly stereospecific (20Nahorski S.R. Potter B.V. Trends Pharmacol. Sci. 1989; 10: 139-144Google Scholar, 21Berridge M.J. Irvine R.F. Nature. 1984; 312: 315-321Google Scholar, 22Hirata M. Yanaga F. Koga T. Ogasawara T. Watanabe Y. Ozaki S. J. Biol. Chem. 1990; 265: 8404-8407Google Scholar). The ability of IP3 to release Ca2+ depends critically upon the positional distribution of the phosphate groups around the inositol ring, suggesting that binding sites for these three phosphate groups make major contributions to the recognition and binding of (1,4,5)IP3. Thus, it has been assumed that there is a pocket of positive charges that facilitate ionic interactions with the negative charges on these three phosphate groups. This hypothetical model is supported by the following evidence. IP3 binding to the platelet membrane is blocked by the specific Arg-modifying reagent, p-hydroxyphenylglyoxal, suggesting the involvement of Arg in the IP3 binding (23O'Rourke F. Feinstein M.B. Biochem. J. 1990; 267: 297-302Google Scholar). IP3 binding to the receptor is competitively blocked by heparin (24Worley P.F. Baraban J.M. Supattapone S. Wilson V.S. Snyder S.H. J. Biol. Chem. 1987; 262: 12132-12136Google Scholar), and the IP3R protein has been purified by heparin affinity column chromatography (2Supattapone S. Worley P.F. Baraban J.M. Snyder S.H. J. Biol. Chem. 1988; 263: 1530-1534Google Scholar, 15Maeda N. Niinobe M. Mikoshiba K. EMBO J. 1990; 9: 61-67Google Scholar). The known binding site for heparin in antithrombin III is highly basic because of enriched Arg or Lys residues (25Smith J.W. Knauer D.J. J. Biol. Chem. 1987; 262: 11964-11972Google Scholar). The IP3 binding to the receptor is augmented with increasing pH over the range 5-9 (24Worley P.F. Baraban J.M. Supattapone S. Wilson V.S. Snyder S.H. J. Biol. Chem. 1987; 262: 12132-12136Google Scholar). A study using NMR spectroscopy showed that IP3 dissociates protons from three phosphate groups over this pH range, indicating that the negative charges of IP3 contribute its binding to the receptor (26White A.M. Varney M.A. Watson S.P. Rigby S. Liu C.S. Ward J.G. Reese C.B. Graham H.C. Williams R.J. Biochem. J. 1991; 278: 759-764Google Scholar). Finally, x-ray crystallographic studies of the pleckstrin homology domain of β spectrin and phospholipase C-δ, which bind IP3 but have no sequence homology with IP3R, in complex with IP3 showed that 2 and 5 positively charged amino acid residues of this domain, respectively, interact with the phosphate groups of IP3 (27Hyvonen M. Macias M.J. Nilges M. Oschkinat H. Saraste M. Wilmanns M. EMBO J. 1995; 14: 4676-4685Google Scholar, 28Ferguson K.M. Lemmon M.A. Schlessinger J. Sigler P.B. Cell. 1995; 83: 1037-1046Google Scholar).Despite the above evidence, the detailed molecular structure of the IP3 binding site of the IP3R remains to be studied. In this study, we developed an Escherichia coli expression system for various recombinant IP3 binding sites of mouse IP3R1. The N-terminal 734 amino acids of IP3R1 (T734) expressed in E. coli exhibited similar binding characteristics to those of the native cerebellar IP3R. Eighteen deletion mutageneses of T734 showed that 353 amino acids (residues 226-578) are directly responsible for the IP3 binding. Furthermore, we performed site-directed mutagenesis on 41 basic amino acid residues within the N-terminal 650 amino acids of T734 and showed that 10 single amino acid substitutions markedly reduced the IP3 binding activity. They were scattered within residues 226-578 and conserved among all members of the IP3R family. Of these, three were critical for IP3 binding and one was involved in binding specificity. We discuss the structure of the IP3 binding site of the IP3R.DISCUSSIONIt has been assumed that IP3R has a pocket with a highly restricted structure that specifically recognizes the IP3 molecule (20Nahorski S.R. Potter B.V. Trends Pharmacol. Sci. 1989; 10: 139-144Google Scholar). What is the minimum number of amino acids required to assure the binding conformation, and which residues are present on the surface of the pocket? To address these questions by molecular biological means, we developed an E. coli expression system in which the N-terminal 734-amino acid residues of mouse IP3R1 (T734) are expressed as soluble proteins. The binding affinity for (1,4,5)IP3 and specificity for various inositol phosphates of the expressed T734 proteins were similar to those of cerebellar IP3R, indicating that the binding sites expressed in this system form a functional conformation resembling the native binding site.Structure of the IP3 Binding Site (Residues 226-578)We found that, at most, 353 amino acid residues (residues 226-578) are sufficient for the IP3 binding with high affinity and specificity. Newton et al. (18Newton C.L. Mignery G.A. Sudhof T.C. J. Biol. Chem. 1994; 269: 28613-28619Google Scholar) could delete the C terminus up to the residue 577. These results indicate that the critical region for the IP3 binding should be localized between the residues 226 and 576. This critical IP3 binding region of 351 amino acids shares 44% identity and 61% similarity with some interspersed diverse sequences in the corresponding region of other members of the IP3R family (Fig. 4). Within this region, only the alternative-splicing segment SI (15 amino acids, residues 318-332) is unlikely to be requisite for the binding, since the IP3R1 SI(−) splicing variant, which lacks this region, retains binding affinity similar to that of the IP3R1 SI(+) variant, which has this region (18Newton C.L. Mignery G.A. Sudhof T.C. J. Biol. Chem. 1994; 269: 28613-28619Google Scholar). 2F. Yoshikawa and T. Furuichi, unpublished data. On the other hand, a deletion of either 3 more N-terminal or 9 more C-terminal amino acids from this 351-amino acid region completely abolished the binding activity. Mignery et al. (7Mignery G.A. Newton C.L. Archer B.T. Sudhof T.C. J. Biol. Chem. 1990; 265: 12679-12685Google Scholar, 16Mignery G.A. Sudhof T.C. EMBO J. 1990; 9: 3893-3898Google Scholar) and Miyawaki et al. (17Miyawaki A. Furuichi T. Ryou Y. Yoshikawa S. Nakagawa T. Saitoh T. Mikoshiba K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4911-4915Google Scholar) have reported that any small deletions within this region cause a failure to bind IP3. Thus, residues 226-576 were sufficient and close to the minimum portion required for specific IP3 binding. Thus, we suggest that this portion forms the IP3 binding core.We mutated 41 basic amino acids (Arg and Lys) within the N-terminal 650-amino acid residues to the neutral amino acid, Gln, either singly or in pairs. Of these mutations, 10 single substitutions of Gln for Arg-241, Lys-249, Arg-265, Arg-269, Arg-504, Arg-506, Lys-508, Arg-511, Arg-568, and Lys-569 markedly reduced the IP3 binding activity. All 10 residues were identical among all IP3Rs, suggesting that they are functionally important. Three of them (Arg-265, Lys-508, and Arg-511) were critical, since the IP3 binding was completely abolished, even by substitution with a single amino acid. The reduction in IP3 binding activity caused by these site-directed substitutions is possibly due to a loss of the original side chain, which directly interacts with IP3, or to disruption of a local or global conformation for the binding. These 10 residues are scattered within the binding core, which is consistent with the results from the deletion studies, and can be classified into four segments (the first containing Arg-241 and Lys-249; the second, Arg-265 and Arg-269; the third, Arg-504, Arg-506, Lys-508, and Arg-511; and the fourth, Arg-568 and Lys-569). We suggest that on the tertiary structure of IP3R, these separated segments are positioned close to each other and form a positively charged pocket for binding to the negatively charged phosphate groups of IP3. The third segment is close to the residues 476-501, which were labeled by photoaffinity ligand, suggesting that it is in the proximity of the ligand binding site (19Mourey R.J. Estevez V.A. Marecek J.F. Barrow R.K. Prestwich G.D. Snyder S.H. Biochemistry. 1993; 32: 1719-1726Google Scholar). Even functionally conservative mutations between Arg and Lys (K508R, R511K, and K569R) markedly reduced the IP3 binding activity like Gln or Ala substitutions, suggesting that the restricted basic amino acid residues in the higher order structure are requisite for the specific interaction with IP3. In comparison with the wild-type T734, the mutant R568Q exhibited lower affinity for (1,4,5)IP3 and a different binding specificity for various inositol phosphates, suggesting that Arg-568 is involved in not only high affinity binding with (1,4,5)IP3 but also determination of binding specificity. Therefore, Arg-568 may be involved in recognition of the functional group at the equatorial position-1 of the inositol ring, since R568Q recognizes (4,5)IP2 and (2,4,5)IP3 with higher affinity but (1,4,5)IP3 and (1,3,4,5)IP4 with lower affinity than the wild type.The Function of the N Terminus (Residues 1-225)This study indicated that the first 225 amino acids are not requisite for the specific IP3 binding, although there is significant sequence homology (64% identity and 76% similarity) in this region of the IP3R family. On the other hand, the deletion of only the first 31 amino acids resulted in a severe reduction in the binding activity. Such contradictory mutational effects were also found in serial N-terminal deletions up to residue 215. However, a deletion of 5 amino acids more toward the C terminus recovered the binding activity (D(1-220)). The binding activity was also recovered in the deletions as far as the residue 225 (D(1-223) and D(1-225)). These can be explained by two models. Serial deletions of the N terminus up to the residue 215 interfere with the higher order structure of the IP3 binding site formed by the residues 226-576, or the residues 216-220 act as a part of the inhibitory determinant when deletions up to residue 215 are performed. Therefore, we tested whether the synthetic peptide (CNTSWKIVLFMK) corresponding to the residues 214-225 inhibits the IP3 binding of the cerebellar IP3R, T734, or the mutant D(1-223). However, as far as we tested, this peptide had no significant inhibitory effect (data not shown) although we could not rule out the possibility that it does not inhibit the binding in an intermolecular manner.The binding affinity for IP3 of the core region (residues 224-579) was more than 10-fold higher than that of T734. This augmented affinity was probably due to deleting the N-terminal 223 amino acids from T734, since the mutant D(1-223) showed similar higher binding affinity (data not shown). Thus, we suggest that the N-terminal 225 amino acids are not directly responsible for, but may modulate, IP3 binding (for example, the binding affinity).Comparison of the N-terminal Portions between IP3R and the Ryanodine Receptor (RyR)RyR is another intracellular Ca2+ release channel originally identified in the sarcoplasmic reticulum of skeletal muscle or cardiac muscle. IP3R has fragmentary sequence homology with RyR in the N-terminal portion, suggesting involvement of this region in common receptor-channel function(s) (6Furuichi T. Yoshikawa S. Miyawaki A. Wada K. Maeda N. Mikoshiba K. Nature. 1989; 342: 32-38Google Scholar, 36Furuichi T. Mikoshiba K. J. Neurochem. 1995; 64: 953-960Google Scholar). This seems to be supported by the fact that a single mutation of Arg-615 of the type 1 RyR (RyR1) to Cys causes porcine malignant hyperthermia (37MacLennan D.H. Phillips M.S. Science. 1992; 256: 789-794Google Scholar). The porcine malignant hyperthermia RyR1 channels are hypersensitive to various modulators, suggesting that the region around Arg-615 is a regulatory domain of channel opening, or that it is involved in binding an unknown channel activator. This region of RyR1 is fragmentarily homologous with the corresponding region (residues 621-659) of IP3R1 (Fig. 4). Besides this region, there are four homologous fragments within the N-terminal 580 amino acids of IP3R1 (residues 115-197, 227-252, 276-319, and 472-516) (6Furuichi T. Yoshikawa S. Miyawaki A. Wada K. Maeda N. Mikoshiba K. Nature. 1989; 342: 32-38Google Scholar), of which three are within the IP3 binding region defined in this study. Among 10 important basic residues for the IP3 binding, three (Arg-241, Arg-504, and Arg-506) are conserved, but three critical residues (Arg-265, Lys-508, and Arg-511) are diverse in the RyR family (Fig. 4). Therefore, these homologous regions may be required for receptor-channel functions common in the intracellular Ca2+ release channel superfamily, such as sensing activation signal(s), modulation, or the transduction of activating signal(s) to channel opening.We defined the importance of basic amino acids for the binding of IP3R1 to IP3 molecule, which is similar to that reported for some pleckstrin homology domains that bind IP3 (27Hyvonen M. Macias M.J. Nilges M. Oschkinat H. Saraste M. Wilmanns M. EMBO J. 1995; 14: 4676-4685Google Scholar, 28Ferguson K.M. Lemmon M.A. Schlessinger J. Sigler P.B. Cell. 1995; 83: 1037-1046Google Scholar). However, the overall constitution of the binding site seems to be different from these, which is reflected in the binding affinity and specificity. On the other hand, IP3R may recognize the hydroxy groups and the inositol ring of IP3 (20Nahorski S.R. Potter B.V. Trends Pharmacol. Sci. 1989; 10: 139-144Google Scholar, 38Hirata M. Watanabe Y. Yoshida M. Koga T. Ozaki S. J. Biol. Chem. 1993; 268: 19260-19266Google Scholar, 39Safrany S.T. Wojcikiewicz R.J.H. Strupish J. Nahorski S.R. Dubreuil D. Cleophax J. Gero S.D. Potter B.V.L. FEBS Lett. 1991; 278: 252-256Google Scholar). The amino acids involved in these interactions in the critical region defined here require further analysis. INTRODUCTIONMany cellular responses to hormones, neurotransmitters, growth factors, etc. are mediated by the intracellular second messenger inositol 1,4,5-trisphosphate (IP3 or (1,4,5)IP3) 1The abbreviations used are: IP3 and (1,4,5)IP3inositol 1,4,5-trisphosphateIP3Rinositol 1,4,5-trisphosphate receptorIP3R1type 1 inositol 1,4,5-trisphosphate receptormAbmonoclonal antibody (1Berridge M.J. Nature. 1993; 361: 315-325Google Scholar)IP1inositol 1-phosphate(1,4)IP2inositol 1,4-bisphosphate(4,5)IP2inositol 4,5-bisphosphate(2,4,5)IP3inositol 2,4,5-trisphosphate(1,3,4,5)IP4inositol 1,3,4,5-tetrakisphosphateRyRryanodine receptor. (1Berridge M.J. Nature. 1993; 361: 315-325Google Scholar). IP3 releases Ca2+ from intracellular stores by binding to the IP3 receptor (IP3R) (2Supattapone S. Worley P.F. Baraban J.M. Snyder S.H. J. Biol. Chem. 1988; 263: 1530-1534Google Scholar), which is a tetrameric IP3-gated Ca2+ release channel (3Ferris C.D. Huganir R.L. Supattapone S. Snyder S.H. Nature. 1989; 342: 87-89Google Scholar, 4Miyawaki A. Furuichi T. Maeda N. Mikoshiba K. Neuron. 1990; 5: 11-18Google Scholar, 5Maeda N. Kawasaki T. Nakade S. Yokota N. Taguchi T. Kasai M. Mikoshiba K. J. Biol. Chem. 1991; 266: 1109-1116Google Scholar). There are at least three types of IP3R derived from distinct genes in mammals (6Furuichi T. Yoshikawa S. Miyawaki A. Wada K. Maeda N. Mikoshiba K. Nature. 1989; 342: 32-38Google Scholar, 7Mignery G.A. Newton C.L. Archer B.T. Sudhof T.C. J. Biol. Chem. 1990; 265: 12679-12685Google Scholar, 8Sudhof T.C. Newton C.L. Archer III, B.T. Ushkaryov Y.A. Mignery G.A. EMBO J. 1991; 10: 3199-3206Google Scholar, 9Blondel O. Takeda J. Janssen H. Seino S. Bell G.I. J. Biol. Chem. 1993; 268: 11356-11363Google Scholar, 10Maranto A.R. J. Biol. Chem. 1994; 269: 1222-1230Google Scholar, 11Yamada N. Makino Y. Clark R.A. Pearson D.W. Mattei M.G. Guenet J.L. Ohama E. Fujino I. Miyawaki A. Furuichi T. Mikoshiba K. Biochem. J. 1994; 302: 781-790Google Scholar, 12Yamamoto-Hino M. Sugiyama T. Hikichi K. Mattei M.G. Hasegawa K. Sekine S. Sakurada K. Miyawaki A. Furuichi T. Hasegawa M. Mikoshiba K. Recept. and Channels. 1994; 2: 9-22Google Scholar). Structural and functional studies on type I IP3R (IP3R1) (2749 amino acids, 313 kDa) have revealed that it is structurally divided into three parts: a large N-terminal cytoplasmic arm (83% of the receptor molecule); a putative six membrane-spanning domains clustered near the C terminus, which are thought to constitute an ion channel by forming a tetramer; and a short C-terminal cytoplasmic tail (13Furuichi T. Kohda K. Miyawaki A. Mikoshiba K. Curr. Opin. Neurobiol. 1994; 4: 294-303Google Scholar, 14Michikawa T. Hamanaka H. Otsu H. Yamamoto A. Miyawaki A. Furuichi T. Tashiro Y. Mikoshiba K. J. Biol. Chem. 1994; 269: 9184-9189Google Scholar).The binding of IP3 to this receptor purified from mouse cerebella is stoichiometric (Kd = ∼100 nM, Hill coefficient = ∼1.0) (2Supattapone S. Worley P.F. Baraban J.M. Snyder S.H. J. Biol. Chem. 1988; 263: 1530-1534Google Scholar, 15Maeda N. Niinobe M. Mikoshiba K. EMBO J. 1990; 9: 61-67Google Scholar). To localize the IP3 binding site, deletion mutagenesis studies showed that IP3R1 binds IP3 within the N-terminal 650 amino acids independently of the tetramer formation (16Mignery G.A. Sudhof T.C. EMBO J. 1990; 9: 3893-3898Google Scholar, 17Miyawaki A. Furuichi T. Ryou Y. Yoshikawa S. Nakagawa T. Saitoh T. Mikoshiba K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4911-4915Google Scholar). Newton et al. (18Newton C.L. Mignery G.A. Sudhof T.C. J. Biol. Chem. 1994; 269: 28613-28619Google Scholar) have reported that the N-terminal 576 amino acids fused to glutathione S-transferase specifically bound IP3 with high affinity, whereas further N- or C-terminal deletions of this region completely abolished the specific binding. Furthermore, Mourey et al. (19Mourey R.J. Estevez V.A. Marecek J.F. Barrow R.K. Prestwich G.D. Snyder S.H. Biochemistry. 1993; 32: 1719-1726Google Scholar) have reported that residues 471-501 in this region were labeled with a photoaffinity ligand. These results indicated that the IP3 binding site is localized within the N-terminal 576 amino acids and consists of some distantly separated motifs.IP3 is characterized by three negatively charged phosphate groups at equatorial positions 1, 4, and 5 of an inositol ring. Ca2+ release experiments using various synthetic inositol phosphate analogues showed that the IP3 recognition site is markedly stereospecific (20Nahorski S.R. Potter B.V. Trends Pharmacol. Sci. 1989; 10: 139-144Google Scholar, 21Berridge M.J. Irvine R.F. Nature. 1984; 312: 315-321Google Scholar, 22Hirata M. Yanaga F. Koga T. Ogasawara T. Watanabe Y. Ozaki S. J. Biol. Chem. 1990; 265: 8404-8407Google Scholar). The ability of IP3 to release Ca2+ depends critically upon the positional distribution of the phosphate groups around the inositol ring, suggesting that binding sites for these three phosphate groups make major contributions to the recognition and binding of (1,4,5)IP3. Thus, it has been assumed that there is a pocket of positive charges that facilitate ionic interactions with the negative charges on these three phosphate groups. This hypothetical model is supported by the following evidence. IP3 binding to the platelet membrane is blocked by the specific Arg-modifying reagent, p-hydroxyphenylglyoxal, suggesting the involvement of Arg in the IP3 binding (23O'Rourke F. Feinstein M.B. Biochem. J. 1990; 267: 297-302Google Scholar). IP3 binding to the receptor is competitively blocked by heparin (24Worley P.F. Baraban J.M. Supattapone S. Wilson V.S. Snyder S.H. J. Biol. Chem. 1987; 262: 12132-12136Google Scholar), and the IP3R protein has been purified by heparin affinity column chromatography (2Supattapone S. Worley P.F. Baraban J.M. Snyder S.H. J. Biol. Chem. 1988; 263: 1530-1534Google Scholar, 15Maeda N. Niinobe M. Mikoshiba K. EMBO J. 1990; 9: 61-67Google Scholar). The known binding site for heparin in antithrombin III is highly basic because of enriched Arg or Lys residues (25Smith J.W. Knauer D.J. J. Biol. Chem. 1987; 262: 11964-11972Google Scholar). The IP3 binding to the receptor is augmented with increasing pH over the range 5-9 (24Worley P.F. Baraban J.M. Supattapone S. Wilson V.S. Snyder S.H. J. Biol. Chem. 1987; 262: 12132-12136Google Scholar). A study using NMR spectroscopy showed that IP3 dissociates protons from three phosphate groups over this pH range, indicating that the negative charges of IP3 contribute its binding to the receptor (26White A.M. Varney M.A. Watson S.P. Rigby S. Liu C.S. Ward J.G. Reese C.B. Graham H.C. Williams R.J. Biochem. J. 1991; 278: 759-764Google Scholar). Finally, x-ray crystallographic studies of the pleckstrin homology domain of β spectrin and phospholipase C-δ, which bind IP3 but have no sequence homology with IP3R, in complex with IP3 showed that 2 and 5 positively charged amino acid residues of this domain, respectively, interact with the phosphate groups of IP3 (27Hyvonen M. Macias M.J. Nilges M. Oschkinat H. Saraste M. Wilmanns M. EMBO J. 1995; 14: 4676-4685Google Scholar, 28Ferguson K.M. Lemmon M.A. Schlessinger J. Sigler P.B. Cell. 1995; 83: 1037-1046Google Scholar).Despite the above evidence, the detailed molecular structure of the IP3 binding site of the IP3R remains to be studied. In this study, we developed an Escherichia coli expression system for various recombinant IP3 binding sites of mouse IP3R1. The N-terminal 734 amino acids of IP3R1 (T734) expressed in E. coli exhibited similar binding characteristics to those of the native cerebellar IP3R. Eighteen deletion mutageneses of T734 showed that 353 amino acids (residues 226-578) are directly responsible for the IP3 binding. Furthermore, we performed site-directed mutagenesis on 41 basic amino acid residues within the N-terminal 650 amino acids of T734 and showed that 10 single amino acid substitutions markedly reduced the IP3 binding activity. They were scattered within residues 226-578 and conserved among all members of the IP3R family. Of these, three were critical for IP3 binding and one was involved in binding specificity. We discuss the structure of the IP3 binding site of the IP3R.
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