rylation may be partly attributable to IICR.Many cellular responses to diverse biological stimuli, such as neurotransmitters, hormones, and growth factors, are mediated by the intracellular second messenger inositol 1,4,5-trisphosphate (IP 3 ).1 IP 3 subsequently induces Ca 2ϩ mobilization from intracellular stores by activating its receptor (IP 3 R) (1, 2). IP 3 R channels form homo-or heterotetramers via the co-assembly of distinct types of IP 3 R subunits (types 1-3) (3) and bind IP 3 in a stoichiometric manner (4 3 R1) is composed of 2749 amino acids (molecular mass about 313 kDa) and is structurally divided into three parts as follows: a large N-terminal cytoplasmic arm region (83% of the receptor molecule); a putative six membrane-spanning region clustered near the C terminus, which is thought to constitute an ion channel by forming a tetramer; and a short C-terminal cytoplasmic tail region (6). In our previous studies (7, 8) on the ligand-binding site of mouse type 1 IP 3 R (mIP 3 R1), we found that the core region, essential for the high affinity of ligand binding, was localized in amino acid residues 226 -576. We also found that the affinity of residues 1-604 (T604) of mIP 3 R1 for IP 3 (K d ϭ 45 nM) was comparable with that of the native cerebellar IP 3 R (83 nM) (4), indicating that T604 could form a well folded or compact conformation for the functional IP 3 -binding pocket (7, 9). The N-terminal residues 1-223, which we assigned as a suppresser region, instead of being directly responsible for IP 3 binding, suppressed the binding activity of the core region (7, 9). Without the putative suppresser region, residues 224 -604 showed markedly higher affinity for IP 3 than did the parental T604 (7). We therefore inferred that such a high affinity IP 3 -binding protein could function as an IP 3 absorbent in living cells and compete with the native IP 3 R for binding to IP 3 .In this paper, we report the development of a novel recombinant IP 3 absorbent, the "IP 3 sponge," which has hyperaffinity for IP 3 (K d ϭ 0.092 nM). The IP 3 sponge was constructed on the basis of the ligand-binding site of mIP 3 R1, and we assumed that this hyperaffinity IP 3 sponge could compete with IP 3 R for the IP 3 signal. We showed that its application actually caused specific inhibition of in vitro IICR in a dose-dependent manner. Moreover, the IP 3 sponge exogenously expressed in HEK293 cells could trap IP 3 produced in response to stimulation with either carbachol (CCh) or ATP, resulting in inhibition of IICR. The IP 3 sponge appeared to be effective in competing for IP 3 with multiple types of tetrameric IP 3 R channels, because HEK293 cells expressed all three IP 3 R subunits (10). The effectiveness of the IP 3 sponge on IP 3 -Ca 2ϩ signaling-dependent cell physiology was verified by providing an invaluable insight into the functional role of IICR in the phosphorylation of cAMPresponse element-binding protein (CREB), which was previously thought to be activated mainly by Ca 2ϩ influx (11,12).
EXPERIMENTAL PROCEDURESConst...