A Novel Recombinant Hyperaffinity Inositol 1,4,5-Trisphosphate (IP3) Absorbent Traps IP3, Resulting in Specific Inhibition of IP3-mediated Calcium Signaling

Abstract

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...

Targeted expression of the inositol 1,4,5-triphosphate receptor (IP 3 R) ligand-binding domain releases Ca 2+ via endogenous IP 3 R channels

“…As shown in Fig. 1A, consistent with earlier studies (18,19), cytoplasmic expression of either the ligand-binding segment of IP 3 R (224 -605) (IP 3 R LBD) or p130PH caused a dose-dependent delay in the onset and the peak of the cytoplasmic Ca 2ϩ increase in response to stimulation of the endogenous P 2y purinergic receptors by ATP. When the Ca 2ϩ peak delays were plotted against the f luorescence intensities for each individual cell expressing one of the two IP 3 -binding proteins, the different IP 3 affinities of the two domains were clearly ref lected in their efficacies in delaying the Ca 2ϩ responses (Fig.…”

Functional Properties of Endogenous Receptor- and Store-operated Calcium Influx Channels in HEK293 Cells

“…Consistent with published observations (34), UTP-induced Ca 2ϩ release was abolished in Ad-InsP 3 R-N-infected cells (Fig. 1C), presumably due to ability of InsP 3 R-N fragment to function as the "InsP 3 sponge" (34). Despite absence of Ca 2ϩ release from InsP 3 -sensitive stores, addition of extracellular Ca 2ϩ resulted in Ca 2ϩ influx in AdInsP 3 R-N-infected cells exposed to UTP (Fig.…”

Cluster Formation of Inositol 1,4,5-Trisphosphate Receptor Requires Its Transition to Open State

“…To construct GFP-IP 3 R1-K508A, a KpnIBglII fragment of G224 (IP 3 sponge), which contains the appropriate substitution (15), was subcloned into pcDNA3.1Zeo(ϩ) vector (Invitrogen) with other parts of GFP-IP 3 R1. To construct GFP-IP 3 R1-D223, an AflII site was introduced at nucleotides 664 -669 of mouse IP 3 R1 by PCR using primers (5Ј-GTGCTTAAGATGAAATGGAGTGATAACAA-AG-3Ј and 5Ј-GCTTCCTTGTCCTCCTTCAG-3Ј for residues 989 -1614 (the AflII site is underlined)) and GFP-IP 3 R1 as a template DNA.…”