Molecular and Functional Evidence for Multiple Ca2+-binding Domains in the Type 1 Inositol 1,4,5-Trisphosphate Receptor

Abstract

Structural and functional analyses were used to investigate the regulation of the inositol 1,4,5-trisphosphate (InsP 3 ) receptor (InsP 3 R) by Ca 2؉ . To define the structural determinants for Ca 2؉ binding, cDNAs encoding GST fusion proteins that covered the complete linear cytosolic sequence of the InsP 3 R-1 were expressed in bacteria. The fusion proteins were screened for Ca 2؉ and ruthenium red binding through the use of 45 The inositol 1,4,5-trisphosphate receptor (InsP 3 R) 1 is an intracellular Ca 2ϩ release channel that is modulated by various physiological ligands such as inositol 1,4,5-trisphosphate (InsP 3 ), Ca 2ϩ , nucleotides, calmodulin (CaM), FK506BP, phosphatases, and kinases (reviewed in Refs. 1 and 2). The InsP 3 R can be divided into three functionally different domains as follows: an N-terminal ligand-binding domain, a modulatory domain, and a channel domain near the C terminus (3, 4). Among the various modulators, Ca 2ϩ itself plays a pivotal role and may be considered as a co-agonist exerting both positive and negative effects on InsP 3 -induced Ca 2ϩ release. Cytosolic Ca 2ϩ has a bell-shaped effect on the InsP 3 R, with low concentrations stimulating the release and high concentrations inhibiting the release (5-8). Both phases are time-dependent, but the inhibition develops more slowly than the stimulation (6, 9 -11). Although these kinetically distinct effects are believed to be crucial for the generation of Ca 2ϩ oscillations and waves (12), little is known about how Ca 2ϩ interacts with the InsP 3 R. Regulation might be exerted by direct binding of Ca 2ϩ to the InsP 3 R and/or via Ca 2ϩ -sensitive protein(s), e.g. via Ca 2ϩ /CaMdependent protein kinase II and protein phosphatase 2B (13) or via a Ca 2ϩ -sensitizing factor (14). Studies in which effects of Sr 2ϩ were compared with those of Ca 2ϩ revealed that at least two interaction sites must exist: a stimulatory site that is modestly sensitive to Sr 2ϩ and an inhibitory site that is nearly insensitive to Sr 2ϩ (15). One Ca 2ϩ -binding domain has been localized on the cytosolic side of the InsP 3 R-1 (16, 17), but whether this domain represents a stimulatory or an inhibitory interaction site is still unclear. Luminal Ca 2ϩ also regulates the InsP 3 -induced Ca 2ϩ release. Loading of the Ca 2ϩ stores results in a relatively more pronounced InsP 3 -induced Ca 2ϩ release (18 -21). A high affinity Ca 2ϩ -binding site was detected on the luminal loop (17), but its functional significance is not yet clear.The aim of the present work was two-fold. First, we wanted to investigate, at the structural level, the presence of direct Ca 2ϩ -binding sites on InsP 3 R-1. Second, we wanted to functionally demonstrate multiple Ca 2ϩ interactions on the InsP 3 R by a kinetic analysis of the effects of Ca 2ϩ and Sr 2ϩ on the InsP 3 -induced Ca 2ϩ release.For the first part of this study we constructed and expressed a number of GST fusion proteins that contain cytosolic fragments of the InsP 3 R sequence. The ability of these fusion proteins to bind ...

IP3 Receptors: Toward Understanding Their Activation

“…Stimulation of IP 3 R by cytosolic Ca 2þ is universally observed even with purified IP 3 R reconstituted into lipid bilayers (Ferris et al 1989;Hirota et al 1995;Michikawa et al 1999), suggesting that this essential Ca 2þ -binding site probably resides within the primary sequence of the IP 3 R. At least seven cytosolic Ca 2þ -binding sites have been identified within IP 3 R1 (Sienaert et al 1996;Sienaert et al 1997), but the physiological relevance of these sites is unresolved. Two of the sites (residues 304-381 and 378-450) are within the IP 3 -binding core, for which there is a high-resolution structure (Bosanac et al 2002).…”

“…However, point mutations of several of these acidic residues had no effect on Ca 2þ -regulation of IP 3 R (Joseph et al 2005). The remaining Ca 2þ -binding sites fall within the central region of the IP 3 R (Sienaert et al 1996;Sienaert et al 1997). The site between residues 1347 -1426 is interesting because its proximity to a calmodulin (CaM)-binding region is reminiscent of RyR, which have two CaM-binding regions within 200 residues of high-affinity Ca 2þ -binding sites, and a third flanked by two high-affinity Ca 2þ -binding sites (Chen and MacLennan 1994).…”

Regulation of cerebellar Ins(1,4,5)P3 receptor by interaction between Ins(1,4,5)P3 and Ca2+

“…We have found that the Ca# + -dependent inhibition of InsP $ binding to cerebellar microsomes might result from a direct interaction of Ca# + with InsP $ R1 [29]. Two of the Ca# + -binding regions identified in the cytoplasmic part of InsP $ R1 are located in the InsP $ -binding domain [25], making these sites reasonable candidates for the mediation of the competitive inhibitory effects of Ca# + described here. Recently, biphasic dependence on Ca# + of InsP $ R channel activity was found to occur with cerebellar receptor isolated and reconstituted in lipid bilayers, suggesting direct effects of Ca# + on this protein [44].…”

“…However, distinct and noninteracting inhibitory sites might also make a significant contribution to the high degree of sigmoidicity of these curves [40]. Several Ca# + -binding sites have been identified in the InsP $ R1 subunit [25].…”

“…The selectivities of stimulation and inhibition of IICR for bivalent cations differ [20][21][22], suggesting that different sites are involved. Ca# + -binding regions have been identified in type 1 [23][24][25] and type 2 InsP $ Rs [23] but their function is unknown. The molecular basis of the Ca# + -dependence might differ for each receptor.…”