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

Abstract

Ca 2ϩ channel ͉ endoplasmic reticulum ͉ red fluorescent protein T he intracellular second messenger, inositol 1,4,5-trisphosphate (IP 3 ) is generated upon stimulation of cell-surface receptors linked to phospholipase C (PLC) activation (1). IP 3 rapidly binds to an intracellular receptor and releases Ca 2ϩ from intracellular Ca 2ϩ stores; hence, both IP 3 and its receptor (IP 3 R) are key components of the signal transduction mechanism that links cell-surface receptors to calcium-regulated intracellular responses (2). All three isoforms of the IP 3 R (types I, II, and III) function as intracellular Ca 2ϩ channels that work as homotetramers or heterotetramers (3). Each receptor subunit has a channel portion containing six transmembrane helices and a pore domain located between TM5 and TM6, close to the C terminus of the protein (4-6). The ligand-binding domain (LBD) of the receptor is located at the N terminus (7) and is separated from the channel domain by a long intervening regulatory region facing the cytoplasm (3, 7). IP 3 binding leads to rapid activation of the channel, but Ca 2ϩ -induced Ca 2ϩ release, similar to that characteristic of the related ryanodine receptors (RyRs), has also been recognized as an important regulatory feature of IP 3 Rs (8). Because of this complex, and often subtype-specific, regulation of IP 3 channels, cells can display complex Ca 2ϩ wave patterns and oscillations after agonist stimulation, the shape and frequency of which can have unique importance in the selective regulation of downstream effectors (9-11).Despite intense studies, little is known about the manner in which the binding of IP 3 to the N-terminal LBD affects the channel gating properties of the molecule. Upon IP 3 binding, the LBD undergoes a significant conformational change as evidenced by the IP 3 -induced alteration of its migration on a size-exclusion column (7) and by its suitability as a FRET-based sensor of IP 3 binding (12). As shown recently, the C-terminal channel domain, isolated from the rest of the receptor, is constitutively active, and the presence of the regulatory domain is required to maintain the suppression of channel activity (13,14). Moreover, elegant cross-linking experiments have shown that the N-terminal domain of the receptor is in juxtaposition with the C-terminal channel domain (15). These data together raised the possibility that the proximity of the LBD to the channel domain may be an important aspect of IP 3 R regulation after binding of IP 3 . The present study was designed to investigate whether the LBD of the IP 3 R acts as a tethered regulatory module that regulates the channel activity via IP 3 -induced conformational changes. For this purpose, we used a molecular approach by which the isolated LBD of type I IP 3 R or its components was tethered to the cytoplasmic surface of the endoplasmic reticulum (ER), and the effects of their expression on Ca 2ϩ signaling was compared with those of the same constructs expressed in the cytoplasm. These experiments revealed that the all-heli...

The inositol 1,4,5-trisphosphate receptor regulates autophagy through its interaction with Beclin 1

“…24 As negative controls, equally targeted versions of RFP (namely Cyto-ERand OMM-RFP) failed to inhibit autophagy in this system, as assessed by measuring GFP-LC3 puncta (Figure 7a and b) or the accumulation of LC3-II (Figure 7e). Moreover, an inactive IP 3 R-LBD mutant (IP 3 R-LBD-9aaER-RFP) in which the insertion of a nonapeptide linker abolishes the interaction with endogenous IP 3 R, 37 failed to affect xestospongin Binduced autophagy (Figure 7b). All the functional variants of the IP 3 R-LBD-RFP (Cyto-, OMM-and ER-targeted) but not IP 3 R-LBD-9aaER-RFP, interacted with Beclin 1 (not shown), and this interaction was reduced in the ER by depletion of Bcl-2 (Figure 7c and d).…”

“…As controls, a plasmid coding for an ER-targeted IP 3 R-LBD that is rendered inactive by the incorporation of a nonapeptide linker sequence (IP 3 R-9aa-LBD-RFP-ER), as well as plasmids encoding RFP alone targeted to the cytosol (RFP-Cyto), the OMM (RFP-OMM) or the ER (RFP-ER) were used. 24,37,52 …”

Visualization and manipulation of phosphoinositide dynamics in live cells using engineered protein domains

“…While several experiments with targeted InsP 3 -binding domains are still in progress, we have reported a serendipitous finding with the ER-targeted InsP 3 -binding domains [86]. Targeted expression of either the PH domain of p130 or the ligand-binding domain (LBD) of the type-I InsP 3 receptor (residues 224-605) inhibited Ca 2+ signaling similarly to their cytoplasmic counterparts (i.e., increasing the lag-time, the rate of rise and impairing the synchronization of the InsP 3 -induced Ca 2+ release).…”

Structural Insights into Ca2+-dependent Regulation of Inositol 1,4,5-Trisphosphate Receptors by CaBP1

“…Our target binding analysis demonstrates that the C-domain of CaBP1 exhibits Ca 2ϩ -induced binding to the N-terminal cytosolic region of InsP 3 R1. We propose that CaBP1 may regulate Ca 2ϩ -dependent channel activity in InsP 3 Rs by promoting a structural interaction between the N-terminal suppressor and ligand-binding core domains that modulates Ca 2ϩ -dependent channel gating (8,33,34).…”