Molecular Cloning of Mouse Type 2 and Type 3 Inositol 1,4,5-Trisphosphate Receptors and Identification of a Novel Type 2 Receptor Splice Variant

Abstract

Inositol 1,4,5-trisphosphate (IP 3 ) 1 is a second messenger generated by the phosphatidylinositol signaling cascade response to hormones, neurotransmitters, and growth factors (1) and causes Ca 2ϩ release from intracellular stores by binding to the IP 3 receptor (IP 3 R), which is an IP 3 -gated intracellular Ca 2ϩ release channel. Ca 2ϩ release in the cytoplasm occurs in complex spatial and temporal patterns, such as Ca 2ϩ waves and Ca 2ϩ oscillations, and regulates many cellular responses, including fertilization, muscle contraction, secretion, cell growth, differentiation, apoptosis, and synaptic plasticity (1).The IP 3 R family consists of three isoforms (IP 3 R1, IP 3 R2, and IP 3 R3) (2), and the primary structure of all three IP 3 R types has been determined in rat (3-5) and human (6 -9). Only a single type of IP 3 R was identified in frog (10), fly (11), starfish (12), lobster (13), and nematode (14). Three isoforms have been found to exist in the mouse (15, 16), but the primary structure of mouse IP 3 R2 and IP 3 R3 has not yet been determined.IP 3 -gated Ca 2ϩ release channels are composed of four subunits (17). The expression levels of the three IP 3 R isoforms are different in each tissue, but most tissues contain multiple isoforms (18). Heterotetrameric channels were detected as well as homotetrameric channels (19), indicating that the structural diversity of the IP 3 -gated channels is greater than the number of genes. There is evidence of functional differences among the three types of IP 3 R in terms of their IP 3 sensitivity (20, 21) and the modulatory effects on them from cytoplasmic Ca 2ϩ (22), ATP (22), calmodulin (23), and cAMP-dependent protein kinase (24). Additional diversity of IP 3 -gated channels is produced by alternative splicing, and alternative splicing segments, designated SI, SII, and SIII, have been found in the mouse (25), rat (26), and human (9) IP 3 R1. These findings suggest that the channels composed of different isoforms possess distinctive functions, but little is known about the physiological significance and exact functional differences arising from the heterotetrameric assembly of IP 3 R.In many cells, the Ca 2ϩ increase triggered by IP 3 is propagated throughout the cytoplasm; however, the diffusion of free Ca 2ϩ is spatially restricted because many immobile or slowly diffusing Ca 2ϩ -binding proteins are present in the cytoplasm (27). Propagation of Ca 2ϩ is thought to be mediated by regenerative Ca 2ϩ release through IP 3 Rs, which are regulated by cytoplasmic Ca 2ϩ (28). Positive feedback regulation of IP 3 R by Ca 2ϩ enables the Ca 2ϩ released by one receptor to excite its

Mechanistic basis of bell-shaped dependence of inositol 1,4,5-trisphosphate receptor gating on cytosolic calcium

“…Therefore, high concentrations of Ca 2+ prevent IP 3 binding to the receptor in this model. This specific property is consistent with the previous experimental observations in which IP 3 binding to recombinant IP 3 R1 expressed in Sf9 cells was examined under various concentrations of Ca 2+ (23,33). Our model shows that IP 3 binding prevents binding of Ca 2+ to the inactivation sites and that, re- ciprocally, Ca 2+ binding to the inactivation sites prevents IP 3 binding.…”

“…S1). The apparent IP 3 binding dissociation constant of vR expressed in Sf9 cells was 21 nM (n = 2), which is consistent with that of wild-type (wt) IP 3 R1 (28.6 nM) (23). Therefore, the N-terminal fusion did not interfere with the IP 3 binding.…”

The Type 2 Inositol (1,4,5)-Trisphosphate (InsP3) Receptor Determines the Sensitivity of InsP3-induced Ca2+ Release to ATP in Pancreatic Acinar Cells

“…Cells were transfected with 2 g of pHcRed1-Nuc (Clontech) alone or in combination with 10 g of pcDNA3-C2 (InsP 3 R2 cDNA in pcDNA3.1) (26). DNA was introduced into cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.…”

“…The three isoforms are also differentially modulated by ATP binding, with InsP 3 R1 and InsP 3 R2 regulated by lower ATP concentrations than InsP 3 R3 (22)(23)(24). Additional functional heterogeneity is brought about by the presence of splice variants (25,26) and the possibility of heterotetrameric channels (27)(28)(29). Even though isoform-specific regulation of InsP 3 R has been well documented, the physiological consequences of this differential modulation are poorly defined.…”

Genetic polymorphism and protein conformational plasticity in the calmodulin superfamily: Two ways to promote multifunctionality

“…For instance, the inositol 1,4,5-trisphosphate receptor (IP 3 R) possesses three isoforms in mammals: Type I IP 3 R is highly expressed in cerebellar Purkinje cells of the central nervous system, whereas type II is predominantly found in cardiac ventricular myocytes (123). These isoforms not only have different subcellular distribution, which is dynamically regulated dependent on physiological conditions, but also generate different splice variants with different biophysical properties in terms of ligand binding (124). Moreover, these CaM targets are often posttranslationally modified, which may affect their ability to interact with CaM.…”