Autophosphorylation of inositol 1,4,5-trisphosphate receptors.

Inositol 1,4,5-trisphosphate (IP3) releases internal stores of calcium by binding to a specific membrane receptor which includes both the IP3 recognition site as well as the associated calcium channel. The IP3 receptor is regulated by ATP, calcium, and phosphorylation by protein kinase A, protein kinase C, and calcium/calmodulin-dependent protein kinase II. Its cDNA sequence predicts at least two consensus sequences where nucleotides might bind, and direct binding of ATP to the IP3 receptor has been demonstrated. In the present study, we demonstrate autophosphorylation of the purified and reconstituted IP3 receptor on serine and find serine protein kinase activity of the IP3 receptor toward a specific peptide substrate. Several independent purification procedures do not separate the IP3 receptor protein from the phosphorylating activity, and many different protein kinase activators and inhibitors do not identify protein kinases as contaminants. Also, renaturation experiments reveal autophosphorylation of the monomeric receptor on polyvinylidene difluoride membranes.     

Reduced [3H]IP3 binding but unchanged IP3 receptor levels in the rat hippocampus CA1 region following transient global ischemia and tolerance induction

Changes in inositol (1,4,5)-trisphosphate (IP3) binding properties and the protein level of the IP3 receptor have been reported in different pathological conditions in the brain, e.g. cerebral ischemia, Alzheimer's disease, and Huntingtons disease. We used the 4-vessel occlusion model in rat brain to investigate the effect of transient ischemia insults on the IP3 receptor mRNA level, the IP3 receptor protein level and [3H]IP3 binding. Recirculation periods were limited (1-72 h) to avoid the development of delayed neuronal death. We found that the IP3 receptor mRNA levels were decreased after damage-inducing ischemia (9 min) in the hippocampus CA1 and CA3 regions. The mRNA levels were unaltered after tolerance-inducing ischemia (3 min). However, [3H]IP3 binding was significantly reduced after both damage- and tolerance-inducing ischemia in the hippocampus CA1 region. Furthermore, all investigated brain areas showed a decreased [3H]IP3 binding when tolerance-inducing ischemia was followed by a second ischemic insult (3 + 8.5 min ischemia). The IP3 receptor protein levels remained constant in all investigated brain areas. These results indicate that a reduced [3H]IP3 binding capability in the particularly vulnerable areas occurs as an early consequence of cerebral ischemia, before IP3 receptor protein levels are reduced in these areas. Structural or conformational changes altering IP3 binding may be of necessity on the pathway leading to down-regulation of IP3 receptor protein levels, as observed by others.     

Mechanism regulating nuclear calcium signaling

Although the outer nuclear membrane is continuous with the endoplasmic reticulum, it is possible to isolate nuclei both intact and free from endoplasmic reticulum contaminants. The outer and the inner nuclear membranes can be purified free from cross-contamination. Evidence in support of autonomous regulation of nuclear calcium signaling relies upon the investigations with isolated nuclei. Mechanisms for generating calcium signaling in the nucleus have been identified. Two calcium transporting systems, an ATP-dependant nuclear Ca(2+)-ATPase and an IP4-mediated inositol 1,3,4,5-tetrakisphosphate receptor, are located on the outer nuclear membrane. Thus, ATP and IP4, depending on external free calcium concentrations, are responsible for filling the nuclear envelope calcium pool. The inositol 1,4,5-trisphosphate receptor is located on the inner nuclear membrane with its ligand binding domain facing toward the nucleoplasm. Likewise, the ryanodine receptor is located on the inner nuclear membrane and its ligand cADP-ribose is generated within the nucleus. A 120 kDa protein fragment of nuclear PLC-gamma1 is stimulated in vivo by epidermal growth factor nuclear signaling coincident with the time course of nuclear membrane epidermal growth factor receptor activation. Stimulated 120 kDa protein fragment interacts with PIKE, a nuclear GTPase, and together they form a complex with PI[3]kinase serving as a module for nuclear PI[3]K stimulation. Thus, the nucleus has its own IP(3) generating system.     

Annexin 7 mobilizes calcium from endoplasmic reticulum stores in brain

Mobilization of intracellular calcium from inositol-1,4,5-triphosphate (IP3)-sensitive endoplasmic reticulum (ER) stores plays a prominent role in brain function. Mice heterozygous for the annexin A7 (Anx7) gene have a profound reduction in IP3 receptor function in pancreatic islets along with defective insulin secretion. We examined IP3-sensitive calcium pools in the brains of Anx7 (+/-) mice by utilizing ATP/Mg(2+)-dependent (45)Ca(2+) uptake into brain membrane preparations and tissue sections. Although the Anx7 (+/-) mouse brain displayed similar levels of IP3 binding sites and thapsigargin-sensitive (45)Ca(2+) uptake as that seen in wild-type mouse brain, the Anx7 (+/-) mouse brain Ca(2+) pools showed markedly reduced sensitivity to IP3. A potent and saturable Ca(2+)-releasing effect of recombinant ANX7 protein was demonstrated in mouse and rat brain membrane preparations, which was additive with that of IP3. We propose that ANX7 mobilizes Ca(2+) from an endoplasmic reticulum-like pool, which can be recruited to enhance IP3-mediated Ca(2+) release.     

Models of IP3 and Ca2+ oscillations: frequency encoding and identification of underlying feedbacks

Hormones that act through the calcium-releasing messenger, inositol 1,4,5-trisphosphate (IP3), cause intracellular calcium oscillations, which have been ascribed to calcium feedbacks on the IP3 receptor. Recent studies have shown that IP3 levels oscillate together with the cytoplasmic calcium concentration. To investigate the functional significance of this phenomenon, we have developed mathematical models of the interaction of both second messengers. The models account for both positive and negative feedbacks of calcium on IP3 metabolism, mediated by calcium activation of phospholipase C and IP3 3-kinase, respectively. The coupled IP3 and calcium oscillations have a greatly expanded frequency range compared to calcium fluctuations obtained with clamped IP3. Therefore the feedbacks can be physiologically important in supporting the efficient frequency encoding of hormone concentration observed in many cell types. This action of the feedbacks depends on the turnover rate of IP3. To shape the oscillations, positive feedback requires fast IP3 turnover, whereas negative feedback requires slow IP3 turnover. The ectopic expression of an IP3 binding protein has been used to decrease the rate of IP3 turnover experimentally, resulting in a dose-dependent slowing and eventual quenching of the Ca2+ oscillations. These results are consistent with a model based on positive feedback of Ca2+ on IP3 production.     

Localisation of the [32P]IP3 binding site on human platelet intracellular membranes isolated by high-voltage free-flow electrophoresis.

This study reports the localisation of the [32P]IP3 binding site on highly purified membrane fractions prepared using high-voltage free-flow electrophoresis. Binding studies on mixed membranes, carried out at 4 degrees C, revealed a binding site with a Kd = 86 nM and beta max = 5.3 pmol/mg protein. The binding was potently inhibited by heparin. High-voltage free-flow electrophoresis was used to further purify surface and intracellular membranes. The intracellular membranes showed a 5-fold enrichment of binding sites with respect to the parent mixed membranes with the same Kd (80 nM), but the surface membranes showed an absence of binding activity. The results indicate the localisation of the IP3 receptor on highly purified intracellular membranes.     

CD44 interaction with ankyrin and IP3 receptor in lipid rafts promotes hyaluronan-mediated Ca2+ signaling leading to nitric oxide production and endothelial cell adhesion and proliferation

In this study, we have showed that aortic endothelial cells (GM7372A cell line) express CD44v10 [a hyaluronan (HA) receptor], which is significantly enriched in cholesterol-containing lipid rafts (characterized as caveolin-rich plasma membrane microdomains). HA binding to CD44v10 promotes recruitment of the cytoskeletal protein, ankyrin and inositol 1,4,5-triphosphate (IP3) receptor into cholesterol-containing lipid rafts. The ankyrin repeat domain (ARD) of ankyrin is responsible for binding IP3 receptor to CD44v10 at lipid rafts and subsequently triggering HA/CD44v10-mediated intracellular calcium (Ca2+) mobilization leading to a variety of endothelial cell functions such as nitric oxide (NO) production, cell adhesion and proliferation. Further analyses indicate (i) disruption of lipid rafts by depleting cholesterol from the membranes of GM7372A cells (using methyl-beta-cyclodextrin treatment) or (ii) interference of endogenous ankyrin binding to CD44 and IP3 receptor using overexpression of ARD fragments (by transfecting cells with ARDcDNA) not only abolishes ankyrin/IP3 receptor accumulation into CD44v10/cholesterol-containing lipid rafts, but also blocks HA-mediated Ca2+ signaling and endothelial cell functions. Taken together, our findings suggest that CD44v10 interaction with ankyrin and IP3 receptor in cholesterol-containing lipid rafts plays an important role in regulating HA-mediated Ca2+ signaling and endothelial cell functions such as NO production, cell adhesion and proliferation.     

Translocation between membranes and cytosol of p42IP4, a specific inositol 1,3,4,5-tetrakisphosphate/phosphatidylinositol 3,4, 5-trisphosphate-receptor protein from brain, is induced by inositol 1,3,4,5-tetrakisphosphate and regulated by a membrane-associated 5-phosphatase.

The highly conserved 42-kDa protein, p42IP4 was identified recently from porcine brain. It has also been identified similarly in bovine, rat and human brain as a protein with two pleckstrin homology domains that binds Ins(1,3,4,5)P4 and PtdIns(3,4,5)P3 with high affinity and selectivity. The brain-specific p42IP4 occurs both as membrane-associated and cytosolic protein. Here, we investigate whether p42IP4 can be translocated from membranes by ligand interaction. p42IP4 is released from cerebellar membranes by incubation with Ins(1,3,4,5)P4. This dissociation is concentration-dependent (> 100 nM), occurs within a few minutes and and is ligand-specific. p42IP4 specifically associates with PtdIns(3, 4,5)P3-containing lipid vesicles and can dissociate from these vesicles by addition of Ins(1,3,4,5)P4. p42IP4 is only transiently translocated from the membranes as Ins(1,3,4,5)P4 can be degraded by a membrane-associated 5-phosphatase to Ins(1,3,4)P3. Then, p42IP4 re-binds to the membranes from which it can be re-released by re-addition of Ins(1,3,4,5)P4. Thus, Ins(1,3,4,5)P4 specifically induces the dissociation from membranes of a PtdIns(3,4,5)P3 binding protein that can reversibly re-associate with the membranes. Quantitative analysis of the inositol phosphates in rat brain tissue revealed a concentration of Ins(1,3,4,5)P4 comparable to that required for p42IP4 translocation. Thus, in vivo p42IP4 might interact with membranes in a ligand-controlled manner and be involved in physiological processes induced by the two second messengers Ins(1,3,4,5)P4 and PtdIns(3,4,5)P3.     

Calcium/calmodulin inhibits direct binding of spectrin to synaptosomal membranes

Brain spectrin, through its beta subunit, binds with high affinity to protein-binding sites on brain membranes quantitatively depleted of ankyrin (Steiner, J., and Bennett, V. (1988) J. Biol. Chem. 263, 14417-14425). In this study, calmodulin is demonstrated to inhibit binding of brain spectrin to synaptosomal membranes. Submicromolar concentrations of calcium are required for inhibition of binding, with half-maximal effects at pCa = 6.5. Calmodulin competitively inhibits binding of spectrin to protein(s) in stripped synaptosomal membranes, with Ki = 1.3 microM in the presence of 10 microM calcium. A reversible receptor-mediated process, and not proteolysis, is responsible for inhibition since the effect of calcium/calmodulin is reversed by the calmodulin antagonist trifluoperazine and by chelation of calcium with sodium [ethylenebis(oxyethylenenitrilo)]tetraacetic acid. The target of calmodulin is most likely the spectrin attachment protein(s) rather than spectrin itself since: (a) membrane binding of the brain spectrin beta subunit, which does not associate with calmodulin, is inhibited by calcium/calmodulin, and (b) red cell spectrin which binds calmodulin very weakly, is inhibited from interacting with membrane receptors in the presence of calcium/calmodulin. Ca2+/calmodulin inhibited association of erythrocyte spectrin with synaptosomal membranes but had no effect on binding of erythrocyte or brain spectrin to ankyrin in erythrocyte membranes. These experiments demonstrate the potential for differential regulation of spectrin-membrane protein interactions, with the consequence that Ca2+/calmodulin can dissociate direct spectrin-membrane interactions locally or regionally without disassembly of the areas of the membrane skeleton stabilized by linkage of spectrin to ankyrin. A membrane protein of Mr = 88,000 has been identified that is dissociated from spectrin affinity columns by calcium/calmodulin and is a candidate for the calmodulin-sensitive spectrin-binding site in brain.     

Inositol 1,4,5-trisphosphate receptor is phosphorylated by cyclic AMP-dependent protein kinase at serines 1755 and 1589.

IP3 activates intracellular calcium release by binding to an intracellular ligand gated calcium permeable channel which has been shown to be regulated by protein kinase A phosphorylation. Two consensus sequences for protein kinase A phosphorylation are predicted by the recently isolated cDNA of the mouse and rat. In the present study we have isolated and sequenced the two peptides in the rat IP3 receptor which are phosphorylated by protein kinase A and demonstrate protein kinase A phosphorylation on S-1755 and S-1589.     

Molecular and binding characteristics of IP3 receptors in bovine spermatozoa

We have shown the presence of inositol 1,4,5-triphosphate (IP3) receptors in bovine spermatozoa. These receptors are mainly localized and functionally associated with the acrosome region. Molecular characterization of these bovine IP3 receptors has shown that the functional size of the IP3 binding domain is a protein of 66+/-2 kDa, in agreement with the size of both bovine adrenal cortex and bovine adrenal medullar chromaffin cells IP3 receptors. In contrast, bovine cerebellum IP3 receptor displays molecular weight of 220+/-5 kDa, a value in agreement with data in the literature. Bovine IP3 receptors have a one-affinity state characterized by a low affinity (Kd 750 nM) and a relatively high density (7.5 pmol/mg protein). They are functional and release internal calcium upon the binding of the second messenger. Moreover, the finding that the specific A1 adenosine receptor agonist R-PIA elicits almost the same effect as IP3 might be of some help in understanding the physiological role of these inhibitory adenosine receptors in mammalian spermatozoa.     

Recombinant p42IP4, a brain-specific 42-kDa high-affinity Ins(1,3,4,5)P4 receptor protein, specifically interacts with lipid membranes containing Ptd-Ins(3,4,5)P3.

We have recently cloned the cDNA of p42IP4, a membrane-associated and cytosolic inositol (1,3,4,5)tetrakisphosphate receptor protein [Stricker, R., Hülser, E., Fischer, J., Jarchau, T., Walter, U., Lottspeich, F. & Reiser, G. (1997) FEBS Lett. 405, 229-236.] p42IP4 is a protein of 374 amino acids with Mr of 42 kDa. The p42IP4 protein has a zinc finger motif at its N-terminus, followed by two pleckstrin homology domains. To characterize further the biochemical and functional properties of p42IP4, it was expressed as a glutathione-S-transferase fusion protein in Sf9 cells using a recombinant baculovirus vector. The protein was affinity adsorbed on glutathione beads, cleaved from glutathione-S-transferase with the protease factor-Xa and purified on heparin agarose. The recombinant purified protein is active because it shows binding affinities similar to those of the native p42IP4, purified from pig cerebellum or rat brain (Ki for inositol(1,3,4,5)P4 of 4.1 nm and 2.2 nm, respectively). Moreover the ligand specificity of the recombinant protein for various inositol polyphosphates is similar to that of the native protein purified from brain. Importantly, we show here that p42IP4 binds phosphatidylinositol(3,4,5)P3 specifically, as the recombinant protein can associate with lipid membranes (vesicles) containing phosphatidylinositol(3,4,5)P3; this binding occurs in a concentration-dependent manner and is blocked by inositol(1,3,4,5)P4. This specific association and the possibility that endogenous p42IP4 can be converted from a membrane-associated state to a soluble state support the hypothesis that p42IP4 might be redistributed between cellular compartments upon hormonal stimulation.     

Solubilization and biochemical characterization of the high affinity [3H]ryanodine receptor from rabbit brain membranes

A high affinity [3H]ryanodine receptor has been solubilized from rabbit brain membranes and biochemically characterized. [3H]Ryanodine binding to rabbit brain membranes is specific and saturable, with a Kd of 1.3 nM. [3H]Ryanodine binding is enriched in membranes from the hippocampus but is significantly lower in membranes from the brain stem and spinal cord. Approximately 60% of [3H]ryanodine-labeled receptor is solubilized from brain membranes using 2.5% CHAPS and 10 mg/ml phosphatidylcholine containing 1 M NaCl. The solubilized brain [3H]ryanodine receptor sediments through sucrose gradients like the skeletal receptor as a large (approximately 30 S) complex. Solubilized receptor is specifically immunoprecipitated by sheep polyclonal antibodies against purified skeletal muscle ryanodine receptor coupled to protein A-Sepharose. [3H]Ryanodine-labeled receptor binds to heparin-agarose, and a protein of approximately 400,000 Da, which is cross-reactive with two polyclonal antibodies raised against the skeletal muscle ryanodine receptor, elutes from the column and is enriched in peak [3H]ryanodine binding fractions. These results suggest that the approximately 400,000-Da protein is the brain form of the high affinity ryanodine receptor and that it shares several properties with the skeletal ryanodine receptor including a large oligomeric structure composed of approximately 400,000-Da subunits.     

Expression of full-length and truncated recombinant human brain type I inositol 1,4,5-trisphosphate receptors in mammalian and insect cells

Intracellular inositol 1,4,5-trisphosphate receptors (IP(3)Rs) form tetrameric Ca2+-release channels that are crucial for Ca2+ signalling in many eukaryotic cells. IP(3)R subunits contain an N-terminal, cytoplasmic, ligand binding domain linked by a modulatory domain to a channel-forming, hydrophobic C-terminal domain. We assembled and sequenced cDNAs encoding the SI-/SII+/SIII+ splice variant of the human brain type I IP(3)R, and functionally expressed the full-length receptor, and a C-terminally truncated receptor lacking the final 20% of the protein, in mammalian and insect cells. Both proteins were insoluble, consistent with in vivo immunofluorescence and ligand binding studies. This contrasted with the behaviour of recombinant FIKBP12 (a soluble control protein). The truncated receptor also fractionated with the "membrane" pellet after alkaline carbonate treatment. We conclude that the human type I IP(3)R forms high MW aggregates or complexes in cells when expressed without the C-terminal hydrophobic domain. This behaviour should be considered when expressing and refolding "soluble" human type I IP(3)R domains for structural studies.     

Inositol 1,4,5-trisphosphate receptor in heart: evidence for its concentration in Purkinje myocytes of the conduction system

Inositol 1,4,5-trisphosphate (IP3) is one of the second messengers capable of releasing Ca2+ from sarcoplasmic reticulum/ER subcompartments. The mRNA encoding the intracellular IP3 receptor (Ca2+ channel) has been detected in low amounts in the heart of various species by Northern blot analysis. The myocardium, however, is a heterogeneous tissue composed of working myocytes and conduction system cells, i.e., myocytes specialized for the beat generation and stimulus propagation. In the present study, the cellular distribution of the heart IP3 receptor has been investigated. [3H]IP3 binding experiments, Western blot analysis and immunofluorescence, with anti-peptide antibodies specific for the IP3 receptor, indicated that the majority of Purkinje myocytes (the ventricular conduction system) express much higher IP3 receptor levels than atrial and ventricular myocardium. Heterogeneous distribution of IP3 receptor immunoreactivity was detected both at the cellular and subcellular levels. In situ hybridization to a riboprobe generated from the brain type 1 IP3 receptor cDNA, showed increased accumulation of IP3 receptor mRNA in the heart conduction system. Evidence for IP3-sensitive Ca2+ stores in Purkinje myocytes was obtained by double immunolabeling experiments for IP3 receptor and cardiac calsequestrin, the sarcoplasmic reticulum intralumenal calcium binding protein. The present findings provide a molecular basis for the hypothesis that Ca2+ release from IP3-sensitive Ca2+ stores evoked by alpha 1-adrenergic stimulation is responsible for the increase in automaticity of Purkinje myocytes (del Balzo, U., M. R. Rosen, G. Malfatto, L. M. Kaplan, and S. F. Steinberg. 1990. Circ. Res. 67:1535-1551), and open new perspectives in the hormonal modulation of chronotropism, and generation of arrhythmias.     

Characterization of the binding sites for the interactions between FKBP12 and intracellular calcium release channels

FKBP12, an FK506 binding protein, interacts with type 1 ryanodine receptor (RyR1) and modulates its calcium channel activity. However, there are many opposing reports of FKBP12's interaction with other related calcium channels, such as type 1 IP(3) receptor and type 3 ryanodine receptor (IP(3)R1 and RyR3). In addition, the involvement of the prolyl-dipeptide motif in the calcium channels and the corresponding binding residues in FKBP12 remain controversial. Through pulldown assays with recombinant proteins, we provide biochemical evidence of the interaction between FKBP12 and RyR1, RyR3 and IP(3)R1. Using NMR chemical shift mapping, we show that the important binding residues in FKBP12 are located in its hydrophobic FK506 binding region. Consistently, we demonstrate that FK506 can competitively inhibit the interaction between FKBP12 and the dipeptide motifs of the calcium channels. We believe our results shed lights on the binding mechanism of calcium channel-FKBP12 interaction.     

Differential Binding Properties of the Peripheral-Type Benzodiazepine Ligands [3H]PK 11195 and [3H]Ro 5-4864 in Trout and Mouse Brain Membranes

High-affinity binding sites for [3H]PK 11195 have been detected in brain membranes of rainbow trout (Salmo gairdneri) and mouse forebrain, where the densities of receptors were 1,030 and 445 fmol/mg of protein, respectively. Ro 5-4864 (4'-chlorodiazepam) was 2,200-fold less potent as a competitor of [3H]PK 11195 binding in the piscine than the murine membranes. Investigation of the regional distribution of these sites in trout yielded a rank order of density of spinal cord greater than olfactory bulb = optic tectum = rhombencephalon greater than cerebellum greater than telencephalon. This site in trout shared some of the characteristics of the peripheral-type benzodiazepine receptor (PTBR) (also known as the mitochondrial benzodiazepine receptor) in rodents, i.e., high affinity for PK 11195 and the endogenous ligand protoporphyrin IX, but was unique in the low affinity of Ro 5-4864 (41 microM) and diazepam and the relatively high affinity of the calcium channel ligand diltiazem and two central benzodiazepine ligands, CGS 8216 and CGS 9896. The differential affinity for the two prototypic PTBR ligands in trout is similar to that previously observed in calf and human brain membranes. Structural differences for the trout sites are indicated by the relative inability of diethyl pyrocarbonate to modify histidine residues of the binding site in trout as compared with mouse membranes. Heterogeneity of binding of the two prototypic PTBR ligands in mouse brain membranes was indicated by additivity studies, equilibrium competition experiments, and saturation isotherms, which together support the hypothesis that Ro 5-4864 discriminates between two [3H]PK 11195 binding sites having high (nanomolar) and low (micromolar) affinity, respectively.     

Distribution of neuronal and non-neuronal spliced variants of type 1 IP(3)-receptor in rat hypothalamus and brain stem

In the nervous system, inositol 1,4,5-trisphosphate (IP(3)) is one of the second messengers produced by PI hydrolysis and triggers IP(3)-receptor (IP(3)R) mediated calcium release from intracellular pools. Throughout the brain, the type 1 IP(3)R is predominantly expressed and its mRNA is widely distributed. Alternative splicing of IP(3)R1 (SI and SII) occurs in two distinct regions. SI splicing in the middle of the ligand binding domain may alter the IP(3) binding activity, while SII splicing probably affects the protein kinase A phosphorylation sites and kinetics. Selective use of IP(3)-receptor subtypes may permit a tissue specific and developmentally specific expression of functionally distinct channels.The present work was focused on detection of the alternatively spliced mRNA of type 1 IP(3)-receptor in individual brain structures and nuclei. Using RT-PCR we detected neuronal (535bp) and non-neuronal (410bp) forms. We identified both spliced variants in the majority of brain structures, except in the cerebellum and medulla. In the cerebellum, the neuronal form of type 1 IP(3)R was found exclusively, while in the medulla, the non-neuronal form was much more abundant. Nevertheless, Western blot analysis and hybridization with specific antibody against IP(3)R revealed no qualitative, but only quantitative differences. Similarly, IP(3) dependent calcium release did not show any differences between the cerebellum and pons. These results demonstrate the distribution of alternatively spliced S2 variants of type 1 IP(3)R in selected brain structures and nuclei. The physiological relevance of these two forms remains to be elucidated by further studies.     

Type-specific inositol 1,4,5-trisphosphate receptor localization in the vomeronasal organ and its interaction with a transient receptor potential channel,TRPC2

The vomeronasal organ (VNO) is the receptor portion of the accessory olfactory system and transduces chemical cues that identify social hierarchy, reproductive status, conspecifics and prey. Signal transduction in VNO neurons is apparently accomplished via an inositol 1,4,5-trisphosphate (IP3)-activated calcium conductance that includes a different set of G proteins than those identified in vertebrate olfactory sensory neurons. We used immunohistochemical (IHC) and SDS-PAGE/western analysis to localize three IP3 receptors (IP3R) in the rat VNO epithelium. Type-I IP3R expression was weak or absent. Antisera for type-II and -III IP3R recognized appropriate molecular weight proteins by SDS-PAGE, and labeled protein could be abolished by pre-adsorption of the respective antibody with antigenic peptide. In tissue sections, type-II IP3R immunoreactivity was present in the supporting cell zone but not in the sensory cell zone. Type-III IP3R immunoreactivity was present throughout the sensory zone and overlapped that of transient receptor potential channel 2 (TRPC2) in the microvillar layer of sensory epithelium. Co-immunoprecipitation of type-III IP3R and TRPC2 from VNO lysates confirmed the overlapping immunoreactivity patterns. The protein-protein interaction complex between type-III IP3R and TRPC2 could initiate calcium signaling leading to electrical signal production in VNO neurons.