Inhibition of cAMP-Dependent PKA Activates ß2-Adrenergic Receptor Stimulation of Cytosolic Phospholipase A2 via Raf-1/MEK/ERK and IP3-Dependent Ca2+ Signaling in Atrial Myocytes.

We previously reported in atrial myocytes that inhibition of cAMP-dependent protein kinase (PKA) by laminin (LMN)-integrin signaling activates ß2-adrenergic receptor (ß2-AR) stimulation of cytosolic phospholipase A2 (cPLA2). The present study sought to determine the signaling mechanisms by which inhibition of PKA activates ß2-AR stimulation of cPLA2. We therefore determined the effects of zinterol (0.1 µM; zint-ß2-AR) to stimulate ICa,L in atrial myocytes in the absence (+PKA) and presence (-PKA) of the PKA inhibitor (1 µM) KT5720 and compared these results with atrial myocytes attached to laminin (+LMN). Inhibition of Raf-1 (10 µM GW5074), phospholipase C (PLC; 0.5 µM edelfosine), PKC (4 µM chelerythrine) or IP3 receptor (IP3R) signaling (2 µM 2-APB) significantly inhibited zint-ß2-AR stimulation of ICa,L in-PKA but not +PKA myocytes. Western blots showed that zint-ß2-AR stimulation increased ERK1/2 phosphorylation in-PKA compared to +PKA myocytes. Adenoviral (Adv) expression of dominant negative (dn) -PKCα, dn-Raf-1 or an IP3 affinity trap, each inhibited zint-ß2-AR stimulation of ICa,L in + LMN myocytes compared to control +LMN myocytes infected with Adv-ßgal. In +LMN myocytes, zint-ß2-AR stimulation of ICa,L was enhanced by adenoviral overexpression of wild-type cPLA2 and inhibited by double dn-cPLA2S505A/S515A mutant compared to control +LMN myocytes infected with Adv-ßgal. In-PKA myocytes depletion of intracellular Ca2+ stores by 5 µM thapsigargin failed to inhibit zint-ß2-AR stimulation of ICa,L via cPLA2. However, disruption of caveolae formation by 10 mM methyl-ß-cyclodextrin inhibited zint-ß2-AR stimulation of ICa,L in-PKA myocytes significantly more than in +PKA myocytes. We conclude that inhibition of PKA removes inhibition of Raf-1 and thereby allows ß2-AR stimulation to act via PKCα/Raf-1/MEK/ERK1/2 and IP3-mediated Ca2+ signaling to stimulate cPLA2 signaling within caveolae. These findings may be relevant to the remodeling of ß-AR signaling in failing and/or aging heart, both of which exhibit decreases in adenylate cyclase activity.

Activation and propagation of Ca(2+) release during excitation-contraction coupling in atrial myocytes.

Fast two-dimensional confocal microscopy and the Ca(2+) indicator fluo-4 were used to study excitation-contraction (E-C) coupling in cat atrial myocytes which lack transverse tubules and contain both subsarcolemmal junctional (j-SR) and central nonjunctional (nj-SR) sarcoplasmic reticulum. Action potentials elicited by field stimulation induced transient increases of intracellular Ca(2+) concentration ([Ca(2+)](i)) that were highly inhomogeneous. Increases started at distinct subsarcolemmal release sites spaced approximately 2 microm apart. The amplitude and the latency of Ca(2+) release from these sites varied from beat to beat. Subsarcolemmal release fused to build a peripheral ring of elevated [Ca(2+)](i), which actively propagated to the center of the cells via Ca(2+)-induced Ca(2+) release. Resting myocytes exhibited spontaneous Ca(2+) release events, including Ca(2+) sparks and local (microscopic) or global (macroscopic) [Ca(2+)](i) waves. The microscopic [Ca(2+)](i) waves propagated in a saltatory fashion along the sarcolemma ("coupled" Ca(2+) sparks) revealing the sequential activation of Ca(2+) release sites of the j-SR. Moreover, during global [Ca(2+)](i) waves, Ca(2+) release was evident from individual nj-SR sites. Ca(2+) release sites were arranged in a regular three-dimensional grid as deduced from the functional data and shown by immunostaining of ryanodine receptor Ca(2+) release channels. The longitudinal and transverse distances between individual Ca(2+) release sites were both approximately 2 microm. Furthermore, electron microscopy revealed a continuous sarcotubular network and one peripheral coupling of j-SR with the sarcolemma per sarcomere. The results demonstrate directly that, in cat atrial myocytes, the action potential-induced whole-cell [Ca(2+)](i) transient is the spatio-temporal summation of Ca(2+) release from subsarcolemmal and central sites. First, j-SR sites are activated in a stochastic fashion by the opening of voltage-dependent sarcolemmal Ca(2+) channels. Subsequently, nj-SR sites are activated by Ca(2+)-induced Ca(2+) release propagating from the periphery.

Calcium transients in 1B5 myotubes lacking ryanodine receptors are related to inositol trisphosphate receptors.

Potassium depolarization of skeletal myotubes evokes slow calcium waves that are unrelated to contraction and involve the cell nucleus (Jaimovich, E., Reyes, R., Liberona, J. L., and Powell, J. A. (2000) Am. J. Physiol. 278, C998-C1010). Studies were done in both the 1B5 (Ry53-/-) murine "dyspedic" myoblast cell line, which does not express any ryanodine receptor isoforms (Moore, R. A., Nguyen, H., Galceran, J., Pessah, I. N., and Allen, P. D. (1998) J. Cell Biol. 140, 843-851), and C(2)C(12) cells, a myoblast cell line that expresses all three isoforms. Although 1B5 cells lack ryanodine binding, they bind tritiated inositol (1,4,5)-trisphosphate. Both type 1 and type 3 inositol trisphosphate receptors were immuno-located in the nuclei of both cell types and were visualized by Western blot analysis. After stimulation with 47 mm K(+), inositol trisphosphate mass raised transiently in both cell types. Both fast calcium increase and slow propagated calcium signals were seen in C(2)C(12) myotubes. However, 1B5 myotubes (as well as ryanodine-treated C(2)C(12) myotubes) displayed only a long-lasting, non-propagating calcium increase, particularly evident in the nuclei. Calcium signals in 1B5 myotubes were almost completely blocked by inhibitors of the inositol trisphosphate pathway: U73122, 2-aminoethoxydiphenyl borate, or xestospongin C. Results support the hypothesis that inositol trisphosphate mediates slow calcium signals in muscle cell ryanodine receptors, having a role in their time course and propagation.

Subunit oligomerization, and topology of the inositol 1,4, 5-trisphosphate receptor.

The inositol 1,4,5-trisphosphate receptor (InsP(3)R) is a tetrameric assembly of highly conserved subunits that contain multiple membrane-spanning sequences in the C-terminal region of the protein. In studies aimed at investigating the oligomerization and transmembrane topology of the type-1 InsP(3)R, a series of membrane-spanning region truncation and deletion plasmids were constructed. These plasmids were transiently transfected in COS-1 cells, and the resulting expression products were analyzed for the ability to assemble into tetrameric structures. The topology of the membrane-spanning region truncations and the full-length receptor was determined by immunocytochemical analysis of transfected COS-1 cells using complete or selective permeabilization strategies. Our results are the first to experimentally define the presence of six membrane-spanning regions. These results are consistent with the current model for the organization of the InsP(3)R in the endoplasmic reticulum and show that the truncation mutants are properly targeted and oriented in the endoplasmic reticulum membrane, thus making them amenable reagents to study receptor subunit oligomerization. Fractionation of soluble and membrane protein components revealed that the first two membrane-spanning regions were necessary for membrane targeting of the receptor. Sedimentation and immunoprecipitation experiments show that assembly of the receptor subunits was an additive process as the number of membrane-spanning regions increased. Immunoprecipitations from cells co-expressing the full-length receptor and carboxyl-terminal truncations reveal that constructs expressing the first two or more membrane-spanning domains were capable of co-assembling with the full-length receptor. Inclusion of the fifth membrane-spanning segment significantly enhanced the degree of oligomerization. Furthermore, a deletion construct containing only membrane-spanning regions 5 and 6 oligomerized to a similar extent as that of the wild type protein. Membrane-spanning region deletion constructions that terminate with the receptor's 145 carboxyl-terminal amino acids were found to have enhanced assembly characteristics and implicate the carboxyl terminus as a determinant in oligomerization. Our results reveal a process of receptor assembly involving several distinct yet additive components and define the fifth and sixth membrane spanning regions as the key determinants in receptor oligomerization.

The N-terminal domain of the IP3 receptor gates store-operated hTrp3 channels.

In the present work, we studied the interaction and effect of several IP3 receptor (IP3R) constructs on the gating of the store-operated (SOC) hTrp3 channel. Full-length IP3R coupled to silent hTrp3 channels in intact cells but did not activate them until stores were depleted of Ca2+. By contrast, constructs containing the IP3-binding domain activated silent hTrp3 channels in unstimulated cells and restored gating of hTrp3 by IP3 in excised plasma membrane patches. We conclude that the N-terminal domain of the IP3R functions as a gate and is sufficient for activation of SOCs. The sensing and transduction domains of the IP3R are required to maintain SOCs in an inactive state.

Location of the permeation pathway in the recombinant type 1 inositol 1,4,5-trisphosphate receptor.

The inositol 1,4,5-trisphosphate receptor (InsP(3)R) forms ligand-regulated intracellular Ca(2+) release channels in the endoplasmic reticulum of all mammalian cells. The InsP(3)R has been suggested to have six transmembrane regions (TMRs) near its carboxyl terminus. A TMR-deletion mutation strategy was applied to define the location of the InsP(3)R pore. Mutant InsP(3)Rs were expressed in COS-1 cells and single channel function was defined in planar lipid bilayers. Mutants having the fifth and sixth TMR (and the interceding lumenal loop), but missing all other TMRs, formed channels with permeation properties similar to wild-type channels (gCs = 284; gCa = 60 pS; P(Ca)/P(Cs) = 6.3). These mutant channels bound InsP(3), but ligand occupancy did not regulate the constitutively open pore (P(o) > 0.80). We propose that a region of 191 amino acids (including the fifth and sixth TMR, residues 2398-2589) near the COOH terminus of the protein forms the InsP(3)R pore. Further, we have produced a constitutively open InsP(3)R pore mutant that is ideal for future site-directed mutagenesis studies of the structure-function relationships that define Ca(2+) permeation through the InsP(3)R channel.

Isoform-specific function of single inositol 1,4,5-trisphosphate receptor channels.

The inositol 1,4,5-trisphosphate receptor (InsP3R) family of Ca2+ release channels is central to intracellular Ca2+ signaling in mammalian cells. The InsP3R channels release Ca2+ from intracellular compartments to generate localized Ca2+ transients that govern a myriad of cellular signaling phenomena (Berridge, 1993. Nature. 361:315-325; Joseph, 1996. Cell Signal. 8:1-7; Kume et al., 1997. Science. 278:1940-1943; Berridge, 1997. Nature. 368:759-760). express multiple InsP3R isoforms, but only the function of the single type 1 InsP3R channel is known. Here the single-channel function of single type 2 InsP3R channel is defined for the first time. The type 2 InsP3R forms channels with permeation properties similar to that of the type 1 receptor. The InsP3 regulation and Ca2+ regulation of type 1 and type 2 InsP3R channels are strikingly different. Both InsP3 and Ca2+ are more effective at activating single type 2 InsP3R, indicating that single type 2 channels mobilize substantially more Ca2+ than single type 1 channels in cells. Furthermore, high cytoplasmic Ca2+ concentrations inactivate type 1, but not type 2, InsP3R channels. This indicates that type 2 InsP3R channel is different from the type 1 channel in that its activity will not be inherently self-limiting, because Ca2+ passing through an active type 2 channel cannot feed back and turn the channel off. Thus the InsP3R identity will help define the spatial and temporal nature of local Ca2+ signaling events and may contribute to the segregation of parallel InsP3 signaling cascades in mammalian cells.

Identification and functional reconstitution of the type 2 inositol 1,4,5-trisphosphate receptor from ventricular cardiac myocytes.

The inositol 1,4,5-trisphosphate receptor (InsP3R) is an intracellular Ca2+ release channel that mediates the rise in cytoplasmic calcium in response to receptor-activated production of InsP3. The InsP3R-mediated signaling pathway appears to be ubiquitous and is involved in many cellular processes including cell division, smooth muscle contraction, and neuronal signaling. Different regions of the heart also express InsP3 receptors. We report here that acutely dissociated ventricular myocytes from ferret and rat hearts express significant levels of InsP3R as indicated by immunoblotting with a receptor consensus antibody. InsP3 binding experiments (KD = 23.6 nM and Bmax = 0.46 pmol/mg) suggest the myocytes contain the high affinity type 2 InsP3 receptor. Exhaustive mRNA screening by polymerase chain reaction, RNase protection, and subsequent DNA sequencing positively identify the InsP3R as type 2. The type 2 receptor from ferret heart was then incorporated into planar lipid bilayers and formed Ca2+-selective, InsP3-activated, heparin-blocked ion channels. We conclude that the predominant InsP3 receptor isoform expressed in cardiac myocytes is type 2 and that it forms a functional InsP3-gated Ca2+ channel when reconstituted in planar lipid bilayers.

Immunogold localization of inositol 1,4,5-trisphosphate receptors and characterization of ultrastructural features of the sarcoplasmic reticulum in phasic and tonic smooth muscle.

Although agonist stimulation leads to an increase in inositol 1,4,5-trisphosphate (InsP3) and decreased calcium in peripherally and centrally located sarcoplasmic reticulum in smooth muscle, the distribution of InsP3 receptors is unknown. InsP3 receptor and the calcium binding protein, calsequestrin were localized by immunolabelling in a tonic and a phasic smooth muscle. InsP3 receptor labelling was predominantly localized at the cell periphery, where most of the sarcoplasmic reticulum is localized in vas deferens (phasic muscle). Elements of central sarcoplasmic reticulum, where present, were also labelled. Distribution of calsequestrin in vas deferens was similar to that of the InsP3 receptor. In aorta (tonic muscle) the InsP3 receptor labelling was proportional to sarcoplasmic reticulum distribution: predominantly central. No labelling of sections or immunoblots was observed with the anti-calsequestrin antibody in aorta. InsP3 and caffeine, but not cyclic ADP-ribose, released intracellular Ca2+ in permeabilized vas deferens and aorta. The ultrastructure of the sarcoplasmic reticulum, investigated in stereo views of semi-thick and thin sections of osmium ferricyanide stained tissue, is shown to have several distinctive features, such as fenestrated sheets (single or in stacks), as well as numerous regions of continuity between central and peripheral sarcoplasmic reticulum, suggesting a single compartment within the smooth muscle cell. Regions of the sarcoplasmic reticulum were closely apposed to and often ensheathed mitochondria. We conclude that InsP3 receptors are present in both the central and the peripheral sarcoplasmic reticulum of tonic and phasic smooth muscle, consistent with electron probe analysis results showing calcium release from both regions.

Co-expression in vertebrate tissues and cell lines of multiple inositol 1,4,5-trisphosphate (InsP3) receptors with distinct affinities for InsP3.

Inositol 1,4,5-trisphosphate (InsP3) is a ubiquitous second messenger in eukaryotic cells that triggers Ca2+ release from intracellular stores. Three types of InsP3 receptors have been identified in mammals. The three receptor types are encoded by homologous genes and are structurally similar, suggesting two alternative hypotheses about the biological significance of multiple InsP3 receptors: (a) the different InsP3 receptors could have similar functions as InsP3-gated Ca2+ channels, and the presence of multiple genes could then serve as a mechanism to allow tissue-specific differential expression of receptors; or (b) the different receptors are co-expressed in cells but have distinct biological roles in these cells. To test these hypothesis, we have investigated the similarities and differences between the expression, alternative splicing, and ligand binding of different receptors. Our results demonstrate co-expression of different InsP3 receptors in almost all tissues and cell lines tested. Although all receptor types exhibit a similar specificity for inositol phosphates, the different receptors have different affinities for InsP3, with a relative order of affinities of type II > type I > type III. These findings suggest that the presence of multiple InsP3-sensitive Ca2+ pools with differential responsiveness to InsP3 may be a general property of all cells mediated by the presence of multiple types of InsP3 receptors.

Inositol 1,4,5-trisphosphate receptor causes formation of ER cisternal stacks in transfected fibroblasts and in cerebellar Purkinje cells.

The inositol 1,4,5-trisphosphate receptor (IP3R) is expressed at very high levels in cerebellar Purkinje cells. Within these neurons, it has a widespread distribution throughout the endoplasmic reticulum (ER) and is present at particularly high concentrations at sites of membrane appositions within peculiar stacks of ER cisternae. Here we report that stacks of ER cisternae, reminiscent of those observed in Purkinje cells, can be induced by overexpression of full-length IP3R, but not of mutant forms of the protein in COS cells. Within these stacks the IP3R forms a crystalline array at apposed cisternal faces. Additionally, we show that Purkinje cell stacks are not permanent structures. Our findings suggest that massive stack formation in purkinje cells represents an adaptive response of the ER to hypoxic conditions and is due to the presence of the high concentration of IP3R in its membranes.

Mechanism of Ca2+ inhibition of inositol 1,4,5-trisphosphate (InsP3) binding to the cerebellar InsP3 receptor.

Ca2+ efficiently inhibits binding of inositol 1,4,5-trisphosphate (InsP3) to the InsP3 receptor in cerebellar membranes but not to the purified receptor. We have now investigated the mechanism of action by which Ca2+ inhibits InsP3 binding. Our results suggest that Ca2+ does not cause the stable association of a Ca(2+)-binding protein with the receptor. Instead, Ca2+ leads to the production of a soluble, heat-stable, low molecular weight substance from cerebellar membranes that competes with InsP3 for binding. This inhibitory substance probably represents endogenously generated InsP3 as judged by the fact that it co-purifies with InsP3 on anion-exchange chromatography, competes with [3H]InsP3 binding in a pattern similar to unlabeled InsP3, and is in itself capable of releasing 45Ca2+ from permeabilized cells. A potent Ca(2+)-activated phospholipase C activity producing InsP3 was found in cerebellar microsomes that exhibited a Ca2+ dependence identical to the Ca(2+)-dependent inhibition of InsP3 binding. Together these results suggest that the Ca(2+)-dependent inhibition of InsP3 binding to the cerebellar receptor is due to activation of a Ca(2+)-sensitive phospholipase C enriched in cerebellum. Nevertheless, Ca2+ probably also modulates the InsP3 receptor function by a direct interaction with the receptor that does not affect InsP3 binding but regulates InsP3-dependent channel gating.

Ca2+ stores in Purkinje neurons: endoplasmic reticulum subcompartments demonstrated by the heterogeneous distribution of the InsP3 receptor, Ca(2+)-ATPase, and calsequestrin.

The nature of second messenger-responsive intracellular Ca2+ stores in neurons remains open for discussion. Here, we demonstrate the existence in Purkinje cells (PCs) of endoplastic reticulum (ER) subcompartments characterized by an uneven distribution of three proteins involved in Ca2+ storage and release: the inositol 1,4,5-trisphosphate (InsP3) receptor, Ca(2+)-ATPase, and calsequestrin. Ca(2+)-ATPase and the InsP3 receptor have a widespread, although not identical, distribution throughout the ER. Calsequestrin is localized throughout the smooth ER and is particularly concentrated in pleiomorphic vesicles with a moderately electron-dense core, which appear to represent a subcompartment of the smooth ER. In double-labeling experiments many of these vesicles were unlabeled by InsP3 receptor antibodies. These results suggest a key role of the ER as an intracellular Ca2+ store and demonstrate a possible structural basis for distinct intracellular Ca2+ pools regulated by different second messengers.

Structure of a novel InsP3 receptor.

Inositol 1,4,5-trisphosphate (InsP3) constitutes a major intracellular second messenger that transduces many growth factor and neurotransmitter signals. InsP3 causes the release of Ca2+ from intracellular stores by binding to specific receptors that are coupled to Ca2+ channels. One such receptor from cerebellum has previously been extensively characterized. We have now determined the full structure of a second, novel InsP3 receptor which we refer to as type 2 InsP3 receptor as opposed to the cerebellar type 1 InsP3 receptor. The type 2 InsP3 receptor has the same general structural design as the cerebellar type 1 InsP3 receptor with which it shares 69% sequence identity. Expression of the amino-terminal 1078 amino acids of the type 2 receptor demonstrates high affinity binding of InsP3 to the type 2 receptor with a similar specificity but higher affinity than observed for the type 1 receptor. These results demonstrate the presence of several types of InsP3 receptor in brain and raise the possibility that intracellular Ca2+ signaling may involve multiple pathways with different regulatory properties dependent on different InsP3 receptors.

rab3A attachment to the synaptic vesicle membrane mediated by a conserved polyisoprenylated carboxy-terminal sequence.

rab3A is a small neuronal GTP-binding protein specifically localized to synaptic vesicles. Membrane-bound rab3A behaves like an intrinsic membrane protein in vitro, but reversibly dissociates from synaptic vesicles after exocytosis in vivo. Here we demonstrate that rab3A is attached to synaptic vesicle membranes by a carboxy-terminal Cys-X-Cys sequence that is posttranslationally modified. This modification is inhibited by compactin in a mevalonate-dependent manner, suggesting that the Cys-X-Cys sequence represents a novel polyisoprenylation sequence. Isolation of a rab3 homolog from D. melanogaster reveals high evolutionary conservation of rab3A, including its carboxy-terminal Cys-X-Cys sequence. The posttranslational modifications of soluble and membrane-bound rab3A are biochemically different, but both require the carboxy-terminal Cys-X-Cys sequence and are faithfully reproduced in nonneuronal cells. Our results suggest that the carboxy-terminal Cys-X-Cys sequence of rab3A is polyisoprenylated and is used as its regulatable membrane anchor. Furthermore, the hydrophobic modification of rab3A and its correct intracellular targeting to synaptic vesicles are independent, presumably consecutive events.

Ryanodine and inositol trisphosphate receptors coexist in avian cerebellar Purkinje neurons.

Two intracellular calcium-release channel proteins, the inositol trisphosphate (InsP3), and ryanodine receptors, have been identified in mammalian and avian cerebellar Purkinje neurons. In the present study, biochemical and immunological techniques were used to demonstrate that these proteins coexist in the same avian Purkinje neurons, where they have different intracellular distributions. Western analyses demonstrate that antibodies produced against the InsP3 and the ryanodine receptors do not cross-react. Based on their relative rates of sedimentation in continuous sucrose gradients and SDS-PAGE, the avian cerebellar InsP3 receptor has apparent native and subunit molecular weights of approximately 1,000 and 260 kD, while those of the ryanodine receptors are approximately 2,000 and 500 kD. Specific [3H]InsP3- and [3H]ryanodine-binding activities were localized in the sucrose gradient fractions enriched in the 260-kD and the approximately 500-kD polypeptides, respectively. Under equilibrium conditions, cerebellar microsomes bound [3H]InsP3 with a Kd of 16.8 nM and Bmax of 3.8 pmol/mg protein; whereas, [3H]ryanodine was bound with a Kd of 1.5 nM and a capacity of 0.08 pmol/mg protein. Immunolocalization techniques, applied at both the light and electron microscopic levels, revealed that the InsP3 and ryanodine receptors have overlapping, yet distinctive intracellular distributions in avian Purkinje neurons. Most notably the InsP3 receptor is localized in endomembranes of the dendritic tree, in both the shafts and spines. In contrast, the ryanodine receptor is observed in dendritic shafts, but not in the spines. Both receptors appear to be more abundant at main branch points of the dendritic arbor. In Purkinje neuron cell bodies, both the InsP3 and ryanodine receptors are present in smooth and rough ER, subsurface membrane cisternae and to a lesser extent in the nuclear envelope. In some cases the receptors coexist in the same membranes. Neither protein is observed at the plasma membrane, Golgi complex or mitochondrial membranes. Both the InsP3 and ryanodine receptors are associated with intracellular membrane systems in axonal processes, although they are less abundant there than in dendrites. These data demonstrate that InsP3 and ryanodine receptors exist as unique proteins in the same Purkinje neuron. These calcium-release channels appear to coexist in ER membranes in most regions of the Purkinje neurons, but importantly they are differentially distributed in dendritic processes, with the dendritic spines containing only InsP3 receptors.

The ligand binding site and transduction mechanism in the inositol-1,4,5-triphosphate receptor.

The inositol-1,4,5-triphosphate (InsP3) receptor consists of a homotetramer of highly conserved 313 kd subunits that contain multiple transmembrane regions in the C-terminal part of the protein. The receptor was expressed in COS cells and its domain structure was studied by mutagenesis. Deletion of the transmembrane regions from the receptor results in the synthesis of a soluble receptor protein that efficiently binds InsP3 but which instead of associating into homotetramers remains monomeric. This result suggests a role for the transmembrane regions in the association of the receptor subunits into tetramers but not in ligand binding. To localize the ligand binding site, further cDNAs encoding truncated receptor proteins were constructed. Assays of InsP3 binding to these truncated InsP3 receptors revealed that sequences in the N-terminal fourth of the InsP3 receptor are sufficient for ligand binding. Accordingly, each subunit of the InsP3 receptor homotetramer contains an independent ligand binding site that is located on the N-terminal ends of each subunit and is separated from the putative channel-forming transmembrane regions by greater than 1400 amino acids. Gel filtration experiments demonstrate a large conformational change of the receptor as a function of ligand binding, suggesting a mechanism by which ligand binding might cause channel opening.

Structure and expression of the rat inositol 1,4,5-trisphosphate receptor.

The complete primary structure of the inositol 1,4,5-trisphosphate receptor from rat brain was elucidated using a series of overlapping cDNA clones. Two different sets of clones that either contain or lack a 45-nucleotide sequence in the amino-terminal third of the protein were isolated, suggesting a differential splicing event that results in the biosynthesis of either a 2734- or 2749-amino acid receptor protein. Hydrophobicity analysis demonstrates the presence of a cluster of hydrophobic sequences in the carboxyl-terminal third of the protein that probably comprise eight transmembrane regions and that may form the calcium channel intrinsic to the receptor. The receptor was universally expressed at low levels in all tissues and cultured cells tested. Transfection of a full-length expression construct of the inositol 1,4,5-trisphosphate receptor into COS cells resulted in the biosynthesis of a 260-kDa protein that bound inositol 1,4,5-trisphosphate and formed high molecular weight complexes similar to the native receptor as analyzed by sucrose gradient centrifugations. On the other hand, the protein product synthesized by a mutant receptor construct in which the amino-terminal 418 amino acids were deleted failed to bind inositol 1,4,5-trisphosphate. The mutant receptor still formed high molecular weight complexes, suggesting that it folded normally and that the amino-terminal sequences of the receptor are part of the ligand binding domain.

Phospholipid binding by a synaptic vesicle protein homologous to the regulatory region of protein kinase C.

Neurotransmitters are released at synapses by the Ca2(+)-regulated exocytosis of synaptic vesicles, which are specialized secretory organelles that store high concentrations of neurotransmitters. The rapid Ca2(+)-triggered fusion of synaptic vesicles is presumably mediated by specific proteins that must interact with Ca2+ and the phospholipid bilayer. We now report that the cytoplasmic domain of p65, a synaptic vesicle-specific protein that binds calmodulin contains an internally repeated sequence that is homologous to the regulatory C2-region of protein kinase C (PKC). The cytoplasmic domain of recombinant p65 binds acidic phospholipids with a specificity indicating an interaction of p65 with the hydrophobic core as well as the headgroups of the phospholipids. The binding specificity resembles PKC, except that p65 also binds calmodulin, placing the C2-regions in a context of potential Ca2(+)-regulation that is different from PKC. This is a novel homology between a cellular protein and the regulatory domain of protein kinase C. The structure and properties of p65 suggest that it may have a role in mediating membrane interactions during synaptic vesicle exocytosis.