Inositol 1,4,5,-tris-phosphate receptor

EVOLUTIONARY HOMOLOGS (part 1/3)

Sequence analysis of Drosophila RyR, IP₃R and SERCA

Sequence analysis of RyR, IP₃R and SERCA was performed as a way to address similarities to and differences from their vertebrate counterparts and within themselves. Computer-generated alignments of 15 RyRs, 13 IP₃Rs and 21 SERCAs were analyzed. The extent of identity between Drosophila RyR and other RyRs considered in this study was the lowest and ranged from 37% (with Homo sapiens RyR type 1) to 45% (with Caenorhabditis elegans unique RyR isoform). It was thus not possible by this means to recognize an accentuated identity among Drosophila RyR and any of the three vertebrate isoforms (Vázquez-Martínez, 2003).

The identity detected between Drosophila IP₃R and other IP₃Rs was intermediate and ranged from 36% (with Caenorhabditis elegans unique IP₃R isoform) to 57% (with Panulirus argus unique IP₃R isoform). In contrast with RyR isoforms, vertebrate IP₃R type 1 isoform showed a slightly higher percentage of identity with the fruit fly receptor (56%), than IP₃R type 2 (53%) and type 3 (50%). SERCA enzymes had the highest percentage of identity within themselves. The range went from 67% (with both Rattus norvergicus and Homo sapiens SERCA type 3) to 81% (with the Procambarus clarkii unique SERCA isoform). Drosophila SERCA had a slightly higher identity with vertebrate type 1 and 2 SERCAs (71-73%) than with type 3 (67-69%) (Vázquez-Martínez, 2003).

Equal-weight ('unrooted') Parsimony and Neighbor Joining analyses of the sequences were performed for RyRs, IP₃Rs and SERCAs. Both programs yielded virtually identical topologies suggesting, as expected, that the three Drosophila proteins grouped together with all other invertebrate genes. Both types of calcium release channels were separated in the phylogram tree very clearly. Drosophila RyR is sister to C. elegans RyR, and both are in a different node from vertebrate RyRs. Type 2 and 3 RyRs were co-segregated in one group and separated from type 1 RyRs (Vázquez-Martínez, 2003).

Drosophila IP₃R was sister to crustacean P. argus IP₃R, whereas the receptor of C. elegans split from all other IP₃Rs. Vertebrate type 1 and 2 IP₃Rs shared a common node. Vertebrate and invertebrate RyRs and IP₃Rs form distinct clades within each type of calcium release channel. Drosophila SERCA grouped with other invertebrates and was closer to vertebrate type 1 SERCAs. Vertebrate type 3 SERCAs were the more distal proteins compared with invertebrate SERCAs (Vázquez-Martínez, 2003).

The homology between the Drosophila RyR and other RyRs, like the C. elegans gene and the three vertebrate ones, was in all cases around 40%. Thus, it is not possible to deduce relationships between the invertebrate receptors and their vertebrate counterparts from sequence data. However, inspection of the phylogram and cladogram indicates that the Drosophila and C. elegans RyRs form a separate group from the vertebrate isoforms. It might seems premature to assign the Drosophila and C. elegans RyRs as type 1 based only in their conspicuous muscular localization, but biochemical data support such a tenet. Taken together, evidence points to a closer relationship between vertebrate RyR type 1 and invertebrate RyR (Vázquez-Martínez, 2003).

IP₃Rs are different: the homology between the Drosophila IP₃R and the IP₃R from C. elegans was smaller (36%) than with the vertebrate isoforms 2 (53%), 3 (50%) and 1 (56%), or the lobster IP₃R (57%). The data may suggest that the Drosophila IP₃R is closer to the vertebrate IP₃R type 1 than the other 2 isoforms. One can speculate that this state of affairs is due to evolutionary divergence since the last common ancestor between nematodes and arthropods/vertebrates happened earlier in time than the split between arthropods and vertebrates. Once the vertebrate lineage split from the arthropods, several duplication events in the vertebrate lineage gave rise to the current three isoforms. It can be added that similarities in homology values among all RyRs considered could be explained by somewhat different structural constraints in RyRs compared to IP₃Rs (Vázquez-Martínez, 2003).

RyRs and IP₃Rs are homologous proteins sharing 30-35% homology at the amino acid level. However, there are three regions where the homology is higher: (1) the first 600 amino acids (numbering based on RyRs sequences), (2) the central region between amino acids 1500 and 2600, and (3) the C-terminal domain starting from residue 3900, containing the transmembrane domains. Both the phylogram and the cladogram show that the RyRs and IP₃Rs from invertebrates are grouped separately from vertebrate isoforms. Vertebrate isoforms could have diverged because they are specialized to fulfill physiological requirements of determined tissues; for example, RyR type 1 allows the excitation-contraction coupling of skeletal muscle, whereas RyR type 2 does the same in cardiac muscle. What is the strategy used in invertebrates? There are three possibilities: (1) that in Drosophila and other invertebrates the specialized roles of each receptor can be accomplished by alternatively spliced forms of the RyR and IP₃R genes; (2) that the intrinsic molecular properties of each receptor enable them to carry out all the different functions encompassed by their vertebrate counterparts, or (3) that owing to the different nature of tissues in vertebrates and invertebrates, such specialized roles are not required (Vázquez-Martínez, 2003).

InsP3R mutation

Signaling through an epidermal growth factor receptor in the nematode C. elegans stimulates a Ca2+ release pathway that is independent of Ras. Activity of LET-23, the C. elegans homolog of the epidermal growth factor receptor (see Drosophila Egf receptor), is required in multiple tissues. RAS activation is necessary and sufficient for certain LET-23 functions. Two sets of experiments suggest that LIN-3 (a C. elegans homolog of the epidermal growth factor) and LET-23 function in the hermaphrodite gonad are RAS independent. (1) Mutations in components of the RAS pathway capable of rescuing defects in vulval development and viability caused by reduction-of-function let-23 mutations are unable to rescue the sterility caused by such mutations. (2) Mutational analysis of the let-23 gene suggests that distinct domains of the receptor mediate LET-23 function in the vulva, versus the hermaphrodite gonad. In particular, while certain putative binding sites for Src Homology-2 domain-containing proteins are required for LET-23 function in viability and vulval induction, a distinct pair of sites is necessary and sufficient for function in the hermaphrodite gonad (Clandinin, 1998).

Mutations in two loci, lfe-1/itr-1 and lfe-2 are tissue-specific suppressors of reduced LIN-3/LET-23-mediated signaling. Mutations in lfe-1/itr-1 are likely to be gain-of-function while a suppressing mutation in lfe-2 is likely loss-of-function. lfe-1/itr-1 and lfe-2 appear to function downstream of let-23 for hermaphrodite fertility because genetic epistasis tests using both a reduction-of-function allele and a null allele of let-23 demonstrate that mutations in lfe-1/itr-1 or lfe-2 can bypass LET-23 function in the gonad. In addition to their suppression phenotype, the activities of lfe-1/itr-1 and lfe-2 appear to be involved in normal hermaphrodite fertility because lfe-1/itr-1(gf); lfe-2(lf) double-mutant animals display ovulation defects similar to those observed in animals bearing reduction of function mutations in either let-23 or lin-3. It is believed that the fertility function of these two genes lies in the adult somatic gonad, based on an analysis of LFE-2. In particular, LFE-2 is expressed in the adult spermatheca, and misexpression of LFE-2 in adult animals is sufficient to induce defects in spermathecal function (Clandinin, 1998).

Molecular characterization of LFE-1/ITR-1 and LFE-2 argues strongly that LET-23 regulates intracellular calcium levels in the spermatheca. LFE-1/ITR-1 encodes a C. elegans homolog of the mammalian IP3R, an established effector of intracellular calcium release. LFE-2 encodes a nematode homolog of IP3 kinase, whose in vivo function in mammalian cells is unknown but which is very likely to play a regulatory role in calcium release based on its substrate specificity. Thus, an inositol trisphosphate receptor can act as a RAS-independent, tissue-specific positive effector of LET-23. An inositol trisphosphate kinase negatively regulates this transduction pathway. Signals transduced by LET-23 control ovulation through changes in spermathecal dilation, possibly dependent upon calcium release regulated by the second messengers IP3 and IP4. It is likely that LET-23 functions by activation of phospholipase Cgamma, which promotes release of intracellular calcium through production of IP3. These results demonstrate that one mechanism by which receptor tyrosine kinases can evoke tissue-specific responses is through activation of distinct signal transduction cascades in different tissues (Clandinin, 1998).

Type 1 InsP3 receptor (InsP3R1) is the major neuronal member of the InsP3R family in the central nervous system, especially enriched in cerebellar Purkinje cells but also concentrated in neurons in the hippocampal CA1 region, caudate-putamen, and cerebral cortex. Most InsP3R1-deficient mice generated by gene targeting die in utero; animals surviving to birth have severe ataxia and tonic or tonic-clonic seizures and die before weaning. An electroencephalogram shows that they suffer from epilepsy, indicating that InsP3R1 is essential for proper brain function. However, observation by light microscope of the hematoxylin-eosin staining of the brain and peripheral tissues of InsP3R1-deficient mice shows no abnormality, and the unique electrophysiological properties of the cerebellar Purkinje cells of InsP3R1-deficient mice are not severely impaired (Matsumoto, 1996).

Domain structure of InsP3R

In an attempt to define structural regions of the type I inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) involved in its intracellular targeting to the endoplasmic reticulum (ER), the use of green fluorescent protein (GFP) was employed to monitor the localization of a truncated InsP3R mutant containing just the putative transmembrane spanning domain and the C-terminal cytoplasmic domain (amino acids 2216-2749), termed InsP3R(ES). A chimeric GFP-InsP3R(ES) fusion protein was expressed in Xenopus laevis oocytes, and fluorescent confocal microscopy was used to monitor its intracellular localization. Intense fluorescence shows up in the perinuclear region and in a reticular-network under the animal pole of the oocyte, consistent with the targeting of expressed GFP-InsP3R(ES) to perinuclear ER and ER under the animal pole. These findings are consistent with the intracellular localization of the endogenous Xenopus InsP3R shown previously. Electron microscopy data indicate that expressed GFP-InsP3R(ES) is in fact targeted to the ER. Sodium carbonate extraction of microsomal membranes and cross-linking experiments indicate that the expressed chimeric protein is in fact membrane anchored and able to form a homotetrameric complex. These data provide evidence that InsP3R(ES) constitutes the membrane spanning domain of the InsP3R and is able to mediate homotetramer formation, without the need for the large N-terminal cytoplasmic domain. The localization of GFP-InsP3R(ES) on the ER indicates that an ER retention/targeting signal is contained within the transmembrane spanning domain of the inositol trisphosphate receptor (Sayers, 1997).

Inositol 1,4,5-trisphosphate receptor (InsP3R) is an inositol InsP3-gated Ca2+ release channel. Type 1 InsP3R (InsP3R1) is the neuronal member of the IP3R family in the CNS and is predominantly expressed in cerebellar Purkinje cells. To elucidate the molecular mechanisms responsible for coupling gene expression to neuronal InsP3/Ca2+ signaling, the structure and function of the 5'-flanking region of the mouse InsP3R1 gene has been studied. The cloned 5'-flanking region has several sequences sharing identity with motifs for known transcriptional regulation. 5'-flanking regions 1N (from -528 to +169) and 4N (from -4,187 to +169) were fused to a beta-galactosidase gene (lacZ) as a reporter marker and their in vivo gene expression characterized. Both 1N and 4N fusion genes function as strong promoters in a neuroblastoma-glioma hybrid cell line NG108-15. Moreover, both 1N and 4N transgenic mouse lines carrying these 1N and 4N fusion genes show characteristic patterns of beta-galactosidase activity in the CNS that are almost consistent with that of the endogenous InsP3R1 protein, thereby suggesting that the 1N region from -528 to +169 contains sequence elements responsible for regulating gene expression in neurons and for specifying predominant expression in cerebellar Purkinje cells (Furutama, 1996).

The amino acid sequence responsible for the calmodulin (CaM)-binding ability of mouse type 1 InsP3 receptor (InsP3R1) was determined. Various fragments of InsP3R1 were examined for their CaM-binding ability. The sequence stretching from Lys-1564 to Arg-1585 is necessary for the binding. The full-length InsP3R1 with replacement of Trp-1576 by Ala has lost its CaM-binding ability. Antibody against residues 1564-1585 of InsP3R1 inhibits cerebellar InsP3R1 from binding CaM. The fluorescence spectrum of the peptide that corresponds to residues 1564-1585 shifts when Ca(2+)-CaM is added. From the change in the fluorescence spectrum, the dissociation constant (KD) between the peptide and CaM was estimated to be 0.7 microM. The submicromolar value of KD suggests an actual interaction within cells takes place between CaM and InsP3R1. The CaM-binding ability of other types of InsP3Rs was also examined. A part of the type 2InsP3R, including the region showing sequence identity with the CaM-binding domain of IP3R1, also binds CaM, while the expressed full-length type 3 InsP3R does not (Yamada, 1995).

To study the Ca2+ regulation of the inositol 1,4,5-trisphosphate receptor (InsP3R) at the molecular level, the mouse type I InsP3R was expressed as a glutathione S-transferase fusion protein. Both cytosolic and luminal Ca2+ binding sites exist. The luminal Ca2+ binding site maps to the nonconserved acidic subregion of the luminal loop between amino acids 2463 and 2528. The cytosolic Ca2+ binding site is localized in a region just preceding the transmembrane domain M1. The Ca2+ binding site maps to a 23-amino acid stretch between amino acids 2124 and 2146. This cytosolic region showed a single high affinity site for Ca2+. Neither of the identified Ca2+ binding regions contain an EF-hand motif. It is concluded that the type I InsP3R has at least two quite distinct types of Ca2+ binding sites, localized in different structural regions of the protein (Sienaert, 1996).

Structural and functional analyses were used to investigate the regulation of the inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) by Ca2+. To define the structural determinants for Ca2+ binding, cDNAs encoding GST fusion proteins that cover the complete linear cytosolic sequence of the InsP3R-1 were expressed in bacteria. The fusion proteins were screened for Ca2+ and ruthenium red binding through the use of 45Ca2+ and ruthenium red overlay procedures. Six new cytosolic Ca2+-binding regions were detected on the InsP3R, in addition to the one described earlier. Strong 45Ca2+ and ruthenium red binding domains are found in the N-terminal region of the InsP3R as follows: two Ca2+-binding domains are located within the InsP3-binding domain, and three Ca2+ binding stretches are located in a 500-amino acid region just downstream of the InsP3-binding domain. A sixth Ca2+-binding stretch is detected in the proximity of the calmodulin-binding domain. Evidence for the involvement of multiple Ca2+-binding sites in the regulation of the InsP3R was obtained from functional studies on permeabilized A7r5 cells, in which the effects of Ca2+ and Sr2+ on the EC50 were examined, as well as the cooperativity of the InsP3-induced Ca2+ release. The activation by cytosolic Ca2+ is due to a shift in EC50 toward lower InsP3 concentrations; this effect is mimicked by Sr2+. The inhibition by cytosolic Ca2+ is caused by a decrease in cooperativity and by a shift in EC50 toward higher InsP3 concentrations. The effect on cooperativity occurs at lower Ca2+ concentrations than the inhibitory effect on the EC50. Sr2+ mimics the effect of Ca2+ on the cooperativity but not the inhibitory effect on the EC50. The different [Ca2+] and [Sr2+] dependencies suggest that three different cytosolic interaction sites are involved. Luminal Ca2+ stimulates the release without affecting the Hill coefficient or the EC50, excluding the involvement of one of the cytosolic Ca2+-binding sites. It is concluded that multiple Ca2+-binding sites are localized on the InsP3R-1 and that at least four different Ca2+-interaction sites may be involved in the complex feedback regulation of the release by Ca2+ (Sienaert, 1997).

To define the structural determinants for inositol 1,4, 5-trisphosphate (InsP3) binding of the type 1 inositol 1,4, 5-trisphosphate receptor (InsP3R1), a means of expressing the N-terminal 734 amino acids of InsP3R1 (T734) that contain the InsP3 binding region was developed in Escherichia coli. The T734 protein expressed in E. coli exhibits a similar binding specificity and affinity for InsP3 as the native InsP3R from mouse cerebellum. Deletion mutagenesis, in which T734 was serially deleted from the N terminus up to residue 215, markedly reduces InsP3 binding activity. However, when deleted a little more toward the C terminus (to residues 220, 223, and 225), the binding activity is retrieved. Further N-terminal deletions over the first 228 amino acids completely abolish it again. C-terminal deletions up to residue 579 do not affect the binding activity, whereas those up to residue 568 completely abolish it. In addition, the expressed 356-amino acid polypeptide (residues 224-579) exhibits specific binding activity. Taken together, residues 226-578 are sufficient and close enough to the minimum region for the specific InsP3 binding, and thus form an InsP3 binding "core." Site-directed mutagenesis was performed on 41 basic Arg and Lys residues within the N-terminal 650 amino acids of T734. Single amino acid substitutions for 10 residues, which are widely distributed within the binding core and conserved among all members of the InsP3R family, significantly reduce the binding activity. Among these residues three (Arg-265, Lys-508, and Arg-511) are critical for the specific binding, and Arg-568 is implicated in the binding specificity for various inositol phosphates. It is suggested that some of these 10 residues form a basic pocket that interacts with the negatively charged phosphate groups of InsP3 (Yoshikawa, 1996).

Subtypes of the type-1 inositol 1,4,5-trisphosphate (InsP3) receptor differ at the mRNA level in two small variably spliced segments. Segment SI encodes for a sequence within the InsP3-binding domain, thus its presence or absence could affect the functions of the receptor. Anti-peptide antibodies were used to confirm the existence of different subtypes of the InsP3 receptor (InsP3R) protein. The antibody against residues 322-332 within the SI region recognizes a 260 kDa polypeptide in membranes prepared from rat cerebellum or cerebral cortex. The cerebellum contains a few percent of the InsP3R protein having the SI region, whereas the cerebral cortex contained a high proportion of receptors with the SI region. These two tissues are representative of both isoforms, SI- or SI+, and display the same [3H]InsP3-binding characteristics. Thus, the SI region is not involved in the basic properties of the receptor. Deletion of the peptide 316-352 containing the SI segment greatly reduces InsP3 binding. Antibodies against the SI region or against residues 337-349 do not modify the binding of [3H]InsP3 in the cortical membranes rich in the SI+ isoform or in cerebellar membranes. These results suggest that the SI region is not part of the binding site. The subcellular distribution of these two isoforms was investigated in rat liver. The two isoforms are identified in different membrane fractions and they follow the same subcellular distribution. It is suggested that the domain with the SI region may be involved in a function other than InsP3-induced Ca2+ release (Lievremont, 1996).

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are a family of intracellular Ca2+ channels that exist as homo- or hetero-tetramers. In order to determine whether the N-terminal ligand-binding domain is in close physical proximity to the C-terminal pore domain, microsomal membranes were prepared from COS-7 cells expressing recombinant type I and type III IP3R isoforms. Trypsin digestion followed by cross-linking and co-immunoprecipitation of peptide fragments suggest an inter-subunit N- and C-terminal interaction in both homo- and hetero-tetramers. This observation is further supported by the ability of in vitro translated C-terminal peptides to interact specifically with an N-terminal fusion protein. Using a ⁴⁵Ca2+ flux assay, functional evidence that the ligand-binding domain of one subunit can gate the pore domain of an adjacent subunit is provided. It is concluded that common structural motifs are shared between the type I and type III IP3Rs and it is proposed that the gating mechanism of IP3R Ca2+ channels involves the association of the N-terminus of one subunit with the C-terminus of an adjacent subunit in both homo- and heterotetrameric complexes (Boehning, 2000).

Many important cell functions are controlled by Ca2+ release from intracellular stores via the inositol 1,4,5-trisphosphate receptor (IP3R), which requires both IP3 and Ca2+ for its activity. Due to the Ca2+ requirement, the IP3R and the cytoplasmic Ca2+ concentration form a positive feedback loop, which has been assumed to confer regenerativity on the IP3-induced Ca2+ release and to play an important role in the generation of spatiotemporal patterns of Ca2+ signals such as Ca2+ waves and oscillations. Glutamate 2100 of rat type 1 IP3R (IP3R1) is a key residue for the Ca2+ requirement. Substitution of this residue by aspartate (E2100D) results in a 10-fold decrease in the Ca2+ sensitivity without other effects on the properties of the IP3R1. Agonist-induced Ca2+ responses are greatly diminished in cells expressing the E2100D mutant IP3R1, particularly the rate of rise of the initial Ca2+ spike is markedly reduced and the subsequent Ca2+ oscillations are abolished. These results demonstrate that the Ca2+ sensitivity of the IP3R is functionally indispensable for the determination of Ca2+ signaling patterns (Miyakawa, 2001).

In a variety of cells, the Ca2+ signalling process is mediated by the endoplasmic-reticulum-membrane-associated Ca2+ release channel, inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R). Being ubiquitous and present in organisms ranging from humans to Caenorhabditis elegans, InsP3R has a vital role in the control of cellular and physiological processes as diverse as cell division, cell proliferation, apoptosis, fertilization, development, behavior, memory and learning. Mouse type I InsP3R (InsP3R1), found in high abundance in cerebellar Purkinje cells, is a polypeptide with three major functionally distinct regions: the amino-terminal InsP3-binding region, the central modulatory region and the carboxy-terminal channel region. A 2.2-Å crystal structure of the InsP3-binding core of mouse InsP3R1 is presented in complex with InsP3. The asymmetric, boomerang-like structure consists of an N-terminal ß-trefoil domain and a C-terminal alpha-helical domain containing an 'armadillo repeat'-like fold. The cleft formed by the two domains exposes a cluster of arginine and lysine residues that coordinate the three phosphoryl groups of InsP3. Putative Ca2+-binding sites are identified in two separate locations within the InsP3-binding core (Bosanac, 2002).

The importance of a direct coupling between InsP3 and Ca2+ binding to the cytoplasmic portion of InsP3R in the regulation of Ca2+ release channel activity has already been established. Truncation mutagenesis studies have identified two Ca2+-binding fragments of InsP3R, one encoding residues 304-381 and the other corresponding to residues 378-450. Residues E425, D426, E428, D442 and D444 have been shown to be essential for Ca2+ coordination. These residues are part of the two surface acidic clusters identified in the present crystal structure of mouse InsP3R1c. The first site, Ca-I, is located in the ß-domain and consists of residues E246, E425, D426 and E428. The second site, Ca-II, located across the two domains, is composed of residues E283, E285, D444 and D448. Interestingly, Ca2+-binding site Ca-II overlaps with the conserved region P-II, suggesting that the binding of Ca2+ to this site is conformationally coupled with the protein-protein interaction involving other protein domain(s). This finding, together with electron microscope studies of InsP3R and biochemical studies, leads to a tempting speculation on the Ca2+-InsP3 coupling mechanism required for channel activation. The role of binding of InsP3 to the core domain (residues 226-576) might include the release of a conformational constraint that prevents Ca2+ from binding to the receptor. The N-terminal InsP3 binding suppressor region (residues 1-225) might be directly involved in this negative regulation of Ca2+ binding to the receptor, in addition to the modulation of InsP3 binding affinity20. It is equally possible that some other part of the InsP3R or an unidentified cellular protein is involved in this InsP3-Ca2+ coupling mechanism (Bosanac, 2002).

Expression and function of multiple InsP3R isoforms

Continued: see Evolutionary Homologs part 2/3 | part 3/3

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