BIOLOGICAL OVERVIEW

The release of intracellular Ca2+ is an intermediate step in many cellular signaling processes. The soluble messenger known as inositol 1,4,5 triphosphate) acts to release intracellular Ca2+ from the intracellular store maintained within the endoplasmic reticulum. The InsP3 receptor (Itp-r83A), here called InsP3R, is a Ca2+ channel that releases intracellular Ca2+ in response to InsP3. This signal transduction pathway is used in processes as diverse as the responses to hormones, growth factors and neurotransmitters, as well as in various vertebrate sensory systems, such as olfaction, gustation and vision (Hasan, 1992 and references).

Before describing the biology of the Drosophila InsP3 receptor, a short diversion will provide a closer look at Ca2+ signaling and intracellular Ca2+ channels. There are two common motifs for Ca2+ release from intracellular storage depots into the cytosol for signal transduction, depending on whether the cells are excitable or non-excitable.

1. Nonexcitable cells: Here the slow inositol (1,4,5)-trisphosphate (InsP3)-mediated pathway predominates. Two receptor classes (the G protein-coupled receptor class of seven transmembrane-spanning receptors and the receptor tyrosine kinases), acting indirectly, release InsP3, which in turn acts as an intracellular second messenger by binding to the specialized tetrameric InsP3 receptor (InsP3R) that spans the endoplasmic reticular membrane and triggers release of Ca2+. G protein-coupled receptors activate phospholipase Cbeta while receptor tyrosine kinases stimulate phospholipase Cgamma to convert the lipid known as phosphatidylinositol (4,5)-biphosphate into the small soluble messenger InsP3 and the membrane bound lipid diacylglycerol. Diacylglycerol serves to activate another signaling cascade by activating the enzyme Protein kinase C (Clapham, 1995).

2. Excitable cells: The Ca2+ release from intracellular storage depots into the cytosol also occurs in excitable cells containing voltage-dependent Ca2+ channels that enable these cells to dramatically increase cytosolic Ca2+ levels. In excitable cells, Ca2+ entering through voltage-dependent Ca2+ channels may directly activate ryanodine receptors, the excitable cell counterparts to the InsP3 receptor, to release Ca2+ from intracellular stores (Clapham, 1995).

The InsP3R of Drosophila (Itp-r83A) is expressed ubiquitously and is involved in multiple aspects of development: it is required for embryonic and larval development, is involved in muscle development and is implicated in chemosensory functions. The InsP3R was identified based on its homology to mammalian InsP3R. The protein is highly conserved and shows the complex structure of a transmembrane protein, with identifiable ligand binding domains, ATP binding domains, a conserved presumptive Ca2+ channel and conserved cysteine residues also found in the ryanodine receptor, but the fly protein shows no Protein kinase A phosphorylation sites, indicating that it is not regulated by the PKA pathway as is the case for the mammalian homologs (Yoshikawa, 1992).

Numerous modulatory signals have been postulated for mammalian InsP3R receptor: the best characterized is known as type 1 InsP3R (Insp3r1). Calmodulin, cyclic GMP-dependent protein kinase and Calcium/calmodulin dependent protein kinase II have all been proposed as modifiers of the activity of the channel protein in addition to ATP, Protein kinase A and ligand. Thus, other second messenger transduction cascades, i.e., cyclic AMP (via PKA), diacyl glycerol (via PKC), and cyclic GMP (via cyclic GMP-dependent protein kinase) all converge on a single modulatory and transducing domain found in the middle portion of the receptor. In addition, alternative splicing results in variations in regulatory regions of the protein. The protein is differentially distributed throughout the adult brain and show preferences for distinct subcellular compartments. Besides being present in cerebellar Purkinje cells, InsP3R1 is present in the olfactory tubercle, cerebral cortex, CA1 region of the hippocampus, caudate-putamen, molecular layer of the cerebellum and choroid plexis (Furuichi, 1995).

Mutants in the Drosophila InsP3R gene arrest development at the second larval instar; many larval tissues are observed that have not proliferated normally in these mutants. Expression during the embryonic phase in mesoderm and sensory organ precursor cells suggests that Insp3R could be involved in embryonic development but that no embryonic phenotype is observed because of the presumed presence of a maternal transcript. If maternal transcripts play a role in embryonic development, then making germ line clones lacking all InsP3R should lead to early lethality. When such clones are generated no viable eggs or embryos are recovered. It is therefore likely that InsP3R larval lethality is a maternal-effect phenomenon due to the preponderance of maternally contributed message. It is concluded that InsP3R is essential for cell proliferation, growth and differentiation. More specific information about the roles of Insp3R in Drosophila development await analysis of mutant clones where the maternal contribution is diminished (Acharya, 1997).

Availability of Insp3R mutants has allowed an assessment of the role of InsP3R in Drosophila photoreceptor signaling. One of the many unanswered questions stimulating research in the field of invertebrate photoreceptor biology has to do with the gating mechanism of light-activated channels. In Drosophila photoreceptor neurons, light activation of rhodopsin activates a Gqalpha, which in turn activates a Phospholipase C (PLC) encoded by the norpA gene. PLC catalyzes the breakdown of PIP2 into InsP3 and diacyl glycerol; these are thought to lead to the eventual opening (and modulation) of the TRP and TRPL membrane channels and the generation of a receptor potential. It has been proposed that the TRP channel functions as a store-operated channel (opened by Ca2+ released from intracellular stores) that responds to a capacitative calcium entry signal. According to this model, light-activation of rhodopsin results in the production of InsP3, which would lead to the emptying of InsP3-sensitive internal calcium stores and the subsequent gating of plasma membrane channels. It has been thought that TRP channels and their vertebrate homologs represent examples of store-operated channels (Acharya, 1997 and references).

However, TRP (transient receptor potential) and TRPL light-activated ion channels, known to be involved in the light sensitivity of the Drosophila retina, do not localize in close proximity to the internal calcium stores, thus eliminating the possibilty of gating by protein-protein interaction via conformation coupling (Niemeyer, 1996). It has also been shown that all light-induced increases in Ca2+ are entirely dependent on the influx of extracellular Ca2+ (Ranganathan, 1994). These results suggest that internal InsP3-sensitive stores are not involved in activation of the light responses (for a contrary result see InsP3R and visual phototransduction). To catagorically test the involvement of InsP3R in light activation, clones of Insp3R negative photoreceptors were generated in an otherwise wild-type background. Mutant photoreceptors and surrounding tissues undergo retinal degeneration, consistent with defective growth of mutant larval tissue. Intracellular recordings were carried out to study light responses in control and mutant photoreceptor cells. InsP3R mutant photoreceptors are indistinguishable from wild-type controls in sensitivity, kinetics of activation and deactivation, and adaptation. Thus, no evidence was found for a role of the InsP3R in Drosophila phototransduction, and models invoking InsP3-induced calcium release in the activation of this pathway are likely to be invalid (Acharya, 1997).

GENE STRUCTURE

Two bands of hybridization, differing very slightly in size, are seen with the Drosophila InsP3R probe. The smaller-sized band (approx. 9.7 kb) is seen only in early embryos of 0-6 hours. A larger band of approx. 10 kb is expressed in mid-late embryos. The basis for the difference between these two bands is unknown (Hasan, 1992). The InsP3R gene is on the third chromsome at position 83A4-5. Two transcriptional units can be detected in this region: InsP3R and the NMDA receptor (Acharya, 1997).

Bases in 5' UTR - 845

PROTEIN STRUCTURE

Amino Acids - 2833

Structural Domains

The Drosophila InsP3R has striking similarity in its overall structure to the mouse InsP3R, although the homologous sequences are interspersed by some stretches of insert, deleted, and diverged sequences. Among the sequence differences, it is notable that a 40-amino acid deletion of the mouse sequence (residues 1692-1731) is found after Lys-1792 in the Drosophila sequence. The amino acids in Drosophila share 57% identity with those of the mouse sequence. The sequence similarity between the mouse InsP3R and the ryanodine receptor is also well conserved overall in Drosophila, with 8 conserved Cys residues (Cys-6555, -1345, -2052, -2123, -2614, -2620, -2697, -2700) in the Drosophila receptor (Yoshikawa, 1992).

A comparison of the InsP3 receptor and the Drosophila ryanodine receptors shows that they share significant homology near their C-terminal amino acids. The 300 C-terminal amino acids of the cloned Drosophila ryanodine receptor and InsP3R are 62.6% similar and 33.6% identical. Rodent and fly ryanodine receptor genes appear equally related to the rodent and fly InsP3 receptor genes, indicating than an ancient duplication event probable gave rise to these two classes of intracellular Ca2+-releasing channel genes (Hasan, 1992).

Hydrophobicity analysis of the InsP3R receptor indicates at least 6 main peaks of hydrophobic amino acids that could represent multiple membrane-spanning sequences (residues 2367-3676). It is likely that the mouse receptor traverses the membrane 6 times as well. The large N-terminal region (83.5% of the protein) is located on the cytoplasmic side, and a short C-terminal region is likewise found on the cytoplasmic face. There is significant homology in the N-terminal regions (residues 1-695 in Drosophila and 1-664 in the mouse), including the N-terminal InsP3-binding region of rodent receptors. The Drosophila and mouse sequences have 12% postively charged amino acids (30 Arg and 51 Lys in 677 amino acids of the Drosophila receptor) that may be involved in binding to a negatively charged InsP3 molecule. These N-terminal homologous regions contain fragmentary but significant similarity to the ryanodine receptor. In contrast to the mouse InsP3R receptor, which is phosphorylated and regulated by protein kinase A, Drosophila InsP3R diverges in the PKA target site and has no potential protein kinase A phosphorylation site (Yoshikawa, 1992).

ATP is known to promote InsP3-induced Ca2+ release in rodents. The ATP binding residues consist of a Gly-rich stretch in Drosophila (residues 2092-97) and in mouse (2016-21). It is assumed that the rodent InsP3Rs form a Ca2+ channel structure in the C-terminal transmembrane regions as a homotetramer. Within this transmembrane region (M1-M6) there is significant homology, in particular from residues 2499 and 2411 onwards, in Drosophila and mouse respectively. The last two membrane-spanning sequences (M5 and M6) are, respectively, 87% and 95% homologous. In addition, they resemble the corresponding sequences M3 and M4 of the ryanodine receptors. Within the luminal loop between M5 and M6, there is a peculiar distribution of negative charges, 20 and 28% in Drosophila and mouse receptors, respectively. These negative charges may be involved in efficient Ca2+ permeation by concentrating Ca2+ near the channel pore (Yoshikawa, 1992).

date revised: 16 July 97  

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