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Store-operated Ca2+ influx has recently been shown to require the activation of two proteins, stromal interaction molecule 1 (STIM1) and Orai1. In mammals the putative channel ion selectivity filter is thought to comprise conserved charged residues in the first and third transmembrane domains of Orai1 in addition to three residues in the first extracellular loop. The latter residues, however, are not conserved in either of the Bombyx mori Orai1 variants or in most insects, suggesting that selectivity is a relatively recent evolutionary event. In B. mori, thapsigargin-mediated STIM1 redistribution is dependent on a cluster of highly conserved basic residues (amino acids 380–385) in the C terminus that likely interact with acidic residues in the Orai1 C terminus. BmSTIM1 redistribution in vitro also occurs downstream of pheromone biosynthesis activating neuropeptide receptor activation. Activation of in vivo RNA interference mechanisms confirmed the physiological role of BmSTIM1 and Orai1 in sex pheromone production.
Sex pheromones provide important olfactory cues regarding the sexual receptiveness of many female moths. De novo synthesis of these volatile compounds in the female pheromone gland (PG) is regulated by pheromone biosynthesis activating neuropeptide (PBAN). PBAN is a C-terminally amidated 33–34 amino acid peptide that stimulates pheromone production via extracellular Ca2+,1 that floods into the cell through store-operated channels (SOCs)2 activated downstream of the PBAN receptor (PBANR).3,4 SOCs have recently been shown to involve stromal interaction molecule 1 (STIM1) and Orai1 (reviewed in refs. 5 and 6). The depletion of ER Ca2+ stores downstream of receptor activation triggers a conformational change in STIM1 and its subsequent redistribution to the plasma membrane where it induces the tetramerization of Orai1 dimers and a concomitant influx of Ca2+.
We recently identified the first lepidopteran homologs of STIM1 and Orai1 and demonstrated their functional role in mediating the pheromonotropic effects of PBAN in the silkmoth, Bombyx mori.7 BmSTIM1 is a 577 amino acid protein with high sequence identity to Drosophila melanogaster STIM. Intriguingly, BmSTIM1 contains the C-terminal domain (residues 340–446), termed by various groups as CAD, SOAR, OASF and Ccb9, necessary for Orai1 activation,8–11 but lacks the Lys-rich region present at the extreme C terminus of mammalian STIM1 necessary for Orai1-independent plasma membrane targeting.8 Two variants of Orai1 (BmOrai1A and BmOrai1B), differentiated by a 37 amino acid N-terminal truncation in BmOrai1B, were identified in B. mori. Both have the glutamic acid residues in the first and third transmembrane domains essential for Ca2+ selectivity,12–15 but lack the conserved triad of residues in the first extracellular loop thought to also play a role in Ca2+ selectivity. In all three variants of human Orai (i.e., Orai1–3) and DmOrai, these key residues consist of glutamic acid, aspartic acid, glutamine, or asparagine. In BmOrai1, however, only the first residue (corresponding to hOrai1 D110, hOrai2 E84, hOrai3 E85, and DmOrai D182) in the triad is present, N55 (Fig. 1). Similar sequence diversity is seen in putative Orai from a number of other insect species (Fig. 1), suggesting that the ion selectivity properties of Orai in insects are likely distinct from that observed in vertebrates. This variation in purported key residues raises questions regarding the evolutionary significance of the ion selectivity filter.
Transient expression of BmSTIM1 and BmOrai1 fluorescent chimeras demonstrated that BmSTIM1, similar to the mammalian STIM1, redistributes from the ER to the plasma membrane following thapsigargin-mediated depletion of ER Ca2+ stores. This redistribution, however, requires co-expression of BmOrai1,7 suggesting that translocation is mediated by BmSTIM1-BmOrai1 interactions. A number of groups have recently shown that a conserved region of the STIM1 C terminus mediates STIM1-Orai1 interactions.8–11 Consistent with those reports, we found that deletion of the analogous region in BmSTIM1 (residues 340–446) abolished the thapsigargin-mediated translocation event.7 Sequence alignment of STIM1 from a variety of species reveals that this region contains a highly conserved cluster of basic amino acids corresponding to BmSTIM1 residues 380–385 (hSTIM1 382–387) (Fig. 2) that could potentially function in mediating Orai1 interactions. Ala substitution of the basic residues within this cluster resulted in a phenotype identical to the Δ340–446 mutant,7 suggesting that BmSTIM1 translocation is dependent on electrostatic interactions, perhaps between this cluster of basic amino acids and acidic amino acids in the Orai1 C terminus as proposed by Calloway and co-workers.16 In support of this, lysines 384–386 have recently been shown to be important for the functional coupling of STIM1 and Orai1.17 C-terminal truncations of BmSTIM1 designed to further examine STIM1-Orai1 interactions unexpectedly identified a translocation modulatory domain between residues 377–478.7 Truncation at residue 478 resulted in normal ER localization of BmSTIM1 under resting conditions, whereas truncation at residue 377 resulted in plasma membrane localization, suggesting that residues 377–478 comprise a modulatory domain that exerts a dominant inhibitory effect on translocation under resting conditions. Smyth and co-workers have recently shown that phosphorylation of STIM1 suppresses SOC function.18 Given the presence of 6 potential phosphorylation sites within the putative translocation modulatory domain, it is appealing to speculate that a similar mechanism may play a role in modulating BmSTIM1 translocation.
Few studies have assessed STIM1 and Orai1 function downstream of receptor activation. Because PBAN stimulates Ca2+ influx in insect cells expressing PBANR,3,4 we sought to examine the role of STIM1 and Orai1 downstream of PBANR activation. In insect cells transiently coexpressing BmPBANR, BmSTIM1 and BmOrai1, PBAN triggered BmSTIM1 redistribution to the plasma membrane, suggesting that STIM1 and Orai1 are activated in response to PBAN binding.7 In vivo RNA interference experiments confirmed these results and firmly established a physiological role for STIM1 and Orai1 in sex pheromone production as dsRNA-mediated knockdown of the respective transcripts significantly reduced the pheromonotropic effects of PBAN.7 These findings, in conjunction with studies in other moths and a recent study examining the role of phospholipase C (personal communication, Hull), has lead us to propose the following PBAN signal transduction model (Fig. 3): PBAN binding promotes Gq α dissociation from PBANR and activation of PLCβ1 to generate inositol 1,4,5-trisphosphate (IP3). Soluble IP3 acts on ER-membrane bound IP3 receptors to promote the depletion of ER Ca2+ stores and the subsequent translocation of STIM1 to the plasma membrane where it interacts with Orai1 to trigger an influx of extracellular Ca2+. It is after this last step that the observed species-specificity of the pheromonotropic control point manifests with cAMP production and activation of an early step in sex pheromone production (i.e., acetyl-CoA carboxylase)19 in heliothine species or activation of a late step (e.g., reductase) in other species such as B. mori.
In addition to offering insights into lepidopteran sex pheromone production, it is our expectation that the molecular components of the B. mori sex pheromone pathway can also serve as a model system for studying various topics at the forefront of modern cell biology including receptoractivated Ca2+ signaling, hormone-regulated lipolysis, and lipogenesis.
Previously published online: www.landesbioscience.com/journals/cib/article/11394