The composite nature of the interaction between nuclear receptors EcR and DHR38.

Ecdysteroids coordinate essential biological processes in Drosophila through a complex of two nuclear receptors, the ecdysteroid receptor (EcR) and the ultraspiracle protein (Usp). Biochemical experiments have shown that, in contrast to Usp, the EcR molecule is characterized by high intramolecular plasticity. To investigate whether this plasticity is sufficient to form EcR complexes with nuclear receptors other than Usp, we studied the interaction of EcR with the DHR38 nuclear receptor. Previous in vitro experiments suggested that DHR38 can form complexes with Usp and thus disrupt Usp-EcR interaction with the specific hsp27pal response element. This article provides the experimental evidence that EcR is able to form complexes with DHR38 as well. The recombinant DNA-binding domains (DBDs) of EcR and DHR38 interact specifically on hsp27pal. However, the interaction between the receptors is not restricted to their isolated DBDs. We pre\xadsent data that indicate that the full-length EcR and DHR38 can also form specific complexes within the nuclei of living cells. This interaction is mediated by the hinge region of EcR, which was recently classified as an intrinsically disordered region. Our results indicate that DHR38 might modulate the activity of the Usp-EcR heterodimer by forming complexes with both of its components.

Overexpression of Orai1 and STIM1 proteins alters regulation of store-operated Ca2+ entry by endogenous mediators.

Orai1 and STIM1 have been identified as the main determinants of the store-operated Ca(2+) entry (SOCE). Their specific roles in SOCE and their molecular interactions have been studied extensively following heterologous overexpression or molecular knockdown and extrapolated to the endogenous processes in naïve cells. Using molecular and imaging techniques, we found that variation of expression levels of Orai1 or STIM1 can significantly alter expression and role of some endogenous regulators of SOCE. Although functional inhibition of Ca(2+)-independent phospholipase A(2) ß (iPLA(2)ß or PLA2g6A), or depletion of plasma membrane cholesterol caused a dramatic loss of endogenous SOCE in HEK293 cells, these effects were attenuated significantly when either Orai1 or STIM1 were overexpressed. Molecular knockdown of iPLA(2)ß impaired SOCE in both control cells and cells overexpressing STIM1. We also discovered important cross-talk between expression of Orai1 and a specific plasma membrane variant of iPLA(2)ß but not STIM1. These data confirm the role of iPLA(2)ß as an essential mediator of endogenous SOCE and demonstrate that its physiological role can be obscured by Orai1 and STIM1 overexpression.

Orai1 and Ca2+-independent phospholipase A2 are required for store-operated Icat-SOC current, Ca2+ entry, and proliferation of primary vascular smooth muscle cells.

Store-operated Ca(2+) entry (SOCE) is important for multiple functions of vascular smooth muscle cells (SMC), which, depending of their phenotype, can resemble excitable and nonexcitable cells. Similar to nonexcitable cells, Orai1 was found to mediate Ca(2+)-selective (CRAC-like) current and SOCE in dedifferentiated cultured SMC and smooth muscle-derived cell lines. However, the role of Orai1 in cation-selective store-operated channels (cat-SOC), which are responsible for SOCE in primary SMC, remains unclear. Here we focus on primary SMC, and assess the role of Orai1 and Ca(2+)-independent phospholipase A(2) (iPLA(2)ß, or PLA2G6) in activation of cat-SOC current (I(cat-SOC)), SOCE, and SMC proliferation. Using molecular, electrophysiological, imaging, and functional approaches, we demonstrate that molecular knockdown of either Orai1 or iPLA(2)ß leads to similar inhibition of the whole cell cat-SOC current and SOCE in primary aortic SMC and results in significant reduction in DNA synthesis and impairment of SMC proliferation. This is the first demonstration that Orai1 and iPLA(2)ß are equally important for cat-SOC, SOCE, and proliferation of primary aortic SMC.

The variety of complexes formed by EcR and Usp nuclear receptors in the nuclei of living cells.

The heterodimer of the ecdysone receptor (EcR) and ultraspiracle (Usp), members of the nuclear receptor superfamily, is considered to be functional receptor for the ecdysteroids that coordinate essential biological processes in insects. In this work we have applied a bimolecular fluorescence complementation (BiFC) method to directly analyze the formation of the EcR/Usp complex. The BiFC experiments were carried out in mammalian cells which are routinely used for heterologous studies of the EcR/Usp complex, including experiments on EcR-based artificial molecular gene switches. BiFC analysis, supported by flow cytometry, revealed that EcR-Usp interaction is nuclei-restricted. If expressed separately, Usp and EcR are able to form nuclear complexes in the absence of the cognate dimerization partner. We have observed that Muristerone A that is widely used for the induction of ecdysteroid-dependent transcription in mammalian cells, does not significantly change the number of EcR/Usp and EcR/EcR complexes, and it does not influence their subcellular localization.

How strict is the correlation between STIM1 and Orai1 expression, puncta formation, and ICRAC activation?

Stromal interaction molecule 1 (STIM1) and Orai1 have been identified as crucial elements of the store-operated Ca(2+) entry (SOCE) pathway, but the mechanism of their functional interaction remains controversial. It is now well established that, upon depletion of the stores, both molecules can accumulate and colocalize in specific areas (puncta) where the endoplasmic reticulum comes in close proximity to the plasma membrane. Some models propose a direct interaction between STIM1 and Orai1 as the most straightforward mechanism for signal transduction from the stores to the plasma membrane. To test some of the predictions of a conformational coupling model, we assessed how tight the relationships are between STIM1 and Orai1 expression, puncta formation, and SOCE activation. Here we present evidence that STIM1 accumulates in puncta equally well in the presence or absence of Orai1 expression, that STIM1 accumulation is not sufficient for Orai1 accumulation in the same areas, and that normal Ca(2+) release-activated Ca(2+) current (I(CRAC)) can be activated in STIM1-deficient cells. These data challenge the idea of direct conformational coupling between STIM1 and Orai1 as a viable mechanism of puncta formation and SOCE activation and uncover greater complexity in their relationship, which may require additional intermediate elements.

Novel role for STIM1 as a trigger for calcium influx factor production.

STIM1 has been recently identified as a Ca(2+) sensor in endoplasmic reticulum (ER) and an initiator of the store-operated Ca(2+) entry (SOCE) pathway, but the mechanism of SOCE activation remains controversial. Here we focus on the early ER-delimited steps of the SOCE pathway and demonstrate that STIM1 is critically involved in initiating of production of calcium influx factor (CIF), a diffusible messenger that can deliver the signal from the stores to plasma membrane and activate SOCE. We discovered that CIF production is tightly coupled with STIM1 expression and requires functional integrity of its intraluminal sterile alpha-motif (SAM) domain. We demonstrate that 1) molecular knockdown or overexpression of STIM1 results in corresponding impairment or amplification of CIF production and 2) inherent deficiency in the ER-delimited CIF production and SOCE activation in some cell types can be a result of their deficiency in STIM1 protein; expression of a wild-type STIM1 in such cells was sufficient to fully rescue their ability to produce CIF and SOCE. We found that glycosylation sites in the ER-resident SAM domain of STIM1 are essential for initiation of CIF production. We propose that after STIM1 loses Ca(2+) from EF hand, its intraluminal SAM domain may change conformation, and via glycosylation sites it can interact with and activate CIF-producing machinery. Thus, CIF production appears to be one of the earliest STIM1-dependent events in the ER lumen, and impairment of this process results in impaired SOCE response.

Novel DNA-binding element within the C-terminal extension of the nuclear receptor DNA-binding domain.

The heterodimer of the ecdysone receptor (EcR) and ultraspiracle (Usp), members of the nuclear receptors superfamily, is considered as the functional receptor for ecdysteroids initiating molting and metamorphosis in insects. Here we report the 1.95 A structure of the complex formed by the DNA-binding domains (DBDs) the EcR and the Usp, bound to the natural pseudopalindromic response element. Comparison of the structure with that obtained previously, using an idealized response element, shows how the EcRDBD, which has been previously reported to possess extraordinary flexibility, accommodates DNA-induced structural changes. Part of the C-terminal extension (CTE) of the EcRDBD folds into an alpha-helix whose location in the minor groove does not match any of the locations previously observed for nuclear receptors. Mutational analyses suggest that the alpha-helix is a component of EcR-box, a novel element indispensable for DNA-binding and located within the nuclear receptor CTE. This element seems to be a general feature of all known EcRs.

EcR and Usp, components of the ecdysteroid nuclear receptor complex, exhibit differential distribution of molecular determinants directing subcellular trafficking.

Ecdysteroids coordinate development, reproduction and other essential biological processes in insects and other arthropods through the receptor which is a heterodimer of two members of the nuclear receptors superfamily, the ecdysteroid receptor (EcR) and the Ultraspiracle (Usp). Although the transcriptionally active EcR/Usp heterocomplex is believed to be the only functional form of the receptor, there are data indicating that EcR may be involved in the mediation of the non-genomic effects outside of the nucleus. Since the nucleocytoplasmic shuttling could be a key element determining participation of the single nuclear receptor molecule both in the genomic and non-genomic functions we have analyzed nuclear import and export properties of the EcR and Usp from Drosophila melanogaster. We show for the first time that both receptors exhibit differential distribution of the nuclear localization and nuclear export signals (NLSs and NESs). In particular, the Usp which exhibits exclusively nuclear localization in all cell types analyzed, contains apparently only NLS activity within the DNA-binding domain. In contrast, the three known EcR isoforms (A, B1 and B2) are mosaics of elements which can potentially mediate their nucleocytoplasmic shuttling. We have found two active NESs in ligand binding domain and NLS activity within the DNA-binding domain of all isoforms. Simultaneously we demonstrate that B1 and A isoforms possess an additional NLS activity localized in AB regions. We speculate that this characteristic, along with the previously reported structural pliability of the EcR molecule, allows the single receptor to evoke many different genomic as well as non-genomic ecdysteroid-dependent responses.

Ultraspiracle promotes the nuclear localization of ecdysteroid receptor in mammalian cells.

The heterodimer consisting of ecdysteroid receptor (EcR) and ultraspiracle (USP), both of which are members of the nuclear receptor superfamily, is considered to be the functional ecdysteroid receptor. Here we analyzed the subcellular distribution of EcR and USP fused to fluorescent proteins. The experiments were carried out in mammalian COS-7, CHO-K1 and HeLa cells to facilitate investigation of the subcellular trafficking of EcR and USP in the absence of endogenous expression of these two receptors. The distribution of USP tagged with a yellow fluorescent protein (YFP-USP) was almost exclusively nuclear in all cell types analyzed. The nuclear localization remained constant for at least 1 day after the first visible signs of expression. In contrast, the intracellular distribution of EcR tagged with a yellow fluorescent protein (YFP-EcR) varied and was dependent on time and cell type, although YFP-EcR alone was also able to partially translocate into the nuclear compartment. Coexpression of YFP-EcR with USP tagged with a cyan fluorescent protein (CFP-USP) resulted in exclusively nuclear localization of both proteins in all cell types analyzed. The USP-induced nuclear localization of YFP-EcR was stable for at least 20 hours. These experiments suggest that USP has a profound effect on the subcellular distribution of EcR.