In search of an autoinhibitory domain that keeps the STIM1-ct in a quiescent state, we identified an inhibitory acidic segment within the first coiled-coil domain of the STIM1 molecule. Mutation of the acidic residues within this domain rendered STIM1 fully constitutively active both in its native full-length form and as a STIM1-ct tail. To our knowledge, this is the first fully constitutively active STIM1 molecule other than the D76A mutant. Importantly, while the D76A mutation triggers the natural STIM1 activation process by decreasing the Ca2+
affinity of the luminal EF-hand domain (4
), the 4EA mutant bypasses this step and becomes active even when its luminal domain is Ca2+
Our attention to this region of STIM1 was drawn by its significant homology with the C-terminal helical tail of the Orai1. This raised the possibility that this segment may mediate an intramolecular interaction with the CAD/SOAR domain of STIM1 as the latter was shown to interact and activate the Orai1 channels (19
). We hypothesize that STIM1 molecules bearing neutralizing mutations within this region (4EA) are unable to keep the STIM1-ct in an inactive state allowing unimpeded access of the CAD/SOAR domain to the Orai1 channels. Together with the importance of the basic stretch within the CAD/SOAR domain in Orai1 activation, shown here and also reported recently (25
), these findings would be consistent with a mechanistic model in which the positive residues of CAD/SOAR form an intramolecular interaction with the acidic segment in the quiescent state of STIM1 molecules. This intramolecular silencing would then be interrupted during normal STIM1 activation and is disrupted in the 4EA mutant form. The high degree of similarity between the acidic inhibitory segment of STIM1 and the C-terminal tail of Orai1, gives support for this model, which is reminiscent of how certain protein kinses are kept inactive by their own pseudosubstrate sequences (28
). Direct evidence to support this model, however, would require demonstration of an interaction between the acidic and basic segments of STIM1, which has so far been unsuccessful in our hand when using separately expressed domains. Nevertheless it is likely that this interaction has significant structural constraints as it depends on a conformation that is regulated by luminal Ca2+
binding of STIM1 (likely being transduced by oligomerizaion). Also, the isolated CAD domain already forms a dimer (19
), which may hampers its interaction with the STIM1 acidic region. More studies are in progress to address this question with recombinant STIM1-ct molecules.
An alternative explanation for the constitutive activity of the 4EA STIM1 mutants could be a simple disruption of the structure of the first coiled-coil domain thereby causing the unmasking of CAD/SOAR domain. Theoretically, the 4EA mutation could even cause the clustering of STIM1 and trigger the activation process that way. However, unlike the D76A mutant the 4EA or 7EA mutant STIM1 molecules did not show massive clustering without the expression of Orai1 molecules and still underwent clustering after store depletion arguing against clustering or even a major structural distortion within the coiled-coil region. Moreover, a major structural defect would more likely render STIM1 inactive then making it constitutively active.
An important question that remains to be answered is how the luminal ER Ca2+
decrease leads to the unmasking of the CAD domain. A major conformational change within the isolated luminal EF hand and SAM domains have been demonstrated upon Ca2+
), and oligomerization of full-length STIM1 in response to Ca2+
depletion has been well documented by FRET analysis (29
). The role of the various STIM domains in this process was thoroughly studied in a recent study by Covington et al.(30
). These authors showed that overexpressed STIM1 molecules could form oligomers even in the resting state but only when they contain the STIM-ct. A STIM1 mutant lacking the entire cytoplasmic domain showed no basal oligomers but still responded to ER Ca2+
depletion with oligomerization. Most importantly, Covington et al. also found that STIM1 truncated after the CC1 domain promoted a constitutive oligomerization that was unresponsive to Ca2+
depletion unless the CAD domain was added. This study clearly established an important role of the CAD domain in regulating oligomerization with data to suggest that a communication exists between the CC1 and CAD domains. In this context it is noteworthy that the 4K mutant STIM1 failed to show clustering after Ca2+
store depletion in our experiments (data not shown). Our rapamycin-inducible conformational change in STIM1-ct suggests that forcing STIM1 molecules to tightly line up with an orderly fashion (their N-termini being fixed) is sufficient to mimic their clustered state. The possibility was considered that a mechanical stretching of STIM1-ct during the ER-PM bridge formation contributes to its activation. While such a mechanism cannot be ruled out (even for the natural activation process), the fact that we observed similar activation with the mitochondrial anchor, which probably does not represent the same pulling force, and with a significantly longer [9x-helical linker, see (26
)] ER-targeted FRB construct (not shown), makes the mechanical pulling a less likely explanation.
Lastly, the similarity between the STIM1 acidic domain and the Orai1 C-tail drew attention to the importance of the acidic residues in the latter during Orai1 activation. We mutated the two DH residues located at the distal end of this acidic cassette in Orai1 and saw no major difference in its activation (not shown). However, Calloway et al. showed that removal of all Glu and Asp residues within this cassette was needed to eliminate Tg-induced Ca2+
). Their recent FRET studies with the polybasic domain mutant STIM1 and acidic mutant Orai1 molecules indicated that the putative electrostatic interaction was important for Orai1 activation but the two molecules were still able to show FRET when these charges were neutralized (25
). Clearly, more studies are needed to fully understand the molecular sequences that transmit the ER luminal Ca2+
change to the Orai1 channels.
Autoinhibitory segments in the cytosolic STIM1 different from the one identified in this study have already been described in three separate studies (19
). One such region is located between residues 470–490 of STIM1 in the two former studies, and between 445–475 in the latter. In our present studies, deletion of this region in the STIM1-ct constructs (as in the 238–463 STIM1 piece) also yielded higher constitutive activity in a number of cells as already described in (19
). A recent elegant study, analyzing the direct interaction between recombinant cytosolic STIM1 pieces and Orai1 channels expressed in yeast, found that the 233–463 piece of STIM1 was a very poor interactor and activator of Orai1 (32
), which is consistent with the transient activation by this fragment in our studies and suggests that sequences between the 463 and 502 contain sites that stabilize STIM1 Orai1 interactions. Recently, this acidic segment encoded by STIM1-(475–483) was found responsible for the fast Ca2+
dependent inactivation of Orai1 (33
). These data together suggest that the 475–483 segment is involved in Ca2+
-dependent inactivation rather than keeping the full-STIM1 molecule inactive in quiescent cells.
Several modifiers are superimposed on the basic activation mechanism of Orai1 by STIM1 suggested in this study. Drosophila STIM roughly corresponds to the minimal segment (1–499) (35
) that still shows almost full Orai1 activation. Nevertheless, STIM1 truncations in the cytoplasmic region that extends beyond residue 502 have identified several other regions within STIM1 that modify the activation and inactivation pattern of ICRAC
. These regions have been reviewed in detail recently (12
). Also, as shown in a recent study, phosphorylation of STIM1 in regions beyond residue 482 can prevent STIM1 activation and being responsible for the inability of Tg to induce Ca2+
influx during the cell cycle (36
). Therefore, more studies are needed to fully understand the complex activation mechanism of STIM1.
In summary, the present studies revealed important regions within the STIM1 molecule suggesting an autoinhibitory intramolecular interaction within the cytoplasmic segment of STIM1 molecules. Identification of an acidic region within the STIM1 coiled-coil domain that keeps STIM1 in an inactive state and a short basic region within the activation domain of STIM1 are suggestive of an intramolecular silencing mechanism. The significant similarity between the acidic inhibitory STIM1 segment and the C-terminal helical segment of Orai1 presumed to be the site of activating interaction with the STIM1 molecule gives support to this model. This mechanism of intramolecular silencing resembles the regulation of kinases by intramolecular pseudosubstrate binding and may reveal a new paradigm in Ca2+ influx regulation.