Essential Role for the CRAC Activation Domain in Store-dependent Oligomerization of STIM1

Oligomerization of the ER Ca(2+) sensor STIM1 is an essential step in store-operated Ca(2+) entry. The lumenal EF-hand and SAM domains of STIM1 are believed to initiate oligomerization after Ca(2+) store depletion, but the contributions of STIM1 cytosolic domains (coiled-coil 1, CC1; coiled-coil 2, CC2; CRAC activation domain, CAD) to this process are not well understood. By applying coimmunoprecipitation and fluorescence photobleaching and energy transfer techniques to truncated and mutant STIM1 proteins, we find that STIM1 cytosolic domains play distinct roles in forming both “resting” oligomers in cells with replete Ca(2+) stores and higher-order oligomers in store-depleted cells. CC1 supports the formation of resting STIM1 oligomers and appears to interact with cytosolic components to slow STIM1 diffusion. On store depletion, STIM1 lacking all cytosolic domains (STIM1-ΔC) oligomerizes through EF-SAM interactions alone, but these oligomers are unstable. Addition of CC1 + CAD, but not CC1 alone, enables the formation of stable store-dependent oligomers. Within the CAD, both CC2 and C-terminal residues contribute to oligomer formation. Our results reveal a new function for the CAD: in addition to binding and activating Orai1, it is directly involved in STIM1 oligomerization, the initial event triggering store-operated Ca(2+) entry.