title: Effects of rapamycin-induced oligomerization of parvalbumin, Stim1 and Orai1 in puncta formation.
abstract: Elevations of cytosolic Ca2+ from the endoplasmic reticulum (ER) regulate a diverse range of cellular processes. When these luminal stores become depleted, the transmembrane ER protein Stim1 oligomerizes and translocates within the ER membrane to puncta junctions to couple with Orai1 channels, activating store-operated calcium entry (SOCE). Stim1 oligomerization and puncta formation have generally been associated with its luminal domains, however, studies have implicated that the cytoplasmic domains may contribute to this oligomerization. Studies have also suggested that intermediate or regulating elements may be required to fine-tune puncta formation and activation of SOCE. Here we made fusion proteins of Stim1 and Orai1 with FRB and FKBP12 domains that associate in the presence of rapamycin. Rapamycin-induced coupling of Stim1 to Stim1, Orai1 to Orai1 and Stim1 to Orai1 was found to be insufficient for puncta formation. Rapamycin was then used to recruit the cytosolic Ca2+ buffer protein parvalbumin (Pav) to Stim1 in order to buffer the local cytosolic Ca2+ near the ER membrane. Interestingly, Pav buffering near the ER caused puncta formation that was indistinguishable from those caused by thapsigargin. Our results suggest that Stim1 oligomerization and puncta formation may be additionally regulated either by local Ca2+ levels near the ER membrane or by as yet unidentified Ca2+-dependent proteins interacting with the cytoplasmic domains of Stim1.
authors.affiliation: Institute of Biomaterials and Biomedical Engineering, Edward S. Rogers, Sr. Department of Electrical and Computer Engineering, University of Toronto, 164 College Street, Toronto, Ontario M5S 3G9, Canada.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Pham
authors.forename: Stanley S C
authors.initials: SS
authors.lastname: Wong
authors.forename: Seema
authors.initials: S
authors.lastname: Nagaraj
authors.forename: Kevin
authors.initials: K
authors.lastname: Truong
mesh_terms: D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects; D002522:Chlorocebus aethiops; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000187:drug effects* / Q000648:ultrastructure; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D010320:Parvalbumins / Q000737:chemistry / Q000378:metabolism; D055503:Protein Multimerization / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D020123:Sirolimus / Q000494:pharmacology; D022061:Tacrolimus Binding Protein 1A / Q000737:chemistry / Q000378:metabolism
chemical_list: D015220:Calcium Channels; D008565:Membrane Proteins; D010320:Parvalbumins; D011993:Recombinant Fusion Proteins; D022061:Tacrolimus Binding Protein 1A; D002118:Calcium; D020123:Sirolimus