Abstract
Upon engagement of the T cell receptor, InsP3 is produced, triggering the release of Ca2+ from ER Ca2+ stores. The resulting ER Ca2+ store depletion is sensed by STIM1, which relocalizes to ER-plasma membrane junctions to activate Ca2+ channels and inhibit Plasma Membrane Ca2+/ATPase (PMCA)-mediated Ca2+ extrusion, thereby enabling sustained increases in cytosolic Ca2+ that drive the process of T cell activation. Partner of STIM1 (POST) is a recently identified STIM1 adaptor protein with multiple binding partners, including STIM1, PMCA and several other transporters, pumps and exchangers. POST is a multi-pass transmembrane protein found in the ER and PM predicted to have 10 transmembrane domains. To assess this predicted topology, we used fluorescence protease protection assays, finding that POST is actually a 9 transmembrane-containing protein with a cytosolic N-terminus and a luminal/extracellular C-terminus. To assess the dynamics of STIM1/POST-mediated control of PMCA activity, we utilized colocalization and FRET. Upon T cell activation, POST migrated and co-localized with both STIM1 and PMCA4 to the immunological synapse. Interestingly, FRET between POST and both proteins was observed, yet FRET between STIM1 and PMCA4 could not be detected. Finally, POST knockdown inhibited activation-dependent inhibition of PMCA4-mediated Ca2+ clearance. These studies provide new insight into the topology and dynamics of POST/STIM1/PMCA4 interactions during T cell activation. Given its numerous targets, further investigations into POST function may reveal additional roles for POST in the T cell activation process.
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