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Ca(2+) signaling mediated by phospholipase C that produces inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] and diacylglycerol (DAG) controls lymphocyte activation. In contrast to store-operated Ca(2+) entry activated by Ins(1,4,5)P(3)-induced Ca(2+) release from endoplasmic reticulum, the importance of DAG-activated Ca(2+) entry remains elusive. Here, we describe the physiological role of DAG-activated Ca(2+) entry channels in B-cell receptor (BCR) signaling. In avian DT40 B cells, deficiency of transient receptor potential TRPC3 at the plasma membrane (PM) impaired DAG-activated cation currents and, upon BCR stimulation, the sustained translocation to the PM of protein kinase Cbeta (PKCbeta) that activated extracellular signal-regulated kinase (ERK). Notably, TRPC3 showed direct association with PKCbeta that maintained localization of PKCbeta at the PM. Thus, TRPC3 functions as both a Ca(2+)-permeable channel and a protein scaffold at the PM for downstream PKCbeta activation in B cells.

Depletion of intracellular calcium (Ca(2+)) stores induces store-operated Ca(2+) (SOC) entry across the plasma membrane (PM). STIM1, a putative Ca(2+) sensor in the endoplasmic reticulum (ER), has been recently shown to be necessary for SOC channel activation. Here we show that STIM1 dynamically moves in tubulovesicular shape on the ER and its subcompartment in resting living cells, whereas, upon Ca(2+) store depletion, it is rapidly redistributed into discrete puncta that are located underneath, but not inserted into the PM. Normal constitutive movement of STIM1 is mediated through the coiled-coil and Ser/Thr-rich C-terminal domains in the cytoplasmic region of STIM1, whereas subsequent inducible puncta formation further requires the sterile alpha motif domain protruding into the ER lumen. Each of these three domains (coiled-coil, Ser/Thr-rich, and sterile alpha motif) was essential for activating SOC channels. Hence, our findings based on structure-function experiments suggest that constitutive dynamic movement of STIM1 in the ER and its subcompartment is obligatory for subsequent depletion-dependent redistribution of STIM1 into puncta underneath the PM and activation of SOC channels.

Depletion of intracellular calcium (Ca2+) stores induces store-operated Ca2+ (SOC) entry across the plasma membrane (PM). STIM1, a putative Ca2+ sensor in the endoplasmic reticulum (ER), has been recently shown to be necessary for SOC channel activation. Here we show that STIM1 dynamically moves in tubulovesicular shape on the ER and its subcompartment in resting living cells, whereas, upon Ca2+ store depletion, it is rapidly redistributed into discrete puncta that are located underneath, but not inserted into the PM. Normal constitutive movement of STIM1 is mediated through the coiled-coil and Ser/Thr-rich C-terminal domains in the cytoplasmic region of STIM1, whereas subsequent inducible puncta formation further requires the sterile α motif domain protruding into the ER lumen. Each of these three domains (coiled-coil, Ser/Thr-rich, and sterile α motif) was essential for activating SOC channels. Hence, our findings based on structure-function experiments suggest that constitutive dynamic movement of STIM1 in the ER and its subcompartment is obligatory for subsequent depletion-dependent redistribution of STIM1 into puncta underneath the PM and activation of SOC channels. [ABSTRACT FROM AUTHOR]