We report here that PLC-gamma isoforms are required for agonist-induced Ca2+ entry (ACE). Overexpressed wild-type PLC-gamma1 or a lipase-inactive mutant PLC-gamma1 each augmented ACE in PC12 cells, while a deletion mutant lacking the region containing the SH3 domain of PLC-gamma1 was ineffective. RNA interference to deplete either PLC-gamma1 or PLC-gamma2 in PC12 and A7r5 cells inhibited ACE. In DT40 B lymphocytes expressing only PLC-gamma2, overexpressed muscarinic M5 receptors (M5R) activated ACE. Using DT40 PLC-gamma2 knockout cells, M5R stimulation of ER Ca2+ store release was unaffected, but ACE was abolished. Normal ACE was restored by transient expression of PLC-gamma2 or a lipase-inactive PLC-gamma2 mutant. The results indicate a lipase-independent role of PLC-gamma in the physiological agonist-induced activation of Ca2+ entry.

The TRPC3 channel, an intensively studied member of the widely expressed transient receptor potential (TRP) family, is a Ca 2؉ -conducting channel activated in response to phospholipase C-coupled receptors. Despite scrutiny, the receptor-induced mechanism to activate TRPC3 channels remains unclear. Evidence indicates TRPC3 channels interact directly with intracellular inositol 1,4,5-trisphosphate receptors (InsP 3 Rs) and that channel activation is mediated through coupling to InsP 3 Rs. TRPC3 channels were expressed in DT40 chicken B lymphocytes in which all three InsP 3 R genes were deleted (DT40InsP 3 R-k/o). Endogenous B-cell receptors (BCR) coupled through Syk kinase to phospholipase C-␥ (PLC-␥) activated the expressed TRPC3 channels in both DT40w/t and DT40InsP 3 R-k/o cells. The diacylglycerol (DAG) analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) also activated TRPC3 channels independently of InsP 3 Rs. BCR-induced TRPC3 activation was blocked by the PLC enzymic inhibitor, U-73122, and also blocked by wortmannin-induced PLC substrate depletion. Neither U-73122 nor wortmannin modified either OAG-induced TRPC3 activation or store-operated channel activation in DT40 cells. Cotransfection of cells with both G protein-coupled M5 muscarinic receptors and TRPC3 channels resulted in successful M5 coupling to open TRPC3 channels mediated by PLC-␤. We conclude that TRPC3 channels are activated independently of InsP 3 Rs through DAG production resulting from receptor-mediated activation of either PLC-␥ or PLC-␤.The TRP 1 channel family comprises a large group of channels mediating an array of signal and sensory transduction pathways (1). The proteins of the mammalian TRPC subfamily are the products of at least seven genes coding for cation channels that appear to be activated in response to PLC-coupled receptors (1-3). These channels are closely related in structure and function to the group of TRP channel proteins, first identified in Drosophila, that mediate the PLC-dependent light-induced current in retinal cells (4,5). Particular interest surrounds the mammalian TRPC subfamily because these channels have been implicated as important mediators of Ca 2ϩ entry (1,3,6). Reports indicate that they may function as "store-operated" channels (6 -12) mediating the process of capacitative Ca 2ϩ entry that is essential for longer term Ca 2ϩ signals and replenishment of Ca 2ϩ stores (13, 14). Of great interest has been the elucidation of a coupling mechanism for the TRPC3 channel involving intracellular InsP 3 Rs (8, 15). Thus, a direct functional communication between TRPC3 channels and InsP 3 Rs has been revealed from reconstitution studies (8,(15)(16)(17). Moreover, recent studies have revealed a physical interaction between the TRPC3 channel and the InsP 3 R (17, 18) and have mapped the interacting loci of the two proteins (19,20). Interactions have also been described between other TRPC channels and InsP 3 Rs (21, 22). Considering these results, it was important to assess the function and coupling of TRPC3 channels expres...

Mutations in presenilin proteins (PS1 and PS2) are associated with most cases of early-onset Alzheimer's disease. Several proteins appear to regulate accumulation of PS proteins in cells. One such protein is ubiquilin-1, which increases levels of coexpressed PS2 protein in a dose-dependent manner. We now report that overexpression of ubiquilin-2, which is 80% identical to ubiquilin-1, also increases the levels of coexpressed PS1 and PS2 proteins in cells. To investigate the mechanism by which ubiquilin proteins increase levels of PS proteins, we examined how overexpression of ubiquilin-1, which possesses all of the key signature motifs present in ubiquilin proteins, affects PS2 gene transcription and PS2 protein turnover and ubiquitination. HeLa cells overexpressing both PS2 and ubiquilin-1 had PS2 mRNA levels lower than HeLa cells overexpressing PS2 alone, indicating that ubiquilin-1 overexpression, in fact, decreases PS2 transcription. Cells overexpressing ubiquilin-1 and PS2 displayed decreased turnover of high molecular weight (HMwt) forms of PS2 but not of full-length PS2 proteins. The reduced turnover of HMwt PS2 proteins appears to be mediated by the binding of the ubiquitin-associated domain (UBA) of ubiquilin to ubiquitin chains conjugated onto PS2 proteins. Immunoprecipitation studies indicated that ubiquilin-1 overexpression decreases ubiquitination of coexpressed PS2 proteins, suggesting that binding of ubiquilin might block ubiquitin chain elongation. Consistent with this model, we found that the UBA domain of ubiquilin-1 binds poly-ubiquitin chains in vitro. In addition, we show that ubiquilin proteins colocalize with ubiquitin-immunoreactive structures in cells and that ubiquilin proteins are present within the inner core of aggresomes, which are structures associated with accumulation of misfolded proteins in cells. Our results suggest that ubiquilin proteins play an important role in regulating PS protein levels in cells.