Cav1⌬51-169 also suppressed thapsigargin-and carbachol-stimulated Ca 2؉ influx and increased the detergent solubility of TRPC1, although plasma membrane lipid raft domains were not disrupted. These data demonstrate that plasma membrane localization of TRPC1 depends on an interaction between its N terminus and Cav1. Thus, our data suggest that Cav1 has an important role in the assembly of SOCE channel(s). SOCE1 is activated in response to stimulation of plasma membrane receptors that are coupled to the mobilization of intracellular Ca 2ϩ . Although there is considerable evidence to suggest that the signal for the activation of SOCE is the depletion of Ca 2ϩ within the endoplasmic reticulum (1), the mechanism involved in activation or inactivation of SOCE is not yet known. A major drawback in understanding the mechanism of SOCE has been the lack of information regarding the identity and regulation of the SOCE channel. Members of the TRPC protein family have been suggested as molecular components of the SOCE channel (2-4). TRPC1 has been reported to be required for SOCE in several cell types (5-8). Our previous studies showed that TRPC1 is a critical component of SOCE in salivary epithelial cells (9 -11). Further, we reported previously that, like the Drosophila TRP (12), TRPC1 and TRPC3 are assembled in signaling complexes that are associated with Cav1 and key Ca 2ϩ signaling proteins such as G␣ q/11 , inositol trisphosphate receptor, phospholipase C␤, PMCA (plasma membrane Ca 2ϩ pump), SERCA (sarco-endoplasmic reticulum Ca 2ϩ pump), and calmodulin (13-15). The Drosophila TRP signalplex is assembled via INAD, a multi-PDZ domain-containing protein. INAD binds to a number of proteins in the complex and determines its localization in the rhabdomeres (4, 12). Interestingly, the components of this signalplex appear to be trafficked independently to the plasma membrane, and interaction with INAD is required for their retention at that location. Although several mammalian PDZ domain-containing proteins have been shown to act as scaffolds for receptor-associated signaling complexes in the plasma membrane (16), little is known about the organization of TRPC channels in mammalian cells (17). Tang et al. (18) have reported that NHERF, a two PDZ domain-containing protein that associates with the actin cytoskeleton via interactions with members of ezrin/radixin/moesin family, also interacts with TRPC4 and TRPC5 and might be involved in their association with phospholipase C isozymes. However, the C-terminal PDZ domain binding motif found in TRPC4 is absent in TRPC1. Thus, the molecular component(s) involved in the assembly of the TRPC1-associated protein complex is not yet known.In recent years, much evidence has emerged which demonstrates that key molecules involved in Ca 2ϩ signaling are associated with caveolar lipid rafts, thus implicating the importance of caveolae in Ca 2ϩ signaling (13, 19 -22). Lipid rafts are detergent-insoluble membrane domains formed by the dynamic clustering of sphingolipids and cholesterol which ...

No abstract

Store-operated Ca 2؉ entry (SOCE) is activated by redistribution of STIM1 into puncta in discrete ER-plasma membrane junctional regions where it interacts with and activates store-operated channels (SOCs). The factors involved in precise targeting of the channels and their retention at these specific microdomains are not yet defined. Here we report that caveolin-1 (Cav1) is a critical plasma membrane scaffold that retains TRPC1 within the regions where STIM1 puncta are localized following store depletion. This enables the interaction of TRPC1 with STIM1 that is required for the activation of TRPC1-SOCE. Silencing Cav1 in human submandibular gland (HSG) cells decreased plasma membrane retention of TRPC1, TRPC1-STIM1 clustering, and consequently reduced TRPC1-SOCE, without altering STIM1 puncta. Importantly, activation of TRPC1-SOCE was associated with an increase in TRPC1-STIM1 and a decrease in TRPC1-Cav1 clustering. Consistent with this, overexpression of Cav1 decreased TRPC1-STIM1 clustering and SOCE, both of which were recovered when STIM1 was expressed at higher levels relative to Cav1. Silencing STIM1 or expression of ⌬ERM-STIM1 or STIM1( 684 EE 685 ) mutant prevented dissociation of TRPC1-Cav1 and activation of TRPC1-SOCE. However expression of TRPC1-( 639 KK 640 ) with STIM1( 684 EE 685 ) restored function and the dissociation of TRPC1 from Cav1 in response to store depletion. Further, conditions that promoted TRPC1-STIM1 clustering and TRPC1-SOCE elicited corresponding changes in SOCE-dependent NFkB activation and cell proliferation. Together these data demonstrate that Cav1 is a critical plasma membrane scaffold for inactive TRPC1. We suggest that activation of TRPC1-SOC by STIM1 mediates release of the channel from Cav1. S tore-operated calcium entry (SOCE) is activated by depletion of endoplasmic reticulum (ER) Ca 2ϩ stores and regulates a variety of critical cellular functions (1). Ca 2ϩ depletion in the ER lumen is detected by the Ca 2ϩ -binding protein STIM1, which oligomerizes into puncta and relocates to discrete ERplasma membrane (ER-PM) junctional regions (2, 3) where it associates with and activates store-operated channels including Orai1 and TRPC1, which are components of CRAC and SOC channels, respectively (4-13). Therefore, the location of these channels in the plasma membrane is likely to be critical for their interaction with peripheral STIM1 and activation. However, mechanisms involved in the precise targeting and retention of the channels at the domains where STIM1 puncta are located are not well-understood.Distinct regions of STIM1 determine aggregation and targeting of the protein to ER-PM junctional domains as well as its clustering with and gating of Orai1 and TRPC1 at these sites. The SAM and coiled-coiled domains are involved in STIM1 aggregation while the polybasic C-terminal region of STIM1 is suggested to target STIM1 to ER-PM junctional regions, which is the likely site for SOCE in native cells (3,(9)(10)(11)14). Thus, it can be predicted that SOCs are either localized in this...

No abstract

We report here an in vivo study of kinesin heavy chain (KHC) functions in yeast. We have identified in Schizosaccharomyces pombe a kinesin motor gene, klp3+, which has the highest homology to the Neurospora crassa KHC. Using indirect immunofluorescence, HA epitope‐tagged Klp3 protein is cytoplasmic and appears as one to a few distinct patches that are coincident with microtubules. The klp3 null allele is viable. In klp3 deleted cells, ER, Golgi and mitochondrial distribution appear normal. Mitochondrial distribution in S. pombe is known to be microtubule‐associated. We show that latrunculin A does not cause mitochondria to aggregate, suggesting that mitochondrial distribution in fission yeast, unlike budding yeast, is not dependent upon actin‐based processes. Neither latrunculin A nor thiabendazole affects ER or Golgi distribution. We also used the vital dye FM4‐64 to visualize the internalization of the dye and its transport to vacuoles in fission yeast in the presence and absence of Klp3. We observed no significant difference between the wild‐type and Klp3 null cells in either the dynamics of endocytosis or the distribution and fusion of vacuoles. The drug brefeldin A causes Golgi‐to‐ER recycling in wild‐type fission yeast cells. Although recycling of Golgi to ER after brefeldin A treatment occurs in klp3 null cells, recycling is defective and the distribution pattern we see is different from that observed in the wild‐type strain. We conclude that Klp3 plays a role in BFA‐induced membrane transport. The nucleotide sequence of S. pombe klp3+ was submitted to GenBank under Accession No. AF154055. Copyright © 2000 John Wiley & Sons, Ltd.