The small GTPases of the Rac/Rho/Cdc42 subfamily are implicated in actin cytoskeleton-membrane interaction in mammalian cells and budding yeast. The in vivo functions of these GTPases in multicellular organisms are not known. We have cloned Drosophila homologs of rac and CDC42, Dracl, and Dcdc42. They share 70% amino acid sequence identity with each other, and both are highly expressed in the nervous system and mesoderm during neuronal and muscle differentiation, respectively. We expressed putative constitutively active and dominant-negative Dracl proteins in these tissues. When expressed in neurons, Dracl mutant proteins cause axon outgrowth defects in peripheral neurons without affecting dendrites. When expressed in muscle precursors, they cause complete failure of, or abnormality in, myoblast fusion. Expressions of analogous mutant Dcdc42 proteins cause qualitatively distinct morphological defects, suggesting that similar GTPases in the same subfamily have unique roles in morphogenesis.

The TRPC (C-type transient receptor potential) class of ion channels has been hypothesized to participate in store-operated Ca 2؉ entry (SOCE). Recently, however, STIM1 and Orai1 proteins have been proposed to form SOCE channels. Whether TRPCs participate in SOCE that is dependent on or regulated by Orai has not been explored. Here we show that Orai1 physically interacts with the N and C termini of TRPC3 and TRPC6, and that in cells overexpressing either TRPC3 or TRPC6 in a store-depletion insensitive manner, these TRPCs become sensitive to store depletion upon expression of an exogenous Orai. Thus, Orai-1, -2, and -3 enhanced thapsigargin-induced calcium entry by 50 -150% in cells stably overexpressing either TRPC3 or TRPC6. Orai1 expression had no significant effect on endogenous, thapsigargin-induced calcium entry in wildtype cells (HEK-293, COS1), in HEK cells expressing a thapsigarginsensitive variant of TRPC3 (TRPC3a), or in HEK cells overexpressing another membrane protein, V1aR. Single-channel cation currents present in membrane patches of TRPC3-overexpressing cells were suppressed by expression of Orai1. We propose that Orai proteins by interacting with TRPCs act as regulatory subunits that confer STIM1-mediated store depletion sensitivity to these channels.capacitative calcium entry ͉ ion channels ͉ thapsigargin ͉ transmembrane signaling

Receptor-operated Ca 2؉ entry (ROCE) and store-operated Ca 2؉ entry (SOCE) into cells are functions performed by all higher eukaryotic cells, and their impairment is life-threatening. The main molecular components of this pathway appear to be known. However, the molecular make-up of channels mediating ROCE and SOCE is largely unknown. One hypothesis proposes SOCE channels to be formed solely by Orai proteins. Another proposes SOCE channels to be composed of both Orai and C-type transient receptor potential (TRPC) proteins. Both hypotheses propose that the channels are activated by STIM1, a sensor of the filling state of the Ca 2؉ stores that activates Ca 2؉ entry when stores are depleted. The role of Orai in SOCE has been proven. Here we show the TRPC-dependent reconstitution of Icrac, the electrophysiological correlate to SOCE, by expression of Orai1; we also show that R91W-Orai1 can inhibit SOCE and ROCE and that Orai1 and STIM1 expression leads to functional expression of Gd-resistant ROCE. Because channels that mediate ROCE are accepted to be formed with the participation of TRPCs, our data show functional interaction between ROCE and SOCE components. We propose that SOCE/Icrac channels are composed of heteromeric complexes that include TRPCs and Orai proteins.capacitative calcium entry ͉ signal transduction ͉ store depletion R eported independently by three laboratories at the beginning of 2006, Orai (also known as CRACM) proteins, especially Orai1, have emerged as the molecular candidates for the underlying structure of store-operated Ca 2ϩ entry (SOCE) channels responsible for the store-depletion activated Ca 2ϩ current, Icrac, without apparent role(s) for transient receptor potential ion channels (TRPCs) (reviewed in refs. 1-3). Before the discovery of Orai genes and their gene products, the only candidates presumed to underlie SOCE had been the members of the TRPC class of ion channels, of which there are seven. This proposition was based on primary research reports from many laboratories showing that expression of the cloned TRPCs enhances store-depletion activated Ca 2ϩ entry (cf. table 1.3 in ref. 4) and/or that injection of specific anti-TRPC antibodies or genetic ablation of TRPC genes reduces SOCE and/or Icrac. Moreover, Zagranichnaya et al. (5) showed partial reduction of SOCE in response to siRNAs that targeted TRPC1, TRPC3, or TRPC7, which, when combined, were partially additive. The idea promoted in the reviews listed above that channels responsible for SOCE and Icrac form without involvement of TRPC proteins is especially difficult to reconcile with the total loss of Icrac in vascular endothelial cells seen in TRPC4 knock-out mice (6) and the 80% loss of SOCE found in submaxillary acinar cells of TRPC1 knockout mice (7).Based on the foregoing data, and recognizing that Orai/ CRACMs are part of SOCE/Icrac channels, we proposed that SOCE/Icrac channels may be heteromeric complexes of TRPC and Orai proteins (8). If this were the case, we reasoned, expression of Orai1 in cells expressing excess ...

TRPC and Orai proteins have both been proposed to form Ca 2؉ -selective, store-operated calcium entry (SOCE) channels that are activated by store-depletion with Ca 2؉ chelators or calcium pump inhibitors. In contrast, only TRPC proteins have been proposed to form nonselective receptor-operated calcium entry (ROCE) cation channels that are activated by Gq/Gi-PLC␤ signaling, which is the physiological stimulus for store depletion. We reported previously that a dominant negative Orai1 mutant, R91W, inhibits Ca 2؉ entry through both SOCE and ROCE channels, implicating Orai participation in both channel complexes. However, the argument for Orai participating in ROCE independently of store depletion is tenuous because store depletion is an integral component of the ROCE response, which includes formation of IP3, a store-depleting agent. Here we show that the R91W mutant also blocks diacylglycerol (DAG)-activated Ca 2؉ entry into cells that stably, or transiently, express DAG-responsive TRPC proteins. This strongly suggests that Orai and TRPC proteins form complexes that participate in Ca 2؉ entry with or without activation of store depletion. To integrate these results with recent data linking SOCE with recruitment of Orai and TRPCs to lipid rafts by STIM, we develop the hypothesis that Orai:TRPC complexes recruited to lipid rafts mediate SOCE, whereas the same complexes mediate ROCE when they are outside of lipid rafts. It remains to be determined whether the molecules forming the permeation pathway are the same when Orai:TRPC complexes mediate ROCE or SOCE.diacylglycerol ͉ STIM1 ͉ store operated calcium entry ͉ transient receptor potential

Phospholipase C⑀ (PLC⑀) is a novel class of phosphoinositide-specific PLC characterized by possession of CDC25 homology and Ras/Rap1-associating domains. We and others have shown that human PLC⑀ is translocated from the cytoplasm to the plasma membrane and activated by direct association with Ras at its Ras/Rap1-associating domain. In addition, translocation to the perinuclear region was induced upon association with Rap1⅐GTP. However, the function of the CDC25 homology domain remains to be clarified. Here we show that the CDC25 homology domain of PLC⑀ functions as a guanine nucleotide exchange factor for Rap1 but not for any other Ras family GTPases examined including Rap2 and Ha-Ras. Consistent with this, coexpression of fulllength PLC⑀ or its N-terminal fragment carrying the CDC25 homology domain causes an increase of the intracellular level of Rap1⅐GTP. Concurrently, stimulation of the downstream kinases B-Raf and extracellular signalregulated kinase is observed, whereas the intracellular level of Ras⅐GTP and Raf-1 kinase activity are unaffected. In wild-type Rap1-overexpressing cells, epidermal growth factor induces translocation of PLC⑀ to the perinuclear compartments such as the Golgi apparatus, which is sustained for at least 20 min. In contrast, PLC⑀ lacking the CDC25 domain translocates to the perinuclear compartments only transiently. Further, the formation of Rap1⅐GTP upon epidermal growth factor stimulation exhibits a prolonged time course in cells expressing fulllength PLC⑀ compared with those expressing PLC⑀ lacking the CDC25 homology domain. These results suggest a pivotal role of the CDC25 homology domain in amplifying Rap1-dependent signal transduction, including the activation of PLC⑀ itself, at specific subcellular locations such as the Golgi apparatus.

Calcium channels in the plasma membrane rarely remain open for much more than a millisecond at any one time, which avoids raising intracellular calcium to toxic levels. However, the dihydropyridinesensitive calcium channels of the CaV1 family, which selectively couple electrical excitation to endocrine secretion, cardiovascular contractility, and neuronal transcription, have a unique second mode of gating, ''mode 2,'' that involves frequent openings of much longer duration. Here we report that two human conditions, cyclosporin neurotoxicity and Timothy syndrome, increase mode 2 gating of the recombinant rabbit CaV1.2 channel. In each case, mode 2 gating depends on a Ser residue at the cytoplasmic end of the S6 helix in domain I (Ser-439, Timothy syndrome) or domain IV (Ser-1517, cyclosporin). Both Ser reside in consensus sequences for type II calmodulin-dependent protein kinase. Pharmacologically inhibiting type II calmodulin-dependent protein kinase or mutating the Ser residues to Ala prevents the increase in mode 2 gating. We propose that aberrant phosphorylation, or ''phosphorylopathy,'' of the CaV1.2 channel protein contributes to the excitotoxicity associated with Timothy syndrome and with chronic cyclosporin treatment of transplant patients.calcium͞calmodulin-dependent protein kinase type II ͉ calcineurin ͉ dihydropyridine ͉ excitotoxicity

A yeast two-hybrid screening for Ras-binding proteins in nematode Caenorhabditis elegans has identified a guanine nucleotide exchange factor (GEF) containing a Ras/Rap1A-associating (RA) domain, termed Ce-RA-GEF. Both Ce-RA-GEF and its human counterpart Hs-RA-GEF possessed a PSD-95/DlgA/ZO-1 (PDZ) domain and a Ras exchanger motif (REM) domain in addition to the RA and GEF domains. They also contained a region homologous to a cyclic nucleotide monophosphate-binding domain, which turned out to be incapable of binding cAMP or cGMP. Although the REM and GEF domains are conserved with other GEFs acting on Ras family small GTP-binding proteins, the RA and PDZ domains are unseen in any of them. Hs-RA-GEF exhibited not only a GTP-dependent binding activity to Rap1A at its RA domain but also an activity to stimulate GDP/GTP exchange of Rap1A both in vitro and in vivo at the segment containing its REM and GEF domains. However, it did not exhibit any binding or GEF activity toward Ras. On the other hand, Ce-RA-GEF associated with and stimulated GDP/GTP exchange of both Ras and Rap1A. These results indicate that Ce-RA-GEF and Hs-RA-GEF define a novel class of Rap1A GEF molecules, which are conserved through evolution.Ras proteins are small guanine nucleotide-binding proteins that serve as molecular switches in regulation of cellular proliferation and differentiation by cycling between the active GTP-bound and the inactive GDP-bound forms (for a review, see Ref. 1). In mammalian cells, the GTP-bound Ras exerts its action by physically associating with and activating effector proteins, such as the serine/threonine kinase Raf-1, through its effector region (amino acid residues 32-40 in human Ha-Ras). In addition to Raf-1 and its isoforms B-Raf and A-Raf, recent searches have identified a number of Ras effectors (or effector candidates) that associate directly with Ras in a GTP-dependent manner (for a review, see Ref.2). Two of them, RalGDS 1

Mammalian Ras proteins regulate multiple effectors including Raf, Ral guanine nucleotide dissociation stimulator (RalGDS), and phosphoinositide 3-kinase. In the nematode Caenorhabditis elegans, LIN-45 Raf has been identified by genetic analyses as an effector of LET-60 Ras. To search for other effectors in C. elegans, we performed a yeast two-hybrid screening for LET-60-binding proteins. The screening identified two cDNA clones encoding a phosphoinositide-specific phospholipase C (PI-PLC) with a predicted molecular mass of 210 kDa, designated PLC210. PLC210 possesses two additional functional domains unseen in any known PI-PLCs. One is the C-terminal Ras-associating domain bearing a structural homology with those of RalGDS and AF-6. This domain, which could be narrowed down to 100 amino acid residues, associated in vitro with human Ha-Ras in a GTP-dependent manner and competed with yeast adenylyl cyclase for binding Ha-Ras. The binding was abolished by specific mutations within the effector region of Ha-Ras. The other functional domain is the N-terminal CDC25-like domain, which possesses a structural homology to guanine nucleotide exchange proteins for Ras. These results strongly suggest that PLC210 belongs to a novel class of PI-PLC, which is a putative effector of Ras.