concentration within intracellular stores is not known, nor is the biochemical machinery that mediates the signaling from Ca 2ϩ stores to the plasma membrane.In this study, we demonstrate that overexpression of calreticulin in L fibroblasts leads to an increase in the Ca 2ϩ content of thapsigargin-sensitive Ca 2ϩ stores and to a decrease in store-operated Ca 2ϩ influx. EXPERIMENTAL PROCEDURESMaterials-Thapsigargin and Geneticin were obtained from Life Technologies (Basel, Switzerland); monensin, ionomycin, and DTPA were obtained from Sigma. 45 Ca 2ϩ was purchased from Du Pont de Nemours/New England Nuclear Inc. (Dreieich, Germany), and fura-2/AM was from Molecular Probes, Inc. (Eugene, OR). Dulbecco's modified Eagle's culture medium and fetal calf serum were obtained from Gibco (Paisley, Scotland). Restriction endonucleases and DNA-modifying enzymes were purchased from Boehringer Mannheim, Life Technologies, Inc., and Bio/Can Scientific. Mouse L fibroblasts were a generous gift of D. Kobasa (Department of Biochemistry, University of Alberta). All other chemicals were of the highest grade available and were obtained from Fluka (Buchs, Switzerland) or Sigma. The medium referred to as "Ca 2ϩ -free medium" contained 138 mM NaCl, 6 mM KCl, 1 mM MgCl 2 , 20 mM glucose, and 20 mM HEPES, pH 7.4. The medium referred to as "Ca 2ϩ medium" contained, in addition, 1.0 mM CaCl 2 . When drugs were added as dimethyl sulfoxide solutions, final concentrations of dimethyl sulfoxide in the recording medium did not exceed 0.25%.Plasmid Construction-To construct pRCR calreticulin expression vector, the DraI/SmaI restriction DNA fragment (nucleotides 20 -1653) of pcDx-CRT (GenBank accession number J05138) (4) was first inserted into SmaI-digested pSVL vector (Pharmacia Biotech Inc.) to generate pSVL-CRT (pSCR-1) calreticulin expression vector. Next, the XhoI/SstI DNA restriction fragment, containing cDNA encoding full-length calreticulin, was excised from pSCR-1 vector and inserted into XhoI/SstI restriction sites of pRc/CMV vector (Invitrogen) to generate pRCR. * This work was supported by Swiss National Foundation Grant 32 30161.90 and by grants from the Medical Research Council of Canada, the Carlos and Elsie de Reuter Foundation, the Sandoz Foundation, the Ernst and Lucie Schmidheiny Foundation, the Société Académique de Genève, the Helmut Horten Foundation, and the Zyma Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.§ To whom correspondence should be addressed.

Stimulation of the T-cell receptor (TCR) activatesCa 2؉ entry across the plasma membrane, which is a key triggering event for the T-cell-associated immune response. We show that TRPC3 channels are important for the TCR-dependent Ca 2؉ entry pathway. The TRPC3 gene was found to be damaged in human T-cell mutants defective in Ca 2؉ influx. Mutations of the TRPC3 gene were accompanied by changes of TRPC3 gene expression. Introduction of the complete human TRPC3 cDNA into those mutants rescued Ca 2؉ currents as well as TCR-dependent Ca 2؉ signals. Our data provide the initial step toward understanding the molecular nature of endogenous Ca 2؉ channels participating in T-cell activation and put forward TRPC3 as a new target for modulating the immune response.

A cDNA clone encoding the human C5a anaphylatoxin receptor has been isolated by expression cloning from a CDM8 expression library prepared from mRNA of human myeloid HL-60 cells differentiated to the granulocyte phenotype with dibutyryladenosine cyclic monophosphate. The cDNA clone was able to transfer to COS-7 cells the capacity to specifically bind iodinated human recombinant C5a. The cDNA was 2.3 kb long, with an open reading frame encoding a 350-residue polypeptide. Cross-linking of iodinated C5a to the plasma membrane of transfected COS cells revealed a complex with an apparent molecular mass of 52-55 kDa, similar to that observed for the constitutively expressed receptor in differentiated HL-60 cells or human neutrophils. Although differentiated HL-60 cells display a single class of binding sites, with a dissociation constant of approximately 800-900 pM, the C5a-R cDNA, expressed in COS cells, generates both high-affinity (1.7 nM) and low-affinity (20-25 nM) receptors. Sequence comparison established that the degree of sequence identity between the C5a receptor and the N-formylpeptide receptor is 34%.

This observation suggests that hyperstimulation likely recruits young follicles that are unable to acquire imprint at KvDMR1 during the course of the maturing process.

The molecular basis of capacitative (or store-operated) Ca 2+ entry is still subject to debate. The transient receptor potential proteins have been hypothesized to be structural components of store-operated Ca 2+ channels and recent evidence suggests that Trp3 and its closely related homolog Trp6 are gated by the N-terminal region of the inositol 1,4,5-triphosphate receptors (InsP 3 R). In this study, we report the existence of two isoforms of the human Trp4 protein, referred to as K K-hTrp4 and L L-hTrp4. The shorter variant L L-hTrp4 is generated through alternative splicing and lacks the C-terminal amino acids G 785Ŝ 868 . Using a yeast two-hybrid assay and glutathione-Stransferase-pulldown experiments, we found that the C-terminus of K K-hTrp4, but not of L L-hTrp4, associates in vitro with the Cterminal domain of the InsP 3 receptors type 1, 2 and 3. Thus, we describe a novel interaction between Trp proteins and InsP 3 R and we provide evidence suggesting that the formation of hTrp4^InsP 3 R complexes may be regulated by alternative splicing. ß