Red fluorescent protein from Discosoma as a fusion tag and a partner for fluorescence resonance energy transfer.

The biochemical and biophysical properties of a red fluorescent protein from a Discosoma species (DsRed) were investigated. The recombinant DsRed expressed in E. coli showed a complex absorption spectrum that peaked at 277, 335, 487, 530, and 558 nm. Excitation at each of the absorption peaks produced a main emission peak at 583 nm, whereas a subsidiary emission peak at 500 nm appeared with excitation only at 277 or 487 nm. Incubation of E. coli or the protein at 37 degrees C facilitated the maturation of DsRed, resulting in the loss of the 500-nm peak and the enhancement of the 583-nm peak. In contrast, the 500-nm peak predominated in a mutant DsRed containing two amino acid substitutions (Y120H/K168R). Light-scattering analysis revealed that DsRed proteins expressed in E. coli and HeLa cells form a stable tetramer complex. DsRed in HeLa cells grown at 37 degrees C emitted predominantly at 583 nm. The red fluorescence was imaged using a two-photon laser (Nd:YLF, 1047 nm) as well as a one-photon laser (He:Ne, 543.5 nm). When fused to calmodulin, the red fluorescence produced an aggregation pattern only in the cytosol, which does not reflect the distribution of calmodulin. Despite the above spectral and structural complexity, fluorescence resonance energy transfer (FRET) between Aequorea green fluorescent protein (GFP) variants and DsRed was achieved. Dynamic changes in cytosolic free Ca2+ concentrations were observed with red cameleons containing yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), or Sapphire as the donor and RFP as the acceptor, using conventional microscopy and one- or two-photon excitation laser scanning microscopy. Particularly, the use of the Sapphire-DsRed pair rendered the red cameleon tolerant of acidosis occurring in hippocampal neurons, because both Sapphire and DsRed are extremely pH-resistant.

Molecular cloning, expression and characterization of a phenol sulfotransferase cDNA from mouse intestine.

A sulfotransferase (ST) cDNA was isolated from a mouse intestinal cDNA library using a probe which was generated by reverse transcription (RT)-PCR based on the conserved amino acid sequences of the ST molecules. The isolated cDNA (1.1 kb) contained an 858 bp open reading frame encoding a 286 amino acid polypeptide with molecular weight of 33439. The deduced amino acid sequence shares 55.1% and 40.2% identity with mouse liver aryl/phenol (mSTp1) and alcohol (mSTa1 or mSTa2) STs, respectively, and it is highly similar to those of rat and human liver phenol ST (P-ST) isozymes, ST1B1 (87.8%) and ST1B2 (71.0%), respectively. RT-PCR analyses showed abundant expression of the P-ST mRNA in the intestine as well as in the liver in the mouse tissues examined (brain, heart, intestine, kidney, liver and lung) of both sexes. E. coli-expressed enzyme is capable of catalyzing the sulfation of 2-naphthol at Km = 3.3 microM and Vmax = 3.33 nmol/min/mg protein and also showed sulfation activity for L-3,4-dihydroxyphenylalanine (L-DOPA) and dopamine. Among food constituents tested, tannic acid and epigallocatechin gallate strongly inhibited the P-ST activity in vitro.

On the nature of rat hepatic and mouse olfactory sulfotransferases.

Rat hydroxysteroid sulfotransferase (HS-SULT) cDNAs, ST-40 and ST-20 are 90% identical in amino acid sequences and show different substrate specificities toward dehydroepiandrosterone (DHEA), androsterone (AD) and cortisol (CS). ST-40 enzyme is active toward the three substrates, whereas ST-20 enzyme is preferentially active for CS. First we prepared mutants of well conserved histidine, lysine and asparagine by site-directed mutagenesis. Secondly we constructed 20 chimeric HS-SULTs by reciprocal exchange of five protein domains between ST-20 and ST-40 enzymes. The studies on the expressed mutant and chimeric enzymes indicate the importance of the C-terminal region for the substrate specificity and the involvement of multiple regions for the enzyme activities. Next we determined the genetic loci of ST-40 and ST-20 by fluorescence in situ hybridization. Biotinylated ST-20 and ST-40 probes gave a pair of fluorescent spots on the same region of rat chromosome 1 and the loci of these genes were localized to the same chromosomal region of 1q21.3 --> q22.1. Finally we studied mouse olfactory phenol SULT (P-SULT). It was immunolocalized in the cytoplasm of mouse olfactory sustentacular cells and mouse nasal cytosols show high SULT activities toward phenolic aromatic odorants. We subsequently isolated a mouse P-SULT cDNA from mouse olfactory cDNA library. It encodes 304 amino acid polypeptide and is 94% identical with rat ST1C1 in amino acid sequences.

Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin.

Important Ca2+ signals in the cytosol and organelles are often extremely localized and hard to measure. To overcome this problem we have constructed new fluorescent indicators for Ca2+ that are genetically encoded without cofactors and are targetable to specific intracellular locations. We have dubbed these fluorescent indicators 'cameleons'. They consist of tandem fusions of a blue- or cyan-emitting mutant of the green fluorescent protein (GFP), calmodulin, the calmodulin-binding peptide M13, and an enhanced green- or yellow-emitting GFP. Binding of Ca2+ makes calmodulin wrap around the M13 domain, increasing the fluorescence resonance energy transfer (FRET) between the flanking GFPs. Calmodulin mutations can tune the Ca2+ affinities to measure free Ca2+ concentrations in the range 10(-8) to 10(-2) M. We have visualized free Ca2+ dynamics in the cytosol, nucleus and endoplasmic reticulum of single HeLa cells transfected with complementary DNAs encoding chimaeras bearing appropriate localization signals. Ca2+ concentration in the endoplasmic reticulum of individual cells ranged from 60 to 400 microM at rest, and 1 to 50 microM after Ca2+ mobilization. FRET is also an indicator of the reversible intermolecular association of cyan-GFP-labelled calmodulin with yellow-GFP-labelled M13. Thus FRET between GFP mutants can monitor localized Ca2+ signals and protein heterodimerization in individual live cells.

Stable overexpression of the type-1 inositol 1,4,5-trisphosphate receptor in L fibroblasts: subcellular distribution and functional consequences.

InsP3 receptor (InsP3R)/Ca(2+)-release channels differ markedly in abundance in different tissues/cell types and InsP3R expression levels may be modulated in response to a variety of external cues. Cell lines overexpressing InsP3Rs will provide useful models for the study of the influence of receptor density and subtype on InsP3-mediated Ca2+ signalling. We have investigated the properties of InsP3Rs in mouse L fibroblast cell lines transfected with either type-1 InsP3R cDNA (L15) or vector control (Lvec). L15 cells express approximately eightfold higher levels of the type-1 InsP3R protein than Lvec cells, as assessed by radioligand binding and immunoblotting. Increased expression was stable since it did not alter over ten cell passages. Both L15 and Lvec cells express predominantly the type-1 InsP3R isoform, indicating that functional differences in the InsP3-mediated Ca2+ signalling in these cell lines are due to alteration in the levels of receptor rather than changes in the isoform expressed. Type-1 InsP3R in L15 cells is largely associated with subcellular membrane fractions bearing the sarco/endoplasmic reticulum Ca2+ ATPase pump, appropriate for rapidly exchanging Ca2+ pools. Functionally, there is an approximately fourfold increase in the sensitivity of permeabilized L15-cell Ca2+ mobilization in response to increasing concentrations of Ins(1,4,5)P3. This study indicates that L15/ Lvec cells provide a suitable model for studying the effects of InsP3R expression level on InsP3-induced Ca2+ mobilization.

Heterotetrameric complex formation of inositol 1,4,5-trisphosphate receptor subunits.

The inositol 1,4,5-trisphosphate receptor (IP3R) exists as a tetrameric complex to form a functional inositol 1,4,5-trisphosphate-gated Ca2+ channel. Molecular cloning studies have shown that there are at least three types of IP3R subunits, designated type 1, type 2, and type 3. The levels of expression of IP3R subunits in various cell lines were investigated by Western blot analysis using type-specific antibodies against 15 C-terminal amino acids of each IP3R subunit. We found that all the three types of IP3R subunits were expressed in each cell line examined, but their levels of expression varied. To determine whether IP3Rs form heterotetramers, we employed immunoprecipitation experiments using Chinese hamster ovary cells (CHO-K1 cells), in which all three types are abundantly expressed. Each type-specific antibody immunoprecipitated not only the respective cognate type but also the other two types. This result suggests that distinct types of IP3R subunits assemble to form heterotetramers in CHO-K1 cells. We also detected heterotetramers in rat liver, in which IP3R type 1 and type 2 are expressed abundantly. Previous studies have shown some functional differences among IP3R types, suggesting the possibility that various compositions of subunits show distinct channel properties. The diversity of IP3R channels may be further increased by the co-assembly of different IP3R subunits to form homo- or heterotetramers.

The calmodulin-binding domain in the mouse type 1 inositol 1,4,5-trisphosphate receptor.

We determined the amino acid sequence responsible for the calmodulin (CaM)-binding ability of mouse type 1 Ins(1,4,5)P3 receptor (IP3R1). We expressed various parts of IP3R1 from deleted cDNA and examined their CaM-binding ability. It was shown that the sequence stretching from Lys-1564 to Arg-1585 is necessary for the binding. The full-length IP3R1 with replacement of Trp-1576 by Ala lost its CaM-binding ability. Antibody against residues 1564-1585 of IP3R1 inhibited cerebellar IP3R1 from binding CaM. The fluorescence spectrum of the peptide that corresponds to residues 1564-1585 shifted when Ca(2+)-CaM was added. From the change in the fluorescence spectrum, we estimated the dissociation constant (KD) between the peptide and CaM to be 0.7 microM. The submicromolar value of KD suggests an actual interaction between CaM and IP3R1 within cells. The CaM-binding ability of other types of IP3Rs was also examined. A part of the type 2IP3R, including the region showing sequence identity with the CaM-binding domain of IP3R1, also bound CaM, while the expressed full-length type 3 IP3R did not.

Differential expression of type 2 and type 3 inositol 1,4,5-trisphosphate receptor mRNAs in various mouse tissues: in situ hybridization study.

The inositol 1,4,5-trisphosphate receptor (IP3R) is an intracellular Ca2+ release channel responsible for mobilizing stored Ca2+. Three different receptor types have been molecularly cloned, and their genes have been classified into a family. The gene for the type 1 receptor (IP3R1) is predominantly expressed in cerebellar Purkinje neurons, but its gene product is localized widely in a variety of tissues; however, there is little information on what types of cells express the other two receptor types, type 2 and type 3 (IP3R2 and IP3R3, respectively). We studied the expression of the IP3R gene family in various mouse tissues by in situ hybridization histochemistry. Compared with IP3R1, the levels of expression of IP3R2 and IP3R3 mRNAs were low in all of the tissues tested. IP3R2 mRNA was localized in the intralobular duct cells of the submandibular gland, the urinary tubule cells of the kidney, the epithelial cells of epididymal ducts and the follicular granulosa cells of the ovary, while the IP3R3 mRNA was distributed in gastric cells, salivary and pancreatic acinar cells and the epithelium of the small intestine. All of these cells which express either IP3R2 or IP3R3 mRNA are known to have a secretory function in which IP3/Ca2+ signalling has been shown to be involved, and thus either IP3R2 or IP3R3 may be a prerequisite to secretion in these cells.

Cloning and expression of a protein kinase C-regulated chloride channel abundantly expressed in rat brain neuronal cells.

cDNA (CIC-3) encoding a protein kinase C-regulated chloride channel was cloned and characterized. The open reading frame encodes 760 amino acids, which possess significantly amino acid identity with previously cloned CIC chloride channels. The chloride currents expressed in Xenopus oocytes injected with CIC-3 cRNA were completely blocked by activation of protein kinase C by 12-O-tetradecanoylphorbol 13-acetate. Abundant expression of CIC-3 mRNA was observed in rat brain, especially in the olfactory bulb, hippocampus, and cerebellum. These findings suggest that CIC-3 may play an important role in neuronal cell function through regulation of membrane excitability by protein kinase C.

Cloning and characterization of human type 2 and type 3 inositol 1,4,5-trisphosphate receptors.

We have cloned cDNAs coding for human type 2 and type 3 and part of type 1 inositol 1,4,5-trisphosphate receptors (IP3Rs). The complete nucleotide sequences for type 2 and type 3 receptors were determined and the pharmacological properties of the latter were characterized. Human type 2 and type 3 IP3Rs are 2701 amino acids and 2671 amino acids long, respectively, and have significant sequence homologies as well as structural similarities including the six membrane-spanning regions near the C-termini when compared with the rat or mouse counterpart. COS-7 cells transfected with human type 3 IP3R showed characteristic inositol 1,4,5-trisphosphate (IP3)-binding properties with Kd values of 28.8 nM. The order of potency of competition with IP3 was Ins(1,4,5)P3 (IP3) > Ins(2,4,5)P3 > Ins(1,3,4,5)P4 > Ins(1,2,3,4,5,6)P6. Type 2 and type 3 IP3Rs were mapped to human chromosomes 12p11 and 6p21, respectively, by in situ hybridization. cDNA cloning of the human IP3Rs allowed us to identify the types of the receptor expressed in various human hematopoietic and lymphoma cell lines. The type 3 receptor was present in all of cell lines tested, while the type 1 or 2 receptor was expressed in only particular cell types. The differential expression of the IP3R types could confer the cell-specific regulation on the IP3/Ca2+ signalling.