Two different allelic mutations in a Finnish family with lecithin:cholesterol acyltransferase deficiency

Lecithin:cholesterol acyltransferase (LCAT) deficiency is a genetic disorder associated with low levels of serum HDL cholesterol. The proband of the Finnish LCAT-deficient family had corneal opacities, proteinuria, anemia with stomatocytosis, low serum HDL cholesterol (0.27 mmol/L), and low LCAT activity. Sequence analysis of his LCAT gene revealed compound heterozygosity for two different mutations: a C insertion in exon 1 between nucleotides 932 and 937 and a C-to-T point mutation in exon 6 at position 4976. The C insertion in exon 1 is predicted to result in premature termination and a truncated polypeptide containing only 16 amino acids. The C-to-T point mutation in exon 6 substitutes cysteine for arginine at residue 399. The functional significance of the Arg399-->Cys mutation was examined by expressing the mutated and wild-type LCAT cDNAs in COS cells. COS cells transfected with mutated and wild-type cDNAs showed comparable levels of mature LCAT mRNA. However, LCAT activity in the cell media of COS cells transfected with the mutant LCAT cDNA was significantly lower than that of COS cells transfected with the wild-type cDNA (1.4% versus 12.0% cholesterol esterified, respectively). A polymerase chain reaction-based duplex assay, in which both mutations can be detected simultaneously, was used for preliminary screening of Finnish subjects with serum HDL levels below 0.9 mmol/L; two additional individuals heterozygous for the Arg399-->Cys mutation were identified.(ABSTRACT TRUNCATED AT 250 WORDS)     

A novel method for quantitative analysis of recombinant secreted proteins using two-dimensional electrophoresis

We describe a two-dimensional polyacrylamide gel electrophoresis (2-D PAGE)-based approach for detecting and quantifying secreted recombinant proteins in media conditioned by baby hamster kidney (BHK) cells. Seven secreted proteins were analyzed in this system: leptin, thrombopoietin, thrombin, glycoprotein 130, soluble interleukin-2 receptor, and two novel sequences obtained from sequence database searches. BHK cells transfected with plasmids encoding each of these proteins, and cells transfected with empty plasmids (control cells), were metabolically labeled and the resulting conditioned media were analyzed by 2-D PAGE. Gel images derived from cells expressing recombinant proteins were compared with images from control cells in order to identify spots corresponding to the expressed proteins. All seven of the test proteins were successfully detected using this method. The sensitivity of the system was evaluated by diluting samples derived from high-expressing clones with conditioned media from control cells. The sensitivity of detection was protein-dependent, but recombinant proteins expressed at levels as low as 10 ng/mL could be detected. Quantification of recombinant protein levels was achieved by measuring spot intensities using phosphorimager analysis. The intensity of spots corresponding to recombinant proteins were compared with the spot intensity of an endogenous BHK protein which had been calibrated to a known standard. Estimates of the levels of expressed protein determined using this technique correlated with the levels determined using standard affinity assays. We conclude that this system provides a reliable method for quantifying levels of protein expression when specific assays are unavailable.     

Characterization of two naturally occurring mutations in the second epidermal growth factor-like domain of factor VII

We investigated the mechanisms responsible for severe factor VII (FVII) deficiency in homozygous Italian patients with either Gly97Cys or Gln100Arg mutations in the second epidermal growth factor domain of FVII. Transient expression of complementary DNA coding for the mutations in COS-1 cells showed impaired secretion of the mutant molecules. Using stably transfected Chinese hamster ovary (CHO) cells, we performed pulse-chase labeling studies, immunohistochemistry, and experiments with inhibitors of protein degradation, showing that FVII-Cys97 did not accumulate intracellularly but was degraded in a pre-Golgi, nonlysosomal compartment by a cysteine protease. In stably transfected CHO cells expressing FVII-Arg100, the level of intracellular FVII was not increased by several inhibitors of protein degradation, but FVII-Arg100 was retained in the endoplasmic reticulum for a longer period of time than wild-type FVII. FVII-Arg100 had a lower apparent molecular weight than did wild-type FVII under nondenaturing conditions, which is attributable to misfolding due to abnormal disulfide bond formation.     

Establishment of a mouse Sertoli cell line producing rat androgen-binding protein (ABP)

The ultimate goal of this study was to compare the fate of rat testicular germ cells cocultured with mouse Sertoli cells that either do or do not produce rat androgen-binding protein (ABP). As a first step, we stably transfected a rat ABP expression construct into an immortalized mouse Sertoli cell line (TM4), which does not produce ABP when growing on plastic without hormones. The transfection of the pRc/CMV- rat ABP cDNA expression vector containing a neomycin resistance gene was made by either the liposome method (Dotap) or by polyethyleneimine transfection (PEI) into TM4 cell cultures. Neomycin-resistant clones were selected by adding Geneticin to the culture medium for 3 weeks. Analysis of over 25 clones revealed the presence of recombinant rat ABP when cell extracts and culture media were probed with a rabbit polyclonal antibody raised against rat testicular ABP, indicating the translation and secretion of a protein similar to rat testicular ABP. Transfected TM4 cells maintain the secretion of rat ABP for more than 40 days, with immunopositive rat ABP localized within cytoplasmic granules in the Golgi region and along cytoplasmic processes in TM4 transfected with either vector. Electron microscopic study revealed a higher development of cytoplasmic organelles involved in protein secretion.     

The vacuolar H(+)-ATPase inhibitor bafilomycin A1 differentially affects proteolytic processing of mutant and wild-type beta-amyloid precursor protein

We analyzed the effect of the vacuolar H(+)-ATPase inhibitor bafilomycin A1 (bafA1) on the processing of beta-amyloid precursor protein (beta APP). In kidney 293 cells stably transfected with the wild-type beta APP cDNA, bafA1 caused a stabilization of mature beta APP and its 10-kDa COOH-terminal fragment. Moreover, it caused a 2-3-fold increase in secretion of soluble APP and amyloid-beta protein (A beta). Interestingly, bafA1 treatment of cells transfected with a mutant beta APP isoform that occurs in a Swedish kindred with familial Alzheimer's disease resulted in a decrease of A beta production and no increase of soluble APP secretion. Identical results were obtained when the effect of bafA1 was analyzed on fibroblasts derived from affected versus unaffected members of the Swedish family. These data demonstrate a differential effect of bafA1 on the production of A beta derived from wild-type or Swedish mutant beta APP. Radiosequencing of A beta derived from bafA1-treated cells expressing wild-type beta APP revealed a marked increase of A beta peptides starting at amino acids phenylalanine 4 and valine -3 and a relative decrease of A beta molecules beginning at the typical NH2 terminus of aspartate 1. Cells transfected with the Swedish mutation and treated with bafA1 did not produce these alternative A beta peptides, so that bafA1 treatment resulted in a decrease of A beta starting at aspartate 1. Our data indicate that multiple proteases are able to cleave A beta at or near its NH2 terminus. Inhibition of the protease cleaving at aspartate 1 by bafA1 and perhaps other similar agents can result in an increase of alternatively cleaved peptides.     

Isolation of genes identified in mouse renal proximal tubule by comparing different gene expression profiles

X-linked chondrodysplasia punctata (CDPX) is a congenital disorder characterized by abnormalities in cartilage and bone development. Mutations leading to amino acid substitutions were identified recently in CDPX patients, in the coding region of the arylsulfatase E (ARSE) gene, a novel member of the sulfatase gene family. Transfection of the ARSE full-length cDNA, in Cos7 cells, allowed us to establish that its protein product is a 60-kD precursor, which is subject to N-glycosylation, to give a mature 68-kD form that, unique among sulfatases, is localized to the Golgi apparatus. Five missense mutations found in CDPX patients were introduced into wild-type ARSE cDNA by site-directed mutagenesis. These mutants were transfected into Cos7 cells, and the arylsulfatase activity and biochemical properties were determined, to study the effect of these substitutions on the ARSE protein. One of the mutants behaves as the wild-type protein. All four of the other mutations resulted in a complete lack of arylsulfatase activity, although the substitutions do not appear to affect the stability and subcellular localization of the protein. The loss of activity due to these mutations confirms their involvement in the clinical phenotype and points to the importance of these residues in the correct folding of a catalytically active ARSE enzyme.     

O-linked glycosylation modifies the association of apolipoprotein A-II to high density lipoproteins

O-linked glycosylation is a common post-translational modification of apolipoproteins, but no structural or functional role for it has been identified. We examined the biosynthesis of apolipoprotein (apo) A-II in Hep G2 cells and in glycosylation-defective Chinese hamster ovary (CHO) cell mutants transfected with apoA-II cDNA. Three monomeric isoforms of apoA-II with an apparent molecular mass of 8.5, 9.8, and 11.4 kDa were synthesized by Hep G2 cells and transfected wild-type CHO cells. The 9.8- and 11.4-kDa isoforms were sialylated but not the 8.5-kDa isoform. Transfected 1dlD cells, which are defective in the biosynthesis of galactose and N-acetylgalactosamine, only produced the 8.5-kDa isoform; however, when grown in media supplemented with these sugars, ldlD cells produced all three isoforms of apoA-II. Pulse-chase analysis of ldlD cells showed that glycosylation was not necessary for secretion of apoA-II. Glycosylation did modify the association of apoA-II with nascent high density lipoprotein (HDL) secreted by Hep G2 cells. The sialylated isoforms were lipid-poor and were present in the lipoprotein-deficient density range, whereas the nonsialylated 8.5-kDa isoform was associated with LpA-I, A-II lipoprotein particles in the HDL density range. ApoA-II from transfected ldlD cells, regardless of glycosylation, were lipid-poor. When preincubated with HDL from serum, however, sialylated apoA-II from both ldlD cells and Hep G2 cells associated with lipoprotein particles within the HDL3 density, whereas nonsialylated apoA-II was found throughout the HDL density range. In summary, O-linked glycosylation is not necessary for the secretion of apoA-II but does modify the association of apoA-II to HDL and may, therefore, play an important role in the metabolism of HDL.     

Human Mig chemokine: biochemical and functional characterization

Mig is a chemokine of the CXC subfamily that was discovered by differential screening of a cDNA library prepared from lymphokine-activated macrophages. The mig gene is inducible in macrophages and in other cells in response to interferon (IFN)-gamma. We have transfected Chinese hamster ovary (CHO) cells with cDNA encoding human Mig and we have derived CHO cell lines from which we have purified recombinant human Mig (rHuMig). rHuMig induced the transient elevation of [Ca2+]i in human tumor-infiltrating T lymphocytes (TIL) and in cultured, activated human peripheral blood-derived lymphocytes. No responses were seen in human neutrophils, monocytes, or Epstein-Barr virus-transformed B lymphoblastoid cell lines. rHuMig was chemotactic for TIL by a modified Boyden chamber assay but rHuMig was not chemotactic for neutrophils or monocytes. The CHO cell lines, IFN-gamma-treated human peripheral-blood monocytes, and IFN-gamma-treated cells of the human monocytic cell line THP-1 all secreted multiple and identical HuMig species as revealed by SDS-PAGE. Using the CHO-derived rHuMig, we have shown that the species' heterogeneity is due to proteolytic cleavage at basic carboxy-terminal residues, and that the proteolysis occurs before and not after rHuMig secretion by the CHO cells. The major species of secreted rHuMig ranged from 78 to 103 amino acids in length, the latter corresponding to the full-length secreted protein predicted from the HuMig cDNA. Carboxy-terminal-truncated forms of rHuMig were of lower specific activity compared to full-length rHuMig in the calcium flux assay, and the truncated species did not block the activity of the full-length species. It is likely that HuMig plays a role in T cell trafficking and perhaps in other aspects of the physiology of activated T cells.     

The human REC2/RAD51B gene acts as a DNA damage sensor by inducing G1 delay and hypersensitivity to ultraviolet irradiation

HsRec2/Rad51B is a 350-amino acid protein with a molecular mass of 38,300 Da that appears to be involved in cell cycle regulation and UV-induced apoptosis. The mouse and human genes were isolated based on their homology to a recombinational repair gene from Ustilago maydis and contain functional domains to hRAD51 and hLIM 15 (M. C. Rice et al., Proc. Natl. Acad. Sci. USA, 94: 7417-7422, 1997). Here, we report the results of studies on the behavior of CHO cells containing a plasmid encoding a wild-type hsRec2/Rad51B, a full-length protein with a single mutation at residue 163, which lies in the putative src site, and a truncated version of hsRec2/Rad51B, containing only the first 100 amino acids at the NH2 terminus. Using fluorescence-activated cell sorting analysis to follow the progression of cells through the cell cycle, we find that stable transfectants constitutively overexpressing the wild-type human Rec2/Rad51B protein exhibit a G1 delay. In addition, when irradiated with UV at a dose of 15 J/m2, CHO cells transfected with the various hREC2/RAD51B vectors exhibited different responses. Cells expressing the wild-type human Rec2/Rad51B underwent apoptosis, with the greatest cell death occurring 24 h after irradiation. The control cells, which contained an empty vector, and the cells expressing truncated hsRec2/Rad51B or the full-length Rec2 with a mutation at residue 163 did not. In summary, these findings of cell cycle slowing and UV-induced apoptosis in CHO cells constitutively expressing the human Rec2/Rad51B protein suggest that hsRec2/Rad51B plays a role in a DNA damage surveillance pathway.     

Requirements for efficient production and transduction of human immunodeficiency virus type 1-based vectors

Phosphatidylglycerophosphate (PGP) synthase catalyzes the first step in the cardiolipin (CL) branch of phospholipid biosynthesis in mammalian cells. In this study, we isolated a Chinese hamster ovary (CHO) cDNA encoding a putative protein similar in sequence to the yeast PGS1 gene product, PGP synthase. The gene for the isolated CHO cDNA was named PGS1. Expression of the CHO PGS1 cDNA in CHO-K1 cells and production of a recombinant CHO PGS1 protein with a N-terminal extension in Escherichia coli resulted in 15-fold and 90-fold increases of PGP synthase specific activity, respectively, establishing that CHO PGS1 encodes PGP synthase. A PGP synthase-defective CHO mutant, PGS-S, isolated previously (Ohtsuka, T., Nishijima, M., and Akamatsu, Y. (1993) J. Biol. Chem. 268, 22908-22913) exhibits striking reductions in biosynthetic rate and cellular content of phosphatidylglycerol (PG) and CL and shows mitochondrial morphological and functional abnormalities. The CHO PGS-S mutant transfected with the CHO PGS1 cDNA exhibited 620-fold and 7-fold higher PGP synthase activity than mutant PGS-S and wild type CHO-K1 cells, respectively, and had a normal cellular content and rate of biosynthesis of PG and CL. In contrast to mutant PGS-S, the transfectant had morphologically normal mitochondria. When the transfectant and mutant PGS-S cells were cultivated in a glucose-depleted medium, in which cellular energy production mainly depends on mitochondrial function, the transformant but not mutant PGS-S was capable of growth. These results demonstrated that the morphological and functional defects displayed by the PGS-S mutant are due directly to the reduced ability to make normal levels of PG and/or CL.     

Dissociation of early folding events from assembly of the human lutropin beta-subunit

The human LH of the anterior pituitary is a member of the glycoprotein hormone family that includes FSH, TSH, and placental CG. All are noncovalently bound heterodimers that share a common alpha-subunit and beta-subunits that confer biological specificity. LHbeta and CGbeta share more than 80% amino acid sequence identity; however, in transfected Chinese hamster ovary (CHO) cells, LHbeta assembles with the alpha-subunit more slowly than does hCGbeta, and only a fraction of the LHbeta synthesized is secreted, whereas CGbeta is secreted efficiently. To understand why the assembly and secretion of these related beta-subunits differ, we studied the folding of LHbeta in CHO cells transfected with either the LHbeta gene alone, or in cells cotransfected with the gene expressing the common alpha-subunit, and compared our findings to those previously seen for CG. We found that the rate of conversion of the earliest detectable folding intermediate of LH, pbeta1, to the second major folding form, pbeta2, did not differ significantly from the pbeta1-to-pbeta2 conversion of CGbeta, suggesting that variations between the intracellular fates of the two beta-subunits cannot be explained by differences in the rates of their early folding steps. Rather, we discovered that unlike CGbeta, where the folding to pbeta2 results in an assembly-competent product, apparently greater than 90% of the LH pbeta2 recovered from LHbeta-transfected CHO cells was assembly incompetent, accounting for inefficient LHbeta assembly with the alpha-subunit. Using the formation of disulfide (S-S) bonds as an index, we observed that, in contrast to CGbeta, all 12 LHbeta cysteine residues formed S-S linkages as soon as pbeta2 was detected. Attempts to facilitate LH assembly with protein disulfide isomerase in vitro using LH pbeta2 and excess urinary alpha-subunit as substrate were unsuccessful, although protein disulfide isomerase did facilitate CG assembly in this assay. Moreover, unlike CGbeta, LHbeta homodimers were recovered from transfected CHO cells. Taken together, these data suggest that differences seen in the rate and extent of LH assembly and secretion, as compared to those of CG, reflect conformational differences between the folding intermediates of the respective beta-subunits.     

beta ig-h3: a transforming growth factor-beta-responsive gene encoding a secreted protein that inhibits cell attachment in vitro and suppresses the growth of CHO cells in nude mice

beta ig-h3 is a novel gene first discovered by differential screening of a cDNA library made from A549 human lung adenocarcinoma cells treated with transforming growth factor-beta 1 (TGF-beta 1). It encodes a 683-amino-acid protein containing a secretory signal sequence and four homologous internal domains. Here we show that treatment of several types of cells, including human melanoma cells, human mammary epithelial cells, human keratinocytes, and human fibroblasts, with TGF-beta resulted in a significant increase in beta ig-h3 RNA. A portion of the beta ig-h3 coding sequence was expressed in bacteria, and antisera against the bacterially produced protein was raised in rabbits. This antisera was used to demonstrate that several cell lines secreted a 68-kD beta IG-H3 protein after treatment with TGF-beta. Transfection of beta IG-H3 expression plasmids into Chinese hamster ovary (CHO) cells led to a marked decrease in the ability of these cells to form tumors in nude mice. The beta IG-H3 protein was purified from media conditioned by recombinant CHO cells, characterized by immunoblotting and protein sequencing and shown to function in an anti-adhesion assay in that it inhibited the attachment of A549, HeLa, and WI-38 cells to plastic in serum-free media. Sequencing of cDNA clones encoding murine beta ig-H3 indicated 90.6% conservation at the amino acid level between the murine and human proteins. Finally, the beta ig-h3 gene was localized to human chromosome 5q31, a region frequently deleted in preleukemic myelodysplasia and leukemia. The corresponding mouse beta ig-h3 gene was mapped to mouse chromosome 13 region B to C1, which confirms a region of conservation on human chromosome 5 and mouse chromosome 13. We suggest that this protein be named p68 beta ig-h3.     

N-glycosylation is not essential for enzyme activity of 11beta-hydroxysteroid dehydrogenase type 2

11Beta-hydroxysteroid dehydrogenase (11beta-HSD) catalyzes the oxidation of cortisol and corticosterone to cortisone and 11-dehydrocorticosterone, respectively. NAD-dependent 11beta-HSD is expressed at high levels in the distal nephron and contributes to mineralocorticoid specificity in that region. The present studies determined whether N-glycosylation is necessary for the activity of NAD-dependent 11beta-HSD (11beta-HSD2). First, cultured human colonic epithelial cells (T84 cells), which express native 11beta-HSD2 activity, were grown in medium with and without tunicamycin, an inhibitor of N-glycosylation. Tunicamycin had no effect on the enzyme activity. Next, the only putative N-glycosylation site (Asn394-Leu395-Ser396) of the cloned human kidney enzyme was eliminated by site-directed mutagenesis. Chinese hamster ovary (CHO) cells transfected with either the wild-type or the mutant cDNA construct showed no difference in the expressed enzyme activity, and Western blot analysis showed that the 11beta-HSD2 protein was the same size in cells expressing either the wild-type or the N394D mutant. Likewise, the molecular mass of the 11beta-HSD2 protein in T84 cells was not altered by treatment with peptide-N-glycosidase F or tunicamycin. We conclude that human 11beta-HSD2 is not a N-glycoprotein and N-glycosylation is not essential for the expression of enzyme activity.     

The use of allele-specific recombinant Fc gamma receptor IIIb antigens for the detection of granulocyte antibodies

The Fcgamma receptor IIIb (FcgammaRIIIb) for the Fc domain of IgG is expressed exclusively on neutrophils. The FcgammaRIIIb bears allotypic polymorphisms referred to as NA1, NA2, and SH, which are known for their frequent involvement in alloimmune and autoimmune neutropenias as well as in transfusion reactions. The bactericidal capacity of isolated neutrophils is easily activatable, and activation results in self-desintegration, thus preventing storage of neutrophils. As a result, only freshly isolated granulocytes can be used for antibody screening, often making it impossible to use typed panel cells. To provide a readily available source of typed panel cells, we therefore established stable mammalian cells expressing recombinant NA1, NA2, and SH antigens. We isolated mRNA from typed neutrophils and then transcribed it in cDNA. The cDNA that codes for the different forms of the FcgammaRIIIb was amplified by polymerase chain reaction and was subsequently subcloned into the mammalian expression vector pcDNA3. Chinese hamster ovary (CHO) cells were transfected with allele-specific constructs, and stable cell lines expressing FcgammaRIIIb were selected by flow cytometry. Because human sera show high background fluorescence with transfectants in flow cytometry, the monoclonal antibody-specific isolation of granulocyte antigens (MAIGA) assay was performed. By MAIGA assay, we tested 14 well-characterized human alloantibodies directed against the antigens NA1, NA2, and SH; 5 FcgammaRIIIb-specific isoantibodies; and 12 FcgammaRIIIb-reactive autoantibodies. Except one NA1- and one SH-specific alloantibody, all other antibodies could be identified by the use of CHO transfectants. In contrast to neutrophils, fixed CHO cells can be stored at 4 degrees C for at least 4 weeks or stored frozen for a longer period. This longer shelf life of the transfected CHO cells compared with isolated neutrophils will simplify the detection of the clinically most important FcgammaRIIIb-reactive alloantibodies and autoantibodies.     

Proteolysis of the carboxyl-terminal GPI signal independent of GPI modification as a mechanism for selective protein secretion

Variable amounts of soluble forms of a variety of glycosyl-phosphatidylinositol (GPI)-anchored proteins occur extracellularly, but the molecular mechanisms governing their release are not entirely clear. When the GPI-anchored folate receptor (FR) type beta was expressed transiently in human 293 fibroblasts, there was a roughly equal distribution of [3H]folic acid binding protein between the cell surface and the medium after 24 h over a wide range of expression levels of FR-beta. The difference in apparent molecular masses between the soluble FR-beta and the PI-PLC-treated membrane protein indicated that the former was not released from the membrane by the action of phospholipase. Brefeldin A inhibited the release of soluble FR-beta from both the transfected 293 cells and stable recombinant CHO (CHO-FR-beta) cells while pre-existing levels of cell surface FR were unaltered suggesting the absence of a precursor-product relationship between the membrane-associated FR-beta and the soluble protein in the medium. [35S]Cysteine pulse-chase analysis was consistent with this finding. Interchanging of carboxyl-terminal peptides between FR-beta and FR-alpha revealed that the nature of the processed signal for GPI modification was responsible for the quantitative membrane anchoring of FR-alpha and the production of soluble FR-beta. When total cell lysates were analyzed by Western blot, a diffuse band of apparent 41 kDa and three additional sharp bands of apparent 35, 33, and 29.3 kDa were seen. The 41 kDa band was identified as the PI-PLC sensitive cell surface receptor. Several mutant constructs of FR-beta, in which the carboxyl-terminal signal for GPI modification was either disrupted or deleted only gave the three lower bands. The three sharp bands from the wild-type and the mutant forms of FR-beta were identified as nonglycosylated (29.3 kDa) or glycosylated polypeptides in which the carboxyl-terminal peptide was at least partially proteolyzed without GPI modification. All of the mutations in the GPI signal resulted in the recovery of [3H]folic acid binding protein in the media which, similar to the wild-type FR recovered from the media, were converted to the 29.3 kDa band by N-glycanase. The results from this study indicate that a carboxyl-terminal peptide in FR-beta is efficiently proteolyzed intracellularly by a pathway that is independent of GPI signal recognition resulting in proper protein folding and secretion. Such carboxyl-terminal sequences could represent a simple adaptation for proteins whose physiologic functions reside both at the cell surface and in extracellular fluids, allowing their selective and tissue-specific release.     

AVP-induced mitogenic responses of Chinese hamster ovary cells expressing human V1A or V1B receptors

Arginine vasopressin (AVP) induces cell proliferation and hypertrophy; however, the human receptor subtype and the intracellular signaling pathways responsible for this mitogenic activity remain unclear. Experiments were conducted to determine which AVP receptor is linked to mitogen-activated protein (MAP) kinase activation and the mitogenic effect seen in Chinese hamster ovary (CHO) cells expressing human V1A or V1B receptors. Adding AVP to CHO cells transfected with human V1A or V1B cDNA significantly and concentration-dependently induced activation of MAP kinase and increased DNA synthesis, as measured by [3H]thymidine incorporation. These effects were inhibited by AVP receptor antagonists and the potency order of antagonists in vitro was similar to that observed in radioligand binding assays. These results suggest that AVP induces the MAP kinase cascade leading to cell proliferation through either human V1A or V1B receptors, and that these cloned, expressed AVP receptors may prove an invaluable tool for probing the physiologic and pathophysiologic effects of AVP.     

Characterization of brain-type ryanodine receptor permanently expressed in Chinese hamster ovary cells

To clarify a function of brain-type ryanodine receptor (RyR3) and its regulation, we established a stable cell line expressing rabbit RyR3 by transfection of Chinese hamster ovary cells (CHO cells) with the cDNA and investigated characteristics of the RyR3. Scatchard analysis of [3H]-ryanodine binding to the membrane from CHO cells expressing RyR3 showed two distinct binding sites. The Kd values of high and low affinity binding sites were 1.92 and 25.9 nM, respectively. [3H]-ryanodine binding to the membrane from CHO cells expressing RyR3 was dependent on pCa. Extracellular Ca2+ (2-10 mM) and high concentration (more than 30 mM) of caffeine activated the RyR3 in CHO cells and increased its intracellular Ca2+ concentration. The enhancement of [3H]-ryanodine binding to the membrane from CHO cells expressing RyR3 was observed by bromoeudistomin D (BED), a caffeine-like powerful Ca2+ releaser, at pCa 5.5. Stably expressed RyR3 in CHO is useful for characterization of its function.