cDNA-transfected Chinese Hamster Ovary Research Articles

Enhancing and Limiting Endothelin‐1 Signaling with a Cell‐penetrating Peptide Mimicking the Third Intracellular Loop of the ETB Receptor

TAT (a 13-mer derived from the HIV-1 Tat protein)-linked cell-permeable peptides deliver plasma membrane impermeable cargos into the cell. We investigated the effect of a TAT-linked intracellular third loop of the endothelin-1 type B receptor on endothelin-1 activation of ERK. The effect of this peptide on ERK activation was determined in ETB receptor cDNA-transfected Chinese hamster ovary cells and in ETA- and ETB-expressing human pulmonary artery smooth muscle cells obtained from a normal and a bone morphogenetic protein-2 receptor, exon 1-8 deletion subject, with pulmonary hypertension. In the Chinese hamster ovary cells the peptide, at optimum 10 μm concentration, suppressed endothelin-1 activation. In the normal human pulmonary artery smooth muscle cells, the peptide marginally enhanced endothelin-1 activation of ERK. However, it markedly enhanced the endothelin-1 activation of ERK in the bone morphogenetic protein-2 receptor human pulmonary artery smooth muscle cells. While the effective concentration for endothelin-1 activation of ERK remained unchanged in the bone morphogenetic protein-2 receptor human pulmonary artery smooth muscle cells, the number of ETB receptors declined by 2/3. These data point to the intracellular third loop peptide as having variable receptor interactive effects with both signal repressive and enhancing capabilities. Peptides that can alter endothelin-1 signal capabilities are potentially important in the study and treatment of pulmonary hypertension.

Binding of Plasmodium falciparum-infected erythrocytes to the membrane-bound form of Fractalkine/CX3CL1.

Plasmodium falciparum-infected erythrocytes (pRBCs) adhere to the endothelium via receptors expressed on the surface of vascular endothelial cells (EC) and sequester in the microvasculature of several organs and block the blood circulation. The sequestration, which involves receptors, may be related to the severity of malaria. Here, we report that pRBCs bind to the membrane-bound form of Fractalkine/CX3CL1 (FKN), which is expressed on the surface of vascular EC in various organs. pRBCs adhered to FKN on the surface of FKN cDNA-transfected Chinese hamster ovary cells (CHO-FKN cells). Both the recombinant human FKN-chemokine domain (FKN-CD) and anti-FKN-CD antibody efficiently blocked adherence of pRBCs to CHO-FKN cells. Similar to binding between FKN and FKN receptor on blood mononuclear cells, two amino acid residues, Lys-7 and Arg-47 within FKN-CD, were critical for FKN-pRBC binding. Immunohistological analysis revealed the expression of FKN on EC at the site of sequestration in the brain of a patient with cerebral malaria. These results suggest that the membrane-bound form of FKN acts as a receptor for pRBCs, and this may contribute to furthering our present understanding of cytoadherence in the pathology of falciparum malaria.

Comparison of Intrinsic Activities of the Putative Sphingosine 1-Phosphate Receptor Subtypes to Regulate Several Signaling Pathways in Their cDNA-transfected Chinese Hamster Ovary Cells

We examined the actions of sphingosine 1-phosphate (S1P) on signaling pathways in Chinese hamster ovary cells transfected with putative S1P receptor subtypes, i.e. Edg-1, AGR16/H218 (Edg-5), and Edg-3. Among these receptor-transfected cells, there was no significant difference in the expressing numbers of the S1P receptors and their affinities to S1P, which were estimated by [(3)H]S1P binding to the cells. In vector-transfected cells, S1P slightly increased cytosolic Ca(2+) concentration ([Ca(2+)](i)) in association with inositol phosphate production, reflecting phospholipase C activation; the S1P-induced actions were markedly enhanced in the Edg-3-transfected cells and moderately so in the AGR16-transfected cells. In comparison with vector-transfected cells, the S1P-induced [Ca(2+)](i) increase was also slightly enhanced in the Edg-1-transfected cells. In all cases, the inositol phosphate and Ca(2+) responses to S1P were partially inhibited by pertussis toxin (PTX). S1P also significantly increased cAMP content in a PTX-insensitive manner in all the transfected cells; the rank order of their intrinsic activity of S1P receptor subtypes was AGR16 > Edg-3 > Edg-1. In the presence of forskolin, however, S1P significantly inhibited cAMP accumulation at a lower concentration (1-100 nM) of S1P in a manner sensitive to PTX in the Edg-1-transfected cells but not in either the Edg-3 or AGR16-transfected cells. As for cell migration activity evaluated by cell number across the filter of blind Boyden chamber, Edg-1 and Edg-3 were equally potent, but AGR16 was ineffective. Thus, S1P receptors may couple to both PTX-sensitive and -insensitive G-proteins, resulting in the selective regulation of the phospholipase C-Ca(2+) system, adenylyl cyclase-cAMP system, and cell migration activity, according to the receptor subtype.

Localization of the neuromedin K receptor (NK3) in the central nervous system of the rat

The distribution of the neuromedin K receptor (NK3; NKR) in the central nervous system was investigated in the adult rat by using in situ hybridization and immunohistochemical techniques. The rabbit anti-NKR antibody was raised against a bacterial fusion protein containing a C-terminal portion of NKR and affinity purified with a Sepharose 4B column conjugated to the fusion protein. Immunoblot analysis was performed to test the reactivity and specificity of the antibody. Crude membrane was prepared from cDNA-transfected Chinese hamster ovary (CHO) cells expressing each of the rat NKR, substance P receptor (NK1; SPR), and substance K receptor (NK2; SKR) and from the hypothalamus, cerebral cortex, and cerebellum. Immunoreactive bands were observed specifically in the NKR-CHO cells, hypothalamus, and cerebral cortex but not in the SPR- or SKR-CHO cells, nor in the cerebellum. Molecular weights of the immunoreactive bands ranged from 73 to 89 kDa and from 59 to 83 kDa in the NKR-CHO cells and tissues, respectively. The distribution of NKR-like immunoreactivity coincided with that of NKR mRNA. The expression of NKR was indicated on neuronal cell bodies and dendrites. NKR was found to be expressed intensely or moderately in neurons in the glomerular and granule cell layers of the main olfactory bulb; glomerular and mitral cell layers of the accessory olfactory bulb; layers IV and V of the cerebral neocortex; medial septal nucleus; nucleus of the diagonal band; bed nucleus of the stria terminalis; globus pallidus; ventral pallidum; paraventricular nucleus; supraoptic nucleus; zona incerta; dorsal, lateral, and posterior hypothalamic areas; amygdaloid nuclei; medial habenular nucleus; ventral tegmental area; midbrain periaqueductal gray; interpeduncular nuclei; substantia nigra pars compacta; linear, median, dorsal, and pontine raphe nuclei; posteromedial tegmental nucleus; sphenoid nucleus; nucleus of the solitary tract; intermediate and rostroventrolateral reticular nuclei; and lamina II of the caudal spinal trigeminal nucleus and spinal dorsal horn. These findings are discussed in relation to the physiological functions associated with neuromedin K. © 1996 Wiley-Liss, Inc.

Localization of the neuromedin K receptor (NK3) in the central nervous system of the rat.

The distribution of the neuromedin K receptor (NK3; NKR) in the central nervous system was investigated in the adult rat by using in situ hybridization and immunohistochemical techniques. The rabbit anti-NKR antibody was raised against a bacterial fusion protein containing a C-terminal portion of NKR and affinity purified with a Sepharose 4B column conjugated to the fusion protein. Immunoblot analysis was performed to test the reactivity and specificity of the antibody. Crude membrane was prepared from cDNA-transfected Chinese hamster ovary (CHO) cells expressing each of the rat NKR, substance P receptor (NK1; SPR), and substance K receptor (NK2; SKR) and from the hypothalamus, cerebral cortex, and cerebellum. Immunoreactive bands were observed specifically in the NKR-CHO cells, hypothalamus, and cerebral cortex but not in the SPR- or SKR-CHO cells, nor in the cerebellum. Molecular weights of the immunoreactive bands ranged from 73 to 89 kDa and from 59 to 83 kDa in the NKR-CHO cells and tissues, respectively. The distribution of NKR-like immunoreactivity coincided with that of NKR mRNA. The expression of NKR was indicated on neuronal cell bodies and dendrites. NKR was found to be expressed intensely or moderately in neurons in the glomerular and granule cell layers of the main olfactory bulb; glomerular and mitral cell layers of the accessory olfactory bulb; layers IV and V of the cerebral neocortex; medial septal nucleus; nucleus of the diagonal band; bed nucleus of the stria terminalis; globus pallidus; ventral pallidum; paraventricular nucleus; supraoptic nucleus; zona incerta; dorsal, lateral, and posterior hypothalamic areas; amygdaloid nuclei; medial habenular nucleus; ventral tegmental area; midbrain periaqueductal gray; interpeduncular nuclei; substantia nigra pars compacta; linear, median, dorsal, and pontine raphe nuclei; posteromedial tegmental nucleus; sphenoid nucleus; nucleus of the solitary tract; intermediate and rostroventrolateral reticular nuclei; and lamina II of the caudal spinal trigeminal nucleus and spinal dorsal horn. These findings are discussed in relation to the physiological functions associated with neuromedin K.

Characterization of the signal transduction of prostaglandin E receptor EP1 subtype in cDNA-transfected Chinese hamster ovary cells

We examined the signal transduction of mouse prostaglandin E receptor EP1 subtype using Chinese hamster ovary cells stably expressing the cloned EP1. Sulprostone, an EP1 agonist, induced a rapid increase in intracellular Ca 2+ concentration in the EP1-expressing cells. Most of the increase was abolished by removal of extracellular Ca 2+, and was insensitive to U-73122, a phospholipase C inhibitor. Sulprostone stimulated phosphatidylinositol hydrolysis, but this stimulation was abolished by removal of extracellular Ca 2+, indicating that EP1-stimulated phosphatidylinositol hydrolysis is the result of extracellular Ca 2+ influx. Thus, the signal transduction of EP1 is extracellular Ca 2+ entry through a pathway independent of phospholipase C activation. We further examined the regulation of the signal transduction of EP1 having potential phosphorylation sites for either protein kinase C or protein kinase A. Short-term exposure of the cells to 12- O-tetradecanoylphorbol 13-acetate (TPA) completely suppressed the sulprostone-induced increase in intracellular Ca 2+- concentration, while forskolin or dibutyryl CAMP did not affect it, suggesting that protein kinase C but not protein kinase A is involved in the regulation of the EP1 signal transduction. Furthermore, long-term exposure to TPA decreased PGE 2 binding activity of EP1 due to the reduction of the EPI mRNA level. Protein kinase C induces short- and long-term desensitization of EP1.

Dra-related X adhesins of gestational pyelonephritis-associated Escherichia coli recognize SCR-3 and SCR-4 domains of recombinant decay-accelerating factor

Bacterial adhesins are important virulence factors that allow colonization of the human urogenital tract by Escherichia coli. Adhesins of the Dr family have been found to be more frequently expressed in strains associated with symptomatic urinary tract infections. Because of the high frequency of symptomatic urinary tract infections during pregnancy, we screened E. coli isolates from 64 gestational pyelonephritis patients for the expression of Dr and X adhesins to address their potential virulence roles in this population. Using PCR and primers for the afaB gene, we detected dra-related operons in 17 isolates (27%). On the basis of the lack of hemagglutination of Dr(a-) erythrocytes containing a point mutation in the decay-accelerating factor (DAF) short consensus repeat-3 (SCR-3) domain, 12 of these strains were categorized as classical Dr adhesins. The hemagglutination of O erythrocytes by Dr+ strains was blocked or reduced by a monoclonal antibody to the DAF SCR-3 domain. The remaining five dra-positive strains agglutinated Dr(a-) erythrocytes. Monoclonal antibody to the DAF SCR-3 domain failed to block O-erythrocyte hemagglutination. Adhesins in these strains did not fulfill criteria for Dr hemagglutinins because of the undefined receptor specificities and were categorized as X. E. coli strains bearing dra-related X adhesins bound to DAF cDNA-transfected Chinese hamster ovary cells. Three of these dra-related X-adhesin-bearing E. coli strains failed to attach to the SCR-3 delta deletion transfectant, which suggested that binding sites were located in the SCR-3 domain but outside the region blocked by the monoclonal anti-SCR-3 immunoglobulin G. The binding sites of the remaining two dra-related X adhesin strains were localized to the SCR-4 domain, as the attachment was shown to be abolished on an SCR-4 delta mutant but unaffected by an SCR-3 delta deletion. The heterogeneity in the binding sites of E. coli DAF (Dr) family adhesins from gestational pyelonephritis isolates may reflect the ability of the adhesins to evolve to recognize alternate peptide epitopes for efficient colonization.

Endothelins stimulate mitogen-activated protein kinase cascade through either ETA or ETB

Endothelin (ET) has been shown to activate mitogen-activated protein kinase (MAPK). However, it has been unclear which of the ET receptors is coupled to MAPK activation. In the present study, we conducted experiments to determine which ET receptor is linked to MAPK activation. We found that both human ETA and ETB were coupled to the MAPK cascade in ETA or ETB cDNA-transfected Chinese hamster ovary cells. ET-1 was more potent than ET-3 in the activation of p42 MAPK, induction of MAPK kinase (MAPKK) gel retardation and uptake of [3H]thymidine in ETA-transfected cells, whereas sarafotoxin (S6c) showed no stimulatory effect on the kinases and [3H]thymidine uptake. ET-1, ET-3, and S6c had approximately the same potency to activate p42 MAPK, MAPKK gel retardation, and [3H]thymidine uptake in ETB-transfected cells. These data suggest that 1) ET isopeptides, through either ETA or ETB receptors, induce the MAPK cascade as well as cell proliferation; and 2) the different potencies of ET isopeptides for activation of the MAPK cascade and induction of cell growth are mainly due to their different affinities toward ETA and ETB.

Prostaglandin F 2α Receptor Is Coupled to Gq in cDNA-transfected Chinese Hamster Ovary Cells

We examined intracellular signal transduction of prostaglandin F 2α (PGF 2α) receptors by transfection and stable expression of the receptor cDNA in Chinese hamster ovary (CHO-PGF 2α) cells. The binding to the membrane was specific for PGF 2α, and the Scatchard plot analysis showed a single high-affinity binding site (Kd=25.2 nM). PGF 2α gradually elevated intracellular Ca 2+ and stimulated phosphoinositide metabolism in CHO-PGF 2α cells. In whole-cell clamp recordings, PGF 2α induced an outward current in the presence of external Ca 2+, but it induced a long-lasting inward Ca 2+ current in a Na +-free solution containing K + channel blockers. G qα antibody applied intracellularly blocked both outward and inward currents induced by 1 μM PGF 2α. These results demonstrate that the PGF 2α receptor is coupled to phosphoinositide metabolism in CHO-PGF 2α cells via G q.

Proteolytic conversion of single chain precursor macrophage-stimulating protein to a biologically active heterodimer by contact enzymes of the coagulation cascade.

Human serum macrophage stimulating protein (MSP) is a disulfide-linked heterodimer that induces motile and phagocytic activity of mouse resident peritoneal macrophages. It is a member of the family of kringle proteins, which typically exist in extracellular fluid as single chain precursors that are activated by proteolytic cleavage. In this work, we expressed [35S]cysteine-labeled recombinant pro-MSP in MSP cDNA-transfected Chinese hamster ovary cells and studied proteolytic processing of pro-MSP and the requirement of cleavage for biological activity. In media containing heat-inactivated fetal bovine serum, the protein was secreted as single chain pro-MSP, which was cleaved over a period of hours to the mature heterodimer. Cleavage was prevented by serine protease inhibitors such as leupeptin or aprotinin; it did not occur if cells were cultured in serum-free medium. Nanomolar concentrations of coagulation proteases kallikrein, factor XIIa or factor XIa cleaved pro-MSP to MSP within 30 min. Pro-MSP had no biological activity. After cleavage by kallikrein, biological activity was quantitatively comparable to that of natural MSP isolated from human plasma. These results support our hypothesis that MSP circulates as the biologically inactive precursor and can be activated by enzymes of the intrinsic coagulation cascade.

Opposite coupling of prostaglandin E receptor EP3C with Gs and G(o). Stimulation of Gs and inhibition of G(o).

We recently identified four isoforms of bovine prostaglandin E receptor EP3 subtype, which are coupled to different signaling pathways; EP3A is coupled to inhibition of adenylate cyclase, while EP3B and EP3C are coupled to its stimulation and EP3D is coupled to phosphatidylinositol turnover, in addition to the adenylate cyclase system (Namba, T., Sugimoto, Y., Negishi, M., Irie, A., Ushikubi, F., Kakizuka, Ito, S., A., Ichikawa, A., and Narumiya, S. (1993) Nature 365, 166-170). We examined here the identity of coupled G proteins and their regulation by one of the isoforms, EP3C, in the membranes of EP3C cDNA-transfected Chinese hamster ovary cells. M&B 28767, an EP3 agonist, stimulated the GTPase activity in the pertussis toxin (PT)-treated cell membrane, but inhibited it in the cholera toxin (CT)-treated cell membrane, while the agonist neither stimulated nor inhibited it in the both PT- and CT-treated cell membrane. In the PT- and CT-treated cell membrane reconstituted with various G proteins, M&B 28767 inhibited the GTPase activity of G(o), but stimulated that of Gs. On the other hand, M&B 28767 did not affect the GTPase activity of Gi1, Gi2, or Gi3. M&B 28767 increased the apparent affinity of G(o) for GDP without any change in that for GTP, as assessed by displacement of [35S]GTP gamma S (guanosine 5'-O-(3-thiotriphosphate)) binding to G(o). In contrast, M&B 28767 increased the apparent affinity of Gs for GTP but decreased that for GDP. These results demonstrated that the EP3 receptor isoform is coupled to two different G proteins, and oppositely regulates their activities, inhibition of G(o), and stimulation of Gs.

Identification of the T cell surface signal-transducing glycoprotein sgp-60 as CD48, a counter-receptor for mouse CD2.

We recently showed that CD48 is the major counter-receptor for CD2 in the murine system. To examine whether sgp-60, which has been proposed as the murine homologue of human LFA-3, is another ligand for mouse CD2, we performed the characterization of sgp-60 by using mouse CD2-human IgG chimeric protein (mCD2Rg) and anti-mouse monoclonal antibodies (mAb). Anti-sgp-60 mAb 5-8A10, as well as anti-mouse CD48 mAb HM48-1 completely inhibited the binding of mCD2Rg at the ligand site. 5-8A10 immunoprecipitated the same molecule as that recognized by HM48-1 and reacted with mouse CD48 cDNA-transfected chinese hamster ovary cells, indicating that sgp-60, recognized by 5-8A10, is identical to mouse CD48. The epitope recognized by 5-8A10 was different from that recognized by HM48-1 and OX78, suggesting that the different T cell activating property of these anti-mouse CD48 mAb depends on the epitopes they recognize. The identification of sgp-60 as CD48 further suggests a role of CD48 in regulating T cell activation.

Functional interaction of prostaglandin E receptor EP 3 subtype with guanine nucleotide-binding proteins, showing low-affinity ligand binding

The functional interaction of prostaglandin E (PGE) receptor EP 3 subtype with GTP-binding proteins (G proteins) was characterized in the membranes prepared from mouse EP 3 receptor cDNA-transfected Chinese hamster ovary cells. PGE 2 inhibited forskolin-stimulated adenylate cyclase activity in CHO cells expressing EP 3 receptor and this inhibition was abolished by pertussis toxin (PT) treatment. The PGE 2 binding to the membranes was increased by GTPγS, and PT treatment also increased the binding activity to the same level as that increased by GTPγS, but the sensitivity of GTPγS was lost. Reconstitution with PT-sensitive G proteins into the ADP-ribosylated membranes reduced the PGE 2 binding activity with the following preference: Gi1 = Gi2 > Gi3 > Go , but GTPγS completely blocked the reduction by G proteins. The G-protein-induced reduction of the binding was due to the increase in K d without the change of B max , and due to suppression of association rate. [ 3H]PGE 2-bound EP 3 receptor solubilized from the ADP-ribosylated membranes in the presence or absence of GTPγS was eluted at the position of M r = approx . 60 kDa, similar to the relative molecular mass of EP 3 receptor deduced from its amino acid sequence. In contrast, [ 3H]PGE 2-bound receptor solubilized from Gi2-reconstituted membranes was eluted at the position of M r = approx . 130 kDa, corresponding to the M r of the complex of EP 3 receptor and Gi2, but GTPγS shifted the position of its elution from M r = 130 to 60 kDa. Furthermore, addition of PGE 2 stimulated the GDP release from G proteins reconstituted into the ADP-ribosylated membranes, and PGE 2 inhibited forskolin-stimulated adenylate cyclase activity in G-protein-reconstituted membranes with a selectivity order of Gi1 = Gi2 > Gi3 > Go . These results indicate that EP 3 receptor can functionally couple to PT-sensitive G proteins and unusually the complex form with G proteins has low affinity for the ligand but the form not associated with G proteins has high affinity.

Participation of prostaglandin E2 in dopamine D2 receptor-dependent potentiation of arachidonic acid release.

Stimulation of dopamine D2 receptors potentiates Ca2+ ionophore- or ATP-induced arachidonic acid (AA) release in D2 receptor cDNA-transfected Chinese hamster ovary (CHO) cells [CHO(D2)]. By using a combination of chromatographic, biochemical, and radioimmunochemical techniques, we show here that prostaglandin (PG) E2 is a major product of AA metabolism in CHO(D2) cells stimulated with the Ca2+ ionophore A23187. Formation of this PG was markedly increased by the concomitant application of quinpirole, a D2 receptor agonist. In addition, PGE2 enhanced D2-dependent amplification of AA release, either when it was added (EC50 = 100 nM) or when it was produced endogenously, as shown by experiments carried out with the cyclooxygenase inhibitor indomethacin. The results suggest that PGE2 may participate in D2 receptor-mediated potentiation of AA release in CHO(D2) cells. They also support a functional role for this PG in the modulation of dopaminergic transmission in areas of the CNS, such as amygdala and hypothalamus, where high levels of both PGE2 and dopamine D2 receptors are found.