Increased AT(1) receptor heterodimers in preeclampsia mediate enhanced angiotensin II responsiveness.

Several examples of functional G-protein-coupled receptor heterodimers have been identified. However, it is not known whether receptor heterodimerization is involved in the pathogenesis of human disorders. Here we show that in preeclamptic hypertensive women, a significant increase in heterodimerization occurs between the AT(1)-receptor for the vasopressor angiotensin II and the B(2)-receptor for the vasodepressor bradykinin. AT(1)-B(2)-receptor heterodimerization in preeclampsia correlated with a 4-5-fold increase in B(2)-receptor protein levels. Expression of the AT(1)-B(2) heterodimer increased the responsiveness to angiotensin II and conferred resistance in AT(1)-receptors to inactivation by reactive oxygen species raised in normotensive and preeclamptic pregnancies. We suggest that AT(1)-B(2) heterodimers contribute to angiotensin II hypersensitivity in preeclampsia. Moreover, we identify preeclampsia as the first disorder associated with altered G-protein-coupled receptor heterodimerization.

The angiotensin II AT2 receptor is an AT1 receptor antagonist.

The vasopressor angiotensin II activates AT(1) and AT(2) receptors. Most of the known in vivo effects of angiotensin II are mediated by AT(1) receptors while the biological functions of AT(2) receptors are less clear. We report here that the AT(2) receptor binds directly to the AT(1) receptor and thereby antagonizes the function of the AT(1) receptor. The AT(1)-specific antagonism of the AT(2) receptor was independent of AT(2) receptor activation and signaling, and it was effective on different cells and on human myometrial biopsies with AT(1)/AT(2) receptor expression. Thus, the AT(2) receptor is the first identified example of a G-protein-coupled receptor which acts as a receptor-specific antagonist.

AT1-receptor heterodimers show enhanced G-protein activation and altered receptor sequestration.

The vasopressor angiotensin II regulates vascular contractility and blood pressure through binding to type 1 angiotensin II receptors (AT1; refs 1, 2). Bradykinin, a vasodepressor, is a functional antagonist of angiotensin II (ref. 3). The two hormone systems are interconnected by the angiotensin-converting enzyme, which releases angiotensin II from its precursor and inactivates the vasodepressor bradykinin. Here we show that the AT1 receptor and the bradykinin (B2) receptor also communicate directly with each other. They form stable heterodimers, causing increased activation of G alpha(q) and G alpha(i) proteins, the two major signalling proteins triggered by AT1. Furthermore, the endocytotic pathway of both receptors changed with heterodimerization. This is the first example of signal enhancement triggered by heterodimerization of two different vasoactive hormone receptors.

Involvement of the amino terminus of the B(2) receptor in agonist-induced receptor dimerization.

The mechanisms and the functional importance of G-protein-coupled receptor dimerization are poorly understood. We therefore analyzed dimerization of the bradykinin B(2) receptor. The binding of the agonist bradykinin to the B(2) receptor endogenously expressed on PC-12 cells led to the formation of receptor dimers, whereas the B(2) antagonist HOE140 did not induce dimerization, suggesting that B(2) receptor dimerization was linked to receptor activation. Addition of a peptide corresponding to the amino terminus of the receptor reduced the amount of detected B(2) receptor dimers, whereas peptides derived from the extracellular loops had no effect. To further analyze the role of the amino terminus of the receptor in receptor dimerization, we created two different rat B(2) receptor variants with truncated amino termini, B(2)(53) and B(2)(65), starting at amino acids 53 and 65. In contrast to the wild-type B(2) receptor and to B(2)(53), bradykinin did not induce dimerization of the B(2)(65) receptor. Both receptor variants were similar to the wild-type B(2) receptor with respect to agonist binding and signal generation. However, B(2)(65) was not phosphorylated, did not desensitize, and was not downregulated upon bradykinin stimulation. Likewise, antibodies directed to the amino terminus of the receptor partially reduced internalization of [(3)H]bradykinin on PC-12 cells. These findings suggest that the amino terminus of the B(2) receptor is necessary for triggering agonist-induced B(2) receptor dimerization, and receptor dimers are involved in receptor-mediated signal attenuation.

Retroviral vectors pseudotyped with lymphocytic choriomeningitis virus.

Pseudotyping can improve retroviral vector stability and transduction efficiency. Here, we describe a novel pseudotype of murine leukemia virus packaged with lymphocytic choriomeningitis virus (LCMV). This pseudotype was stable during ultracentrifugation and infected several cell lines from different species. Moreover, LCMV glycoproteins were not cell toxic.

Rapid and efficient cloning of proviral flanking fragments by kanamycin resistance gene complementation.

We have developed a technique for the rapid cloning of unknown flanking regions of transgenic DNA. We complemented a truncated kanamycin resistance gene of a bacterial plasmid with a neomycin resistance gene fragment from a gene transfer vector. Optimized transformation conditions allowed us to directly select for kanamycin-resistant bacteria. We cloned numerous proviral flanking fragments from growth factor-independent cell mutants that were obtained after infection with a replication incompetent retroviral vector and identified integrations into the cyclin D2 and several unknown genomic sequences. We anticipate that our method could be adapted to various vector systems that are used to tag and identify genes and to map genomes.

Detection and spatial distribution of IL-2 receptors on mouse T-lymphocytes by immunogold-labeled ligands.

To identify the plasma membrane (PM) structures implicated in T-cell activation, we studied the distribution of interleukin-2 receptors (IL-2R) and the surface topography of lymphocytes by affinity labeling in electron microscopy (EM). In particular, we analyzed the distribution of the IL-2R alpha-chain on CTLL-2 cells (a murine cytotoxic T-cell lymphoma line). Some of our experiments were extended to the functionally and morphologically distinct cell line EL4 (a routine helper T-cell lymphoma line). As affinity ligands we used a rat monoclonal antibody (clone 7D4) reactive with the routine alpha-chain of IL-2R and recombinant mouse IL-2 (rIL-2). The distribution of IL-2R was visualized on the cell surface by ligands coupled to colloidal gold particles of different sizes. Unfixed cells were labeled with gold probes and attached to concanavalin A (ConA)-pretreated coverslips. Subsequently, the cells were prepared for EM. Examination of ultrathin sections and large surface replicas revealed a high degree of variability in cell morphology and in the density of the randomly distributed gold-labeled ligands among CTLL cells. According to their typical appearance, lymphocytes with strong receptor expression can be easily identified within the cell population. In contrast, the label on many mitogen-activated EL4 cells showed a cap-like polar distribution. The results suggest the existence of diverse distribution patterns of IL-2R on CTLL and EL4 cells. These differences are believed to reflect the different physiological roles played by T-cell subsets in the immune system.

Excision of specific DNA-sequences from integrated retroviral vectors via site-specific recombination.

Vectors for gene transfer and gene therapy were developed which combine the advantages of the integrase and recombinase systems. This was achieved by inserting two loxP sites for specific DNA excision into an MESV based retroviral vector. We show that this 'retroviral lox system' allows the infection of cells and the expression of transferred genes. In addition, we constructed an efficient retrovirus-based expression system for a modified Cre recombinase. Functional tests for DNA excision from integrated retroviral lox vectors were performed by the use of a negative selectable marker gene (thymidine kinase). Cre expression in cells infected with retroviral lox vectors and subsequent BrdU selection for cells in which site-specific recombination has occurred results in large numbers of independent cell clones. These results were confirmed by detailed molecular analysis. In addition we developed retroviral suicide vectors in which the enhancer/promoter elements of both LTRs were replaced by lox sequences. We show that lox-sequences located in the LTRs of retroviral vectors are stable during retroviral replication. Potential applications of this system would be the establishment of revertants of retrovirus-infected cells by controlled excision of nearly the complete proviral DNA.

Fibroblasts as efficient antigen-presenting cells in lymphoid organs.

Only so-called "professional" antigen-presenting cells (APCs) of hematopoietic origin are believed capable of inducing T lymphocyte responses. However, fibroblasts transfected with viral proteins directly induced antiviral cytotoxic T lymphocyte responses in vivo, without involvement of host APCs. Fibroblasts induced T cells only in the milieu of lymphoid organs. Thus, antigen localization affects self-nonself discrimination and cell-based vaccine strategies.

Homodimeric murine interleukin-3 agonists indicate that ligand dimerization is important for high-affinity receptor complex formation.

Homodimeric murine interleukin 3 (mIL-3) agonists were generated by intermolecular cystine-bonding. Steady-state binding assays and association kinetics performed at 4 degrees C using these agonists revealed specific binding to both the high- and low-affinity receptor. DSS-mediated crosslinking studies performed at 4 degrees C with agonist concentrations compatible with high-affinity receptor complex formation allowed to detect protein complexes of the alpha chain, the beta chain(s) and the high-affinity receptor complex migrating with apparent molecular weights of 90 kDa, 140 kDa, and above 180 kDa, respectively. In contrast, monomeric mIL-3 was crosslinked to the alpha chain receptor only unless high concentrations were used. Binding studies performed at 4 degrees C revealed a positive cooperative interaction of monomeric mIL-3 with the low-affinity receptor. Proliferation studies and association kinetics performed at 37 degrees C showed that under physiological conditions these agonists were at least 2- to 3-fold more potent than monomeric mIL-3. We therefore propose that dimerization of mIL-3 may be involved in high-affinity receptor complex formation.

Intermolecular cystine-bonding of murine interleukin 2 indicates that ligand dimerization is important for the formation of the high-affinity receptor complex.

Interleukin 2 is thought to be active as a monomeric protein. As the nonessential Cys-140 of murine interleukin 2 (mIL2) is located in the hydrophobic interface of the amphiphilic F domain it was successfully used to stabilize hydrophobic amino acid contacts between two mIL2 cores yielding biologically active cystine-bonded dimeric mIL2. (3H) thymidine incorporation assays with intermolecular cystine-bonded or monomeric mIL2 revealed almost identical median effective concentrations (EC50) and high-affinity dissociation constants (Kdh), respectively. Comparative binding and internalization assays suggest that one cystine-bonded dimeric or two monomeric mIL2 molecules bind to the high-affinity receptor complex. Furthermore, DSS concentration-dependent crosslinking studies using monomeric mIL2 revealed four membrane-derived protein-complexes with apparent molecular weights of about 70 kDa, 85 kDa, 95 kDa and 100 kDa, respectively, showing that both mIL2 receptor chains may be crosslinked to a monomeric or dimeric ligand molecule, respectively. We therefore propose that dimerization of murine interleukin 2 occurring either in solution at concentrations above the low-affinity dissociation constant or at the low-affinity receptor is important for regulation of high-affinity complex formation and signal transduction.

IFN-gamma production in tissues of mice during acute infection with lymphocytic choriomeningitis virus.

As shown by a single-cell solid-phase ELISA, splenocytes as well as liver lymphoid cells from unmanipulated specific-pathogen free mice synthesized and released IFN-gamma. Synthesis of this lymphokine could not be demonstrated either on the transcriptional level by Northern blotting or immunocytochemically. Thus, IFN-gamma is constitutively produced in mice, although in low quantities. During acute infection with lymphocytic choriomeningitis virus, IFN-gamma mRNA became detectable in spleen and brain but not in the liver. In spleens and livers of these mice, the numbers of cells synthesizing the lymphokine were increased and many were seen in foot tissue undergoing a delayed-type hypersensitivity reaction after intraplantar inoculation of the virus. In contrast, few IFN-gamma-producing cells were found in the inflammatory infiltrates of leptomeninges and choroid plexus after intracerebral infection.

Mechanism of recovery from acute virus infection. VIII. Treatment of lymphocytic choriomeningitis virus-infected mice with anti-interferon-gamma monoclonal antibody blocks generation of virus-specific cytotoxic T lymphocytes and virus elimination.

In acutely infected mice the lymphocytic choriomeningitis (LCM) virus multiplies to high titers in essentially all tissues. Around day 6, virus clearance sets in, which has previously been shown to be mediated by CD8+ cytotoxic T lymphocytes (CTL), probably by releasing (or inducing other cells to release) anti-viral cytokines. To ascertain whether interferon-gamma plays a role, infected mice were injected once i.v. with monoclonal antibody known to neutralize this lymphokine, and the effect this had on both termination of the infection and development of LCM virus-specific CTL was determined. Administration 1 day after infection blocked virus elimination from spleen and liver and decreased the generation of CTL; also, limiting dilution analysis revealed absence of activation of CTL precursors. In contrast, when the antibody was given 3 days after or 1 day before the virus, neither clearance nor generation of CTL was measurably affected. Furthermore, the antiviral effect of immune spleen cells after their transfer into infected recipients was not altered by treatment of the latter with monoclonal antibody. We conclude that in the generation of LCM virus-specific CTL an early event is dependent on constitutively produced interferon-gamma; when its activity is blocked, CTL do not mature, resulting in the mouse's inability to terminate the infection.

Homologous interference of lymphocytic choriomeningitis virus involves a ribavirin-susceptible block in virus replication.

Depending on the multiplicity of infection (MOI), infection of L929 cells results in either productive lymphocytic choriomeningitis virus replication or homologous interference M. Bruns, A. Gessner, H. Lother, and F. Lehmann-Grube, Virology 166:133-139, 1988). As shown in this communication, productive lymphocytic choriomeningitis virus replication as observed at a low MOI was effectively inhibited by ribavirin. In contrast, virus yields increased if cells were infected with a high MOI and in the presence of 5 microM of the antiviral compound. This drug-dependent release of infectious virus was preceded by enhanced nucleoprotein (NP) synthesis, a change in intracellular NP distribution, and by an onset of glycoprotein synthesis. It is therefore proposed that this block in viral replication is brought about by a posttranslational effect on a viral gene product, probably the NP, present in reasonably large quantities both during homologous interference as well as persistent infection.

Host cell-dependent homologous interference in lymphocytic choriomeningitis virus infection.

The generation of virus progeny as well as transcription, translation, and replication of the viral small RNA (S-RNA), which codes for the nucleoprotein (NP) and the glycoprotein precursor (GPC), was followed in L and MDCK cells after infection with multiplicities (m.o.i.) ranging from 0.01 to 100. In L cells, the yields of both plaque-forming units and interfering particles varied inversely with the m.o.i. Northern blot analysis revealed that early after infection with high multiplicity NP-mRNA was present, but later few or no signals of any specificity were registered. After low m.o.i. the results were negative at 8 hr, but large quantities of mRNAs for NP and GPC as well as viral genomic S-RNA and genomic-sized complementary S-RNA had been synthesized at 48 hr. In MDCK cells, throughout the range of m.o.i. both entities attained lower levels and most were generated at m.o.i. one. The degree of hybridization correlated roughly with the quantity of infectious virus to which the cells had been exposed. In the cells of both lines the NP-mRNA corresponded to the synthesis of its translation product, but once produced, most of it appeared to be retained in the phosphorylated form. We assume that the homologous interference seen in L cells after infection with high m.o.i. results from a host-dependent inhibition of viral transcription and replication mediated by NP.

Transcripts within the replication origin, oriC, of Escherichia coli.

Transcription start and termination sites were mapped in the E. coli replication origin, oriC. Outward transcription from within oriC (promoters Pori-r and Pori-l) was found to start in vivo at position 178 for Pori-l and at positions 294 and 304 for Pori-r, respectively. These transcripts were terminated after 100-150 bases, at terminators designated Tori-l and Tori-r. Transcription from the 16 kd promoter, which lies clockwise adjacent to oriC and promotes transcription toward oriC, started at position 757 and gave transcripts with 3' ends at several positions within and to the left of the minimal replication origin. However, the majority of transcripts traversed the whole oriC region, and were not terminated within the DNA segment tested. Transcription of the chromosomal 16 kd gene was negatively regulated by DnaA protein and positively affected by dam methylation. The possible function of these transcripts is discussed.

Regulation of transcription of the chromosomal dnaA gene of Escherichia coli.

By comparative S1 analysis we investigated the in vivo regulation of transcription of the chromosomal dnaA gene coding for a protein essential for the initiation of replication at the chromosomal origin. Inactivation of the protein in dnaA mutants results in derepression, whereas excess DnaA protein (presence of a DnaA overproducing plasmid) leads to repression of dnaA transcription. Both dnaA promoters are subject to autoregulation allowing modulation of transcriptional efficiency by at least 20-fold. Increasing the number of oriC sequences (number of DnaA binding sites) in the cell by introducing oriC plasmids leads to a derepression of transcription. Autoregulation and binding to oriC suggest that the DnaA protein exerts a major role in the regulation of the frequency of initiation at oriC. The efficiency of transcription of the dnaA2 promoter is reduced in the absence of dam methylation, which is involved in the regulation of oriC replication.

AsnC: an autogenously regulated activator of asparagine synthetase A transcription in Escherichia coli.

The regulation of the asparagine synthetase A gene of Escherichia coli was studied in vitro with a coupled transcription-translation system. It was shown that the 17-kilodalton gene, which is transcribed divergently from the adjacent asnA gene, codes for an activator of asnA transcription. The synthesis of the 17-kilodalton protein, which we now call AsnC, is autogenously regulated. The stimulating effect of AsnC on asnA transcription is abolished by asparagine, while the autoregulation of asnC is not affected by asparagine. The N-terminal part of the asnC protein, inferred from the DNA sequence, is homologous to the DNA-binding domain of regulatory proteins like catabolite gene activator, cro, and cI. This homology and direct repeats found in the region of the two asn promoters suggest that the asnC protein regulates transcription by binding to DNA. The asn promoters were defined by mapping of the mRNA start sites of in vitro-generated transcripts.

Effect of dam methylation on the activity of the E. coli replication origin, oriC.

Methylation of GATC sites by the dam methylase is required for efficient initiation of DNA replication at the replication origin, oriC, of Escherichia coli. This is demonstrated by the inability of minichromosomes to be maintained in dam mutant strains. The requirement for methylated GATC sites is less stringent in vitro than in vivo. The time required for complete methylation of the origin region apparently determines the minimal spacing of replication forks on the chromosome.

dnaA protein-regulated transcription: effects on the in vitro replication of Escherichia coli minichromosomes.

Both initiation of replication and initiation of transcription are influenced by dnaA protein, when minichromosomes are assayed in vitro for dnaA protein complementation. This dnaA protein effect is seen only if minichromosomes are used containing the 16-kd promoter, from which transcription is directed into the minimal origin. Determination of the 16-kd promoter activity both in vivo and in vitro showed that this strong promoter is specifically repressed by dnaA protein. The 16-kd promoter is thus an integral regulatory region of oriC.