Fluorescence resonance energy transfer analysis of alpha 2a-adrenergic receptor activation reveals distinct agonist-specific conformational changes.

Several lines of evidence suggest that G-protein-coupled receptors can adopt different active conformations, but their direct demonstration in intact cells is still missing. Using a fluorescence resonance energy transfer (FRET)-based approach we studied conformational changes in alpha(2A)-adrenergic receptors in intact cells. The receptors were C-terminally labeled with cyan fluorescent protein and with fluorescein arsenical hairpin binder at different sites in the third intracellular loop: N-terminally close to transmembrane domain V (I3-N), in the middle of the loop (I3-M), or C-terminally close to transmembrane domain VI (I3-C). All constructs retained normal ligand binding and signaling properties. Changes in FRET between the labels were determined in intact cells in response to different agonists. The full agonist norepinephrine evoked similar FRET changes for all three constructs. The strong partial agonists clonidine and dopamine induced partial FRET changes for all constructs. However, the weak partial agonists octopamine and norphenephrine only induced detectable changes in the construct I3-C but no change in I3-M and I3-N. Dopamine-induced FRET-signals were approximately 1.5-fold slower than those for norepinephrine in I3-C and I3-M but >3-fold slower in I3-N. Our data indicate that the different ligands induced conformational changes in the receptor that were sensed differently in different positions of the third intracellular loop. This agrees with X-ray receptor structures indicating larger agonist-induced movements at the cytoplasmic ends of transmembrane domain VI than V and suggests that partial agonism is linked to distinct conformational changes within a G-protein-coupled receptor.

Downregulation of soluble guanylyl cyclase in young and aging spontaneously hypertensive rats.

Endothelial dysfunction, as observed in hypertension and atherosclerosis, is associated with a reduction in the bioavailability of endothelium-derived nitric oxide (NO). We tested the hypothesis that alterations in the soluble guanylyl cyclase (sGC) pathway may also contribute to the pathogenesis of hypertension. Therefore, we investigated the expression and activity of sGC in young (6 weeks) and aging (17 months) spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto rats (WKY). Endothelium-independent relaxation of aortic rings in response to the sGC activator YC-1 was attenuated in SHR, and expression of both alpha(1) and beta(1) subunits of heterodimeric sGC and the basal contents of cGMP were reduced specifically in SHR aorta. Moreover, mRNA expression of the cGMP receptor and effector protein cGMP-dependent protein kinase type Ialpha (cGKIalpha) was also reduced. Interestingly, downregulation of both sGC and cGKIalpha expression was observed in young, ie, normotensive SHR, whereas impairment of the endothelium-independent relaxation was found only in aging SHR. Accordingly, similar cGMP levels were reached in response to YC-1 in young SHR and young WKY, suggesting a compensatory increased sensitivity or effectiveness of the sGC pathway in young SHR. In aging SHR, however, increased sensitivity to YC-1 no longer compensated for the impairment of endothelium-independent relaxation, suggesting that other mechanisms were involved. In fact, endothelium-independent relaxations were partially restored by superoxide dismutase, suggesting a pathophysiological role of superoxide production, particularly at later disease stages. Thus, tissue-specific downregulation of components of the sGC/cGMP pathway is an early event in the pathogenesis of hypertension.

Effects of the soluble guanylyl cyclase activator, YC-1, on vascular tone, cyclic GMP levels and phosphodiesterase activity.

The vasomotor and cyclic GMP-elevating activity of YC-1, a novel NO-independent activator of soluble guanylyl cyclase (sGC), was studied in isolated rabbit aortic rings and compared to that of the NO donor compounds sodium nitroprusside (SNP) and NOC 18. Similarly to SNP and NOC 18, YC-1 (0.3-300 microM) caused a concentration-dependent, endothelium-independent relaxation that was greatly reduced by the sGC inhibitor 1-H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ 10 microM; 59% inhibition of dilation induced by 100 microM YC-1) suggesting the activation of sGC as one mechanism of action. Preincubation with YC-1 (3 and 30 microM) significantly increased the maximal dilator responses mediated by endogenous NO in aortic rings that was released upon exposure to acetylcholine, and enhanced the dilator response to the exogenous NO-donors, SNP and NOC 18, by almost two orders of magnitude. Vasoactivity induced by SNP and YC-1 displayed different kinetics as evidenced by a longlasting inhibition by YC-1 (300 microM) on the phenylephrine (PE)-induced contractile response, which was not fully reversible even after extensive washout (150 min) of YC-1, and was accompanied by a long-lasting elevation of intracellular cyclic GMP content. In contrast, SNP (30 microM) had no effect on the vasoconstrictor potency of PE, and increases in intravascular cyclic GMP levels were readily reversed after washout of this NO donor compound. Surprisingly, YC-1 not only activated sGC, but also affected cyclic GMP metabolism, as it inhibited both cyclic GMP break down in aortic extracts and the activity of phosphodiesterase isoforms 1-5 in vitro. In conclusion, YC-1 caused persistent elevation of intravascular cyclic GMP levels in vivo by activating sGC and inhibiting cyclic GMP break down. Thus, YC-1 is a highly effective vasodilator compound with a prolonged duration of action, and mechanisms that are unprecedented for any previously known sGC activator.

Homodimerization of soluble guanylyl cyclase subunits. Dimerization analysis using a glutathione s-transferase affinity tag.

Soluble guanylyl cyclase (sGC) is an alpha/beta-heterodimeric hemoprotein that, upon interaction with the intercellular messenger molecule NO, generates cGMP. Although the related family of particulate guanylyl cyclases (pGCs) forms active homodimeric complexes, it is not known whether homodimerization of sGC subunits occurs. We report here the expression in Sf9 cells of glutathione S-transferase-tagged recombinant human sGCalpha1 and beta1 subunits, applying a novel and rapid purification method based on GSH-Sepharose affinity chromatography. Surprisingly, in intact Sf9 cells, both homodimeric GSTalpha/alpha and GSTbeta/beta complexes were formed that were catalytically inactive. Upon coexpression of the respective complementary subunits, GSTalpha/beta or GSTbeta/alpha heterodimers were preferentially formed, whereas homodimers were still detectable. When subunits were mixed after expression, e.g. GSTbeta and beta or GSTalpha and beta, no dimerization was observed. In conclusion, our data suggest the previously unrecognized possibility of a physiological equilibrium between homo- and heterodimeric sGC complexes.

Human soluble guanylate cyclase: functional expression and revised isoenzyme family.

Soluble guanylate cyclase (sGC), a heterodimeric (alpha/beta) haem protein that converts GTP to the second messenger cGMP, functions as the receptor for nitric oxide (NO) and nitrovasodilator drugs. Three distinct cDNA species of each subunit (alpha1-alpha3, beta1-beta3) have been reported from various species. From human sources, none of these have been expressed as functionally active enzyme. Here we describe the expression of human alpha/beta heterodimeric sGC in Sf9 cells yielding active recombinant enzyme that was stimulated by the nitrovasodilator sodium nitroprusside or the NO-independent activator 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1). At the protein level, both alpha and beta subunits were detected in human tissues, suggesting co-expression also in vivo. Moreover, resequencing of the human cDNA clones [originally termed alpha3 and beta3; Giuili, Scholl, Bulle and Guellaen (1992) FEBS Lett. 304, 83-88] revealed several sequencing errors in human alpha3; correction of these eliminated major regions of divergence from rat and bovine alpha1. As human beta3 also displays more than 98% similarity to rat and bovine beta1 at the amino acid level, alpha3 and beta3 represent the human homologues of rat and bovine alpha1 and beta1, and the isoenzyme family is decreased to two isoforms for each subunit (alpha1, alpha2; beta1, beta2). Having access to the human key enzyme of NO signalling will now permit the study of novel sGC-modulating compounds with therapeutic potential.

Nic96p is required for nuclear pore formation and functionally interacts with a novel nucleoporin, Nup188p.

The amino-terminal domain of Nic96p physically interacts with the Nsp1p complex which is involved in nucleocytoplasmic transport. Here we show that thermosensitive mutations mapping in the central domain of Nic96p inhibit nuclear pore formation at the nonpermissive temperature. Furthermore, the carboxyterminal domain of Nic96p functionally interacts with a novel nucleoporin Nup188p in an allele-specific fashion, and when ProtA-Nup188p was affinity purified, a fraction of Nic96p was found in physical interaction. Although NUP188 is not essential for viability, a null mutant exhibits striking abnormalities in nuclear envelope and nuclear pore morphology. We propose that Nic96p is a multivalent protein of the nuclear pore complex linked to several nuclear pore proteins via its different domains.

Nuclear uptake control of NF-kappa B by MAD-3, an I kappa B protein present in the nucleus.

I kappa B proteins specifically inhibit the DNA binding of NF-kappa B/Rel transcription factors. An additional role as inhibitors of nuclear uptake was supposed by subcellular fractionation and enucleation experiments. Using indirect immunofluorescence labeling of cells, we show here that the DNA-binding p50 and p65 subunits of NF-kappa B, as well as the I kappa B protein MAD-3, all occur in the nucleus when overexpressed on their own. Nuclear uptake of p65 and, to a lesser extent of p50, was, however, suppressed when MAD-3 was coexpressed. Likewise, nuclear uptake of MAD-3 was blocked by overexpressed p65 or p50. This directly demonstrates that I kappa B is a nuclear uptake regulatory protein and that the various subunits of NF-kappa B can mutually control their access to the nucleus. In the presence of MAD-3, antibodies specific for peptides overlapping the nuclear location signal (NLS) sequences of p65 and p50 could not recognize their epitopes on NF-kappa B, suggesting that the I kappa B protein rendered the signals inaccessible for NLS receptors.

Intramolecular masking of the nuclear location signal and dimerization domain in the precursor for the p50 NF-kappa B subunit.

We show that the non-DNA-binding precursor for the p50 subunit (p110), like NF-kappa B, is subject to control of nuclear uptake. In contrast to p50, p110 was excluded from nuclei and unable to associate detectably with p50 or p65 NF-kappa B subunits. The nuclear location signal in the N-terminal half of p110 was not accessible for monospecific antibodies. Removal of only 191 amino acids from the C-terminus of p110 restored antibody accessibility as well as nuclear uptake. The C-terminal half of p110, which is linked to the p50 portion via a glycine-rich hinge, could also noncovalently bind to p50. This helps to explain why p50, after cleavage of the precursor in intact cells, was still retained in an inactive form in the cytoplasm. Our study describes a novel mechanism of nuclear uptake control by masking of a nuclear location signal through a remote domain within a precursor molecule.

Purified I kappa B-beta is inactivated upon dephosphorylation.

In uninduced cells, the NF-kappa B transcription factor resides in the cytoplasm in complex with an inhibitory protein, I kappa B. I kappa B is a specific inhibitor of DNA binding and apparently prevents nuclear uptake of NF-kappa B. Stimulation of cells, for instance with the cytokine tumor necrosis factor, releases I kappa B and allows nuclear translocation and DNA binding of NF-kappa B to regulatory DNA sequences in many genes. We recently reported on the purification of a major form of I kappa B, referred to as I kappa B-alpha, with a molecular size of 37 kDa. Here, we purified and characterized I kappa B-beta, a chromatographically distinct second form of I kappa B. I kappa B-beta has a size of 43 kDa and, as I kappa B-alpha, an acidic isoelectric point between 4.8 and 5.0. Both forms of I kappa B were inactivated by a treatment with protein kinases A and C in vitro. In contrast to I kappa B-alpha, I kappa B-beta lost its inhibiting activity upon a treatment with phosphatase. Phosphatase treatment also released active NF-kappa B from its inactive complex with I kappa B-beta suggesting that the activation of NF-kappa B in intact cells might not only rely on phosphate transfer onto I kappa B but also on phosphate removal from one form of I kappa B.

[Premature return of the drivers' license--temporal limits according to section 69 a Abs. 7 StGB]. / Vorzeitige Wiedererteilung der Fahrerlaubnis--zeitliche Grenzen gem. section 69 a Abs. 7 StGB.

1) Text of law and kind of crime (driving under the influence of alcohol) don't allow a reduction of the minimal period of revocation. 2) A new definition of section 69 a Abs. 7 StGB and a reduction respectively a renunciation of minimal period of revocation should give possibility to courts and reprieval authorities to ensure the inclusion of a large number of persons suitable for additional training and in cases of total abstinence traffic authority should regard the aptitude for participation in traffic as regranted.

A novel complex between the p65 subunit of NF-kappa B and c-Rel binds to a DNA element involved in the phorbol ester induction of the human urokinase gene.

The NF-kappa B subunits, p50 and p65, have extensive sequence homology with the c-rel proto-oncogene and the Drosophila morphogen dorsal. It has recently been shown that in vitro translated c-Rel can bind to DNA and form a complex with p50. However, the conditions for DNA binding of c-Rel in vivo and its DNA sequence specificity have not been established. Here we report the identification a novel heterodimeric complex that binds to a kappa B-like, phorbol ester (TPA) responsive DNA sequence, 5'-GGGAAAGTAC-3', in the 5' flanking region of the human urokinase (uPA) gene. This sequence was shown to bind two protein complexes, LC and UC. LC was indistinguishable from NF-kappa B as it reacted with antibodies recognizing the p50 subunit of NF-kappa B, and was shown by UV crosslinking to contain the p50 and p65 subunits of NF-kappa B. UC, on the other hand, strongly reacted with anti-v-Rel, but not with the anti-p50 antibodies, and was shown by crosslinking to contain 75 kDa and 85 kDa protein-DNA adducts. The 75 kDa and the 85 kDa adducts could be immunoprecipitated only by anti-p65 and anti-c-Rel antibodies, respectively, showing that c-Rel formed a heterodimer with p65. Both protein complexes were present in inactive forms in HeLa cell cytosol, and their nuclear translocation was induced by TPA. DNA binding of UC and LC could, furthermore, be inhibited by I kappa B-alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

[Reducing the duration of revoked driving permit penalty in alcoholic drivers following psychological remedial traffic education]. / Abkürzung der Fahrerlaubnissperre bei Alkoholtätern nach verkehrspsychologischer Nachschulung.

1) Also new facts--driver improvement, longstanding good reputation in road traffic (minimum 25 years)--are only conditionally acceptable to substantiate a reduction in the blocking period in terms of section 69 a section 7 StGB. 2) A high blood alcohol level (as from 0.2 g %) precludes the reduction in the blocking period for obtaining a driving licence of drunken drivers. 3) Attending a post-schooling course may, in individual cases, as a new fact able to justify a reduction of blocking period. 4) In many cases the court is insisting behind own behaviour prognosis on a specialist for psychological selection tests.

[Driving fitness of apprehended alcoholics--fitness test and remedial psychological traffic education]. / Zur Fahreignung von Alkoholtätern--Eignungstest und verkehrspsychologische Nachschulung.

1) No Therapy Without Screening: According to the prevailing opinion of jurists and psychologists) who are occupied with problems concerning post-schooling and of the traffic authorities in charge of issuing driving licences screening is absolutely necessary with regard to traffic-psychological post-schoolings as characteristical maladjustments of either reparable or irreparable nature can only be detected at such screening). Only medical and psychological screening can help to achieve the necessary clarification.) Screening is frequently connected with counselling for therapy, as it is for example stressed in the names of the institutions concerned in the länder of Rhineland-Palatine, North Rhine-Westphalia and Lower Saxony). 2) Exclusion of Drink-Drivers: Screening is particularly essential to exclude the so called drink-drivers who are alcoholics from post-schooling. The necessity of such an exclusion is stressed by Müller's) latest investigation of traffic--accident data in correlation to the blood-alcohol concentration, and it must be assumed that the percentage of undetected drink-drivers with a medium blood-alcohol concentration is extremely high. One has to agree with Himmelreich) that models for post-schooling must not exclude alcoholics and criminal offenders. In this case one has to differentiate between the problem drinker) who can be post-schooled and the drink-driver described by Winkler.) Bode) and Scherer) with rigid drinking habits, a raised disposition for risk) and a reduced responsibility according to sections 20, 21 of the German Criminal Law (StGB) to such an extend that a consciousness of guilt can be ruled out). Already after the preliminary examination of such alcoholics) it has to be assumed that an immediate traffic-psychological post-schooling will hardly be successful because of the lack or considerably reduced ability of responsibility. Post-schoolings do not have compensatory effects when regular alcohol abuse (drink-driver) is concerned. In extreme cases a detoxication and the attendance of sessions of a self-help group would have to take place first. Only afterwards a traffic-psychological post-schooling can be carried out as a long term therapy. 3) Models for the Post-schooling of Individuals Tested: The "Saarbrücken model" of a collaboration of the medical-psychological institutes of the technical supervision union (TUV) and independent psychologists allows a culmination of different post-schooling and control measures with drink-drivers after negative examinations: medical tests, laboratory, chemical checks of the liver data, traffic-psychological post-schoolings of individuals and the attendance of sessions of self-help groups.(ABSTRACT TRUNCATED AT 400 WORDS)

DNA binding of purified transcription factor NF-kappa B. Affinity, specificity, Zn2+ dependence, and differential half-site recognition.

A rapid purification procedure for the NF-kappa B transcription factor from the cytosol of human placenta is demonstrated which exploits the insensitivity of the NF-kappa B.DNA complex toward the intercalating agent chloroquine. Purified NF-kappa B required 100 mM KCl or NaCl and a pH of 7.5 to optimally bind to DNA. Equilibrium of binding was reached within less than 5 min in the absence of competitor DNA and after 1 h in the presence of 0.1 mg/ml poly(dI-dC). DNA binding of NF-kappa B was specifically blocked by the chelating agent 1,10-orthophenantroline and could only be reconstituted by addition of Zn2+. Under optimal binding conditions, the dissociation constant for the complex of the purified NF-kappa B with its most frequent cognate DNA motif 5'-GGGACTTTCC-3' was in the range of 10(-12) to 10(-13) M. Various other cis-acting kappa B motifs were recognized by NF-kappa B with lower affinities. A comparative analysis of known NF-kappa B-binding sites and competition experiments with synthetic polynucleotides and oligonucleotides encompassing only one half-site or single-stranded kappa B motifs suggested that the two DNA-binding monomers in the NF-kappa B protein complex can interact differentially with the half-sites of the decameric cognate motif.

[Some focal points for penalty apportionment and penalty suspension in the assessment of alcohol-related road traffic accidents]. / Einige Schwerpunkte für die Strafzumessung und Strafaussetzung zur Bewährung bei Alkoholdelikten im Strassenverkehr.

According to a great number of judgements by law courts the authors show different criterions for and against fixing a prison sentence and for and against suspension of punishment for probation in cases of drunken driving. The criterions depend partly on the way of committing an offence, partly on the person of the offender and partly even on his actions after the offence and before punishment. On principle an execution of punishment is necessary in cases of mortal drunken driving.

Purified human I kappa B can rapidly dissociate the complex of the NF-kappa B transcription factor with its cognate DNA.

I kappa B is an inhibitory protein that stabilizes the inducible cytoplasmic form of the NF-kappa B transcription factor. We have purified I kappa B-alpha, a major form of I kappa B with an apparent molecular size of 37 kd, from cytosol of human placenta. A second chromatographically distinct form, I kappa B-beta, was partially purified and found to be more basic and 3-8 kd larger than the alpha form. The occurrence of distinct forms of I kappa B could explain how NF-kappa B can be activated in response to various agents that signal via different intracellular messenger systems. Both I kappa B-alpha and -beta exclusively inactivated NF-kappa B containing the non-DNA binding 65 kd subunit and, within minutes, could dissociate a high affinity complex of NF-kappa B with its cognate DNA. On the assumption that free I kappa B-alpha and -beta can enter the nucleus, these proteins could rapidly release NF-kappa B from high affinity binding sites in enhancer and promoter elements, thereby terminating NF-kappa B-dependent initiation of gene expression.