Functional expression of a novel human neurokinin-3 receptor homolog that binds [3H]senktide and [125I-MePhe7]neurokinin B, and is responsive to tachykinin peptide agonists

In 1992, Xie et al. identified a cDNA sequence in the expression cloning search for the κ opioid receptor. When the cDNA was expressed in Cos-7 cells, binding of opioid compounds was observed to be of low affinity and without κ, μ, or δ selectivity [Xie, G.-X., Miyajima, A. and Goldstein, A. (1992) Proc. Natl. Acad. Sci. USA 89, 4124–4128]. This cDNA was highly homologous to the human neurokinin-3 (NK-3) receptor sequence, and displayed lower homology to NK-1 and NK-2 sequences. This sequence was stably expressed in Chinese hamster ovary cells, which do not express neurokinin receptors naturally, and ligand binding and second messenger characteristics were compared with a human NK-3 receptor. The NK-3 receptor homolog bound [³H]senktide with a K_d of 39 nM, similar to that of the NK-3 receptor. The rank order of tachykinin peptides competing for [³H]senktide binding at the NK-3 receptor homolog was [MePhe⁷]neurokinin B > senktide > substance P = neurokinin A > neurokinin B. This cell line also bound [¹²⁵I-MePhe⁷]neurokinin B; however, neurokinin B was an effective competitor. Tachykinin peptides stimulated both inositol phospholipid hydrolysis and arachidonic acid release at NK-3 and NK-3 receptor homolog cell lines, with similar rank orders of potency of [MePhe⁷]neurokinin B = neurokinin B = senktide > NKA = substance P. These results indicate that expression of the NK-3 receptor homolog cDNA in the Chinese hamster ovary cell system induces the expression of a receptor site with many similarities but certain key differences from that of the human NK-3 receptor. The results are discussed with reference to the existence of a novel human tachykinin receptor.     

Demonstration of a novel circulating anti-prostacyclin receptor antibody

Although coronary artery disease (CAD) is appreciated to be accelerated in patients with chronic spinal cord injury (SCI), the underlying mechanism of CAD in SCI remains obscure. We have recently shown that platelets from subjects with SCI develop resistance to the inhibitory effect of prostacyclin (PGI₂) on the platelet stimulation of thrombin generation. The loss of the inhibitory effect was due to the loss of high-affinity prostanoid receptors, which may contribute to atherogenesis in SCI. Incubation of normal, non-SCI platelets in SCI plasma (n = 12) also resulted in the loss of high-affinity binding of PGI₂ (Kd₁ = 9.1 ± 2.0 nM; n₁ = 170 ± 32 sites per cell vs. Kd₁ = 7.2 ± 1.1 nM; n₁ = 23 ± 8 sites per cell), with no significant change in the low-affinity receptors (Kd₂ = 1.9 ± 0.1 μM; n₂ = 1,832 ± 232 sites per cell vs. Kd₂ = 1.6 ± 0.1 μM; n₂ = 1,740 ± 161 sites per cell) as determined by Scatchard analysis of the binding of [³H]PGE₁. The loss of high-affinity PGI₂ binding led to the failure of PGI₂ to inhibit the platelet-stimulated thrombin generation. The increase of cellular cyclic AMP level, mediated through the binding of PGI₂ to low-affinity receptors in platelets, was unaffected in SCI platelets. PAGE and immunoblot of SCI plasma showed the presence of an IgG band, which specifically blocked the binding of [³H]PGE₁ to the high-affinity PGI₂ receptors of normal platelets. PAGE of the reduced IgG band, the amino acid sequence of the novel band as a heavy chain of IgG that inhibits the binding of [³H]PGE₁ to the high-affinity platelet PGI₂ receptor, demonstrates that the specific recognition and inhibition of high-affinity PGI₂ binding to platelets was due to an anti-prostacyclin receptor antibody present in SCI plasma.     

Impaired locomotor activity and exploratory behavior in mice lacking histamine H1 receptors

From pharmacological studies using histamine antagonists and agonists, it has been demonstrated that histamine modulates many physiological functions of the hypothalamus, such as arousal state, locomotor activity, feeding, and drinking. Three kinds of receptors (H₁, H₂, and H₃) mediate these actions. To define the contribution of the histamine H₁ receptors (H1R) to behavior, mutant mice lacking the H1R were generated by homologous recombination. In brains of homozygous mutant mice, no specific binding of [³H]pyrilamine was seen. [³H]Doxepin has two saturable binding sites with higher and lower affinities in brains of wild-type mice, but H1R-deficient mice showed only the weak labeling of [³H]doxepin that corresponds to lower-affinity binding sites. Mutant mice develop normally, but absence of H1R significantly increased the ratio of ambulation during the light period to the total ambulation for 24 hr in an accustomed environment. In addition, mutant mice significantly reduced exploratory behavior of ambulation and rearings in a new environment. These results indicate that through H1R, histamine is involved in circadian rhythm of locomotor activity and exploratory behavior as a neurotransmitter.     

Phytosulfokine-α, a sulfated pentapeptide, stimulates the proliferation of rice cells by means of specific high- and low-affinity binding sites

Peptide growth factors were isolated from conditioned medium derived from rice (Oryza sativa L.) suspension cultures and identified to be a sulfated pentapeptide [H-Tyr(SO₃H)-Ile-Tyr(SO₃H)-Thr-Gln-OH] and its C-terminal-truncated tetrapeptide [H-Tyr(SO₃H)-Ile-Tyr(SO₃H)-Thr-OH]. These structures were identical to the phytosulfokines originally found in asparagus (Asparagus officinalis L.) mesophyll cultures. The pentapeptide [phytosulfokine-α (PSK-α)] very strongly stimulated colony formation of rice protoplasts at concentrations above 10⁻⁸ M, indicating a similar mode of action in rice of phytosulfokines. Binding assays using ³⁵S-labeled PSK-α demonstrated the existence of both high- and low-affinity specific saturable binding sites on the surface of rice cells in suspension. Analysis of [³⁵S]PSK-α binding in differential centrifugation fractions suggested association of the binding with a plasma membrane-enriched fraction. The apparent K_d values for [³⁵S]PSK-α binding were found to be 1 × 10⁻⁹ M for the high-affinity type and 1 × 10⁻⁷ M for the low-affinity type, with maximal numbers of binding sites of 1 × 10⁴ sites per cell and 1 × 10⁵ sites per cell, respectively. Competition studies with [³⁵S]PSK-α and several synthetic PSK-α analogs demonstrated that only peptides that possesses mitogenic activity can effectively displace the radioligand. These results suggest that a signal transduction pathway mediated by peptide factors is involved in plant cell proliferation.     

A point mutation in the γ2 subunit of γ-aminobutyric acid type A receptors results in altered benzodiazepine binding site specificity

Benzodiazepines allosterically modulate γ-aminobutyric acid (GABA) evoked chloride currents of γ-aminobutyric acid type A (GABA_A) receptors. Coexpression of either rat γ₂ or γ₃, in combination with α₁ and β₂ subunits, results both in receptors displaying high [³H]Ro 15-1788 affinity. However, receptors containing a γ₃ subunit display a 178-fold reduced affinity to zolpidem as compared with γ₂-containing receptors. Eight chimeras between γ₂ and γ₃ were constructed followed by nine different point mutations in γ₂, each to the homologous amino acid residue found in γ₃. Chimeric or mutant γ subunits were coexpressed with α₁ and β₂ in human embryonic kidney 293 cells to localize amino acid residues responsible for the reduced zolpidem affinity. Substitution of a methionine-to-leucine at position 130 of γ₂ (γ₂M130L) resulted in a 51-fold reduction in zolpidem affinity whereas the affinity to [³H]Ro 15-1788 remained unchanged. The affinity for diazepam was only decreased by about 2-fold. The same mutation resulted in a 9-fold increase in Cl 218872 affinity. A second mutation (γ₂M57I) was found to reduce zolpidem affinity by about 4-fold. Wild-type and γ₂M130L-containing receptors were functionally expressed in Xenopus oocytes. Upon mutation allosteric coupling between agonist and modulatory sites is preserved. Dose–response curves for zolpidem and for diazepam showed that the zolpidem but not the diazepam apparent affinity is drastically reduced. The apparent GABA affinity is not significantly affected by the γ₂M130L mutation. The identified amino acid residues may define part of the benzodiazepine binding pocket of GABA_A receptors. As the modulatory site in the GABA_A receptor is homologous to the GABA site, and to all agonist sites of related receptors, γ₂M130 may either point to a homologous region important for agonist binding in all receptors or define a new region not underlying this principle.     

[3Fe-4S] to [4Fe-4S] cluster conversion in Desulfovibrio fructosovorans [NiFe] hydrogenase by site-directed mutagenesis

The role of the high potential [3Fe-4S]^1+,0 cluster of [NiFe] hydrogenase from Desulfovibrio species located halfway between the proximal and distal low potential [4Fe-4S]^2+,1+ clusters has been investigated by using site-directed mutagenesis. Proline 238 of Desulfovibrio fructosovorans [NiFe] hydrogenase, which occupies the position of a potential ligand of the lacking fourth Fe-site of the [3Fe-4S] cluster, was replaced by a cysteine residue. The properties of the mutant enzyme were investigated in terms of enzymatic activity, EPR, and redox properties of the iron-sulfur centers and crystallographic structure. We have shown on the basis of both spectroscopic and x-ray crystallographic studies that the [3Fe-4S] cluster of D. fructosovorans hydrogenase was converted into a [4Fe-4S] center in the P238 mutant. The [3Fe-4S] to [4Fe-4S] cluster conversion resulted in a lowering of approximately 300 mV of the midpoint potential of the modified cluster, whereas no significant alteration of the spectroscopic and redox properties of the two native [4Fe-4S] clusters and the NiFe center occurred. The significant decrease of the midpoint potential of the intermediate Fe-S cluster had only a slight effect on the catalytic activity of the P238C mutant as compared with the wild-type enzyme. The implications of the results for the role of the high-potential [3Fe-4S] cluster in the intramolecular electron transfer pathway are discussed.     

Cyp7b, a novel brain cytochrome P450, catalyzes the synthesis of neurosteroids 7α-hydroxy dehydroepiandrosterone and 7α-hydroxy pregnenolone

Steroids produced locally in brain (neurosteroids), including dehydroepiandrosterone (DHEA), influence cognition and behavior. We previously described a novel cytochrome P450, Cyp7b, strongly expressed in rat and mouse brain, particularly in hippocampus. Cyp7b is most similar to steroidogenic P450s and potentially could play a role in neurosteroid metabolism. To examine the catalytic activity of the enzyme mouse Cyp7b cDNA was introduced into a vaccinia virus vector. Extracts from cells infected with the recombinant showed NADPH-dependent conversion of DHEA (K_m, 13.6 μM) and pregnenolone (K_m, 4.0 μM) to slower migrating forms on thin layer chromatography. The expressed enzyme was less active against 25-hydroxycholesterol, 17β-estradiol and 5α-androstane-3β,17β-diol, with low to undetectable activity against progesterone, corticosterone, and testosterone. On gas chromatography and mass spectrometry of the Cyp7b metabolite of DHEA the retention time and fragmentation patterns were identical to those obtained with authentic 7α-hydroxy DHEA. The reaction product also comigrated on thin layer chromatography with 7α-hydroxy DHEA but not with 7β-hydroxy DHEA; when [7α-³H]pregnenolone was incubated with Cyp7b extracts the extent of release of radioactivity into the medium suggested that hydroxylation was preferentially at the 7α position. Brain extracts also efficiently liberated tritium from [7α-³H]pregnenolone and converted DHEA to a product with a chromatographic mobility indistinguishable from 7α-hydroxy DHEA. We conclude that Cyp7b is a 7α-hydroxylase participating in the synthesis, in brain, of neurosteroids 7α-hydroxy DHEA, and 7α-hydroxy pregnenolone.     

Ca2+-independent inhibition of inositol trisphosphate receptors by calmodulin: Redistribution of calmodulin as a possible means of regulating Ca2+ mobilization

The interactions between calmodulin, inositol 1,4,5-trisphosphate (InsP₃), and pure cerebellar InsP₃ receptors were characterized by using a scintillation proximity assay. In the absence of Ca²⁺, ¹²⁵I-labeled calmodulin reversibly bound to multiple sites on InsP₃ receptors and Ca²⁺ increased the binding by 190% ± 10%; the half-maximal effect occurred when the Ca²⁺ concentration was 184 ± 14 nM. In the absence of Ca²⁺, calmodulin caused a reversible, concentration-dependent (IC₅₀ = 3.1 ± 0.2 μM) inhibition of [³H]InsP₃ binding by decreasing the affinity of the receptor for InsP₃. This effect was similar at all Ca²⁺ concentrations, indicating that the site through which calmodulin inhibits InsP₃ binding has similar affinities for calmodulin and Ca²⁺-calmodulin. Calmodulin (10 μM) inhibited the Ca²⁺ release from cerebellar microsomes evoked by submaximal, but not by maximal, concentrations of InsP₃. Tonic inhibition of InsP₃ receptors by the high concentrations of calmodulin within cerebellar Purkinje cells may account for their relative insensitivity to InsP₃ and limit spontaneous activation of InsP₃ receptors in the dendritic spines. Inhibition of InsP₃ receptors by calmodulin at all cytosolic Ca²⁺ concentrations, together with the known redistribution of neuronal calmodulin evoked by protein kinases and Ca²⁺, suggests that calmodulin may also allow both feedback control of InsP₃ receptors and integration of inputs from other signaling pathways.     

Promiscuous coassembly of serotonin 5-HT3 and nicotinic α4 receptor subunits into Ca2+-permeable ion channels

Serotonin (5-hydroxytryptamine) type 3 receptors (5-HT₃R) and nicotinic acetylcholine receptors are structurally and functionally related proteins, yet distinct members of the family of ligand-gated ion channels. For most members of this family a diversity of heteromeric receptors is known at present. In contrast, known 5-HT₃R subunits are all homologs of the same 5-HT₃R-A subunit and form homopentameric receptors. Here we show, by heterologous expression followed by immunoprecipitation, that 5-HT₃R and nicotinic acetylcholine receptor α4 subunits coassemble into a novel type of heteromeric ligand-gated ion channel, which is activated by 5-HT. The Ca²⁺ permeability of this heteromeric ion channel is enhanced as compared with that of the homomeric 5-HT₃R channel. Heteromeric 5-HT₃/α4 and homomeric 5-HT₃Rs have similar pharmacological profiles, but distinct sensitivities to block by the antagonist d-tubocurarine. Coassembly of subunits beyond the boundaries of ligand-gated ion channel families may constitute an important mechanism contributing to the diverse properties and functions of native neurotransmitter receptors.     

Fatty acid-induced β cell apoptosis: A link between obesity and diabetes

Like obese humans, Zucker diabetic fatty (ZDF) rats exhibit early β cell compensation for insulin resistance (4-fold β cell hyperplasia) followed by decompensation (>50% loss of β cells). In prediabetic and diabetic ZDF islets, apoptosis measured by DNA laddering is increased 3- and >7-fold, respectively, compared with lean ZDF controls. Ceramide, a fatty acid-containing messenger in cytokine-induced apoptosis, was significantly increased (P < 0.01) in prediabetic and diabetic islets. Free fatty acids (FFAs) in plasma are high (>1 mM) in prediabetic and diabetic ZDF rats; therefore, we cultured prediabetic islets in 1 mM FFA. DNA laddering rose to 19.6% vs. 4.6% in lean control islets, preceded by an 82% increase in ceramide. C₂-Ceramide without FFA induced DNA laddering, but fumonisin B₁, a ceramide synthetase inhibitor, completely blocked FFA-induced DNA laddering in cultured ZDF islets. [³H]Palmitate incorporation in [³H]ceramide in ZDF islets was twice that of controls, but [³H]palmitate oxidation was 77% less. Triacsin C, an inhibitor of fatty acyl-CoA synthetase, and troglitazone, an enhancer of FFA oxidation in ZDF islets, both blocked DNA laddering. These agents also reduced inducible nitric oxide (NO) synthase mRNA and NO production, which are involved in FFA-induced apoptosis. In ZDF obesity, β cell apoptosis is induced by increased FFA via de novo ceramide formation and increased NO production.     

A single serine residue controls the cation dependence of substrate transport by the rat serotonin transporter

The serotonin transporter (SERT) is a member of the Na⁺/Cl⁻-dependent neurotransmitter transporter family and constitutes the target of several clinically important antidepressants. Here, replacement of serine-545 in the recombinant rat SERT by alanine was found to alter the cation dependence of serotonin uptake. Substrate transport was now driven as efficiently by LiCl as by NaCl without significant changes in serotonin affinity. Binding of the antidepressant [³H]imipramine occurred with 1/5th the affinity, whereas [³H]citalopram binding was unchanged. These results indicate that serine-545 is a crucial determinant of both the cation dependence of serotonin transport by SERT and the imipramine binding properties of SERT.     

Overexpression of the SUP45 gene encoding a Sup35p-binding protein inhibits the induction of the de novo appearance of the [PSI+] prion

[PSI⁺], a non-Mendelian element found in some strains of Saccharomyces cerevisiae, is presumed to be the manifestation of a self-propagating prion conformation of eRF3 (Sup35p). Translation termination factor eRF3 enhances the activity of release factor eRF1 (Sup45p). As predicted by the prion model, overproduction of Sup35p induces the de novo appearance of [PSI⁺]. However, another non-Mendelian determinant, [PIN⁺], is required for this induction. We now show that SUP45 overexpression inhibits the induction of [PSI⁺] by Sup35p overproduction in [PIN⁺] strains, but has no effect on the propagation of [PSI⁺] or on the [PIN] status of the cells. We also show that SUP45 overexpression counteracts the growth inhibition usually associated with overexpression of SUP35 in [PSI⁺] strains. We argue that excess Sup45p inhibits [PSI⁺] seed formation. Because Sup45p complexes with Sup35p, we hypothesize that excess Sup45p may sequester Sup35p, thereby reducing the opportunity for Sup35p conformational flips and/or self-interactions leading to prion formation. This in vivo yeast result is reminiscent of the in vitro finding by investigators of Alzheimer disease that apolipoprotein E inhibits amyloid nucleation, but does not reduce seeded growth of amyloid.     

5-HT4 and 5-HT2 receptors antagonistically influence gap junctional coupling between rat auricular myocytes

5-hydroxytryptamine-4 (5-HT₄) receptors have been proposed to contribute to the generation of atrial fibrillation in human atrial myocytes, but it is unclear if these receptors are present in the hearts of small laboratory animals (e.g. rat). In this study, we examined presence and functionality of 5-HT₄ receptors in auricular myocytes of newborn rats and their possible involvement in regulation of gap junctional intercellular communication (GJIC, responsible for the cell-to-cell propagation of the cardiac excitation). Western-blotting assays showed that 5-HT₄ receptors were present and real-time RT-PCR analysis revealed that 5-HT_4b was the predominant isoform. Serotonin (1 μM) significantly reduced cAMP concentration unless a selective 5-HT₄ inhibitor (GR113808 or ML10375, both 1 μM) was present. Serotonin also reduced the amplitude of L-type calcium currents and influenced the strength of GJIC without modifying the phosphorylation profiles of the different channel-forming proteins or connexins (Cxs), namely Cx40, Cx43 and Cx45. GJIC was markedly increased when serotonin exposure occurred in presence of a 5-HT₄ inhibitor but strongly reduced when 5-HT_2A and 5-HT_2B receptors were inhibited, showing that activation of these receptors antagonistically regulated GJIC. The serotoninergic response was completely abolished when 5-HT₄, 5-HT_2A and 5-HT_2B were simultaneously inhibited. A 24 h serotonin exposure strongly reduced Cx40 expression whereas Cx45 was less affected and Cx43 still less. In conclusion, this study revealed that 5-HT₄ (mainly 5-HT_4b), 5-HT_2A and 5-HT_2B receptors coexisted in auricular myocytes of newborn rat, that 5-HT₄ activation reduced cAMP concentration, I_CaL and intercellular coupling whereas 5-HT_2A or 5-HT_2B activation conversely enhanced GJIC.     

The [URE3] prion is an aggregated form of Ure2p that can be cured by overexpression of Ure2p fragments

The [URE3] nonchromosomal genetic element is a prion of Ure2p, a regulator of nitrogen catabolism in Saccharomyces cerevisiae. Ure2p^1–65 is the prion domain of Ure2p, sufficient to propagate [URE3] in vivo. We show that full length Ure2p–green fluorescent protein (GFP) or a Ure2p^1–65-GFP fusion protein is aggregated in cells carrying [URE3] but is evenly distributed in cells lacking the [URE3] prion. This indicates that [URE3] involves a self-propagating aggregation of Ure2p. Overexpression of Ure2p^1–65 induces the de novo appearance of [URE3] by 1,000-fold in a strain initially [ure-o], but cures [URE3] from a strain initially carrying the [URE3] prion. Overexpression of several other fragments of Ure2p or Ure2-GFP fusion proteins also efficiently cures the prion. We suggest that incorporation of fragments or fusion proteins into a putative [URE3] “crystal” of Ure2p poisons its propagation.     

UDP activates a mucosal-restricted receptor on human nasal epithelial cells that is distinct from the P2Y2 receptor

The presence of the P2Y₂ (P_2U-purinergic) receptor on the apical surface of airway tissue raises the possibility that aerosolized UTP might be used therapeutically to induce Cl⁻ secretion in individuals with cystic fibrosis. However, the duration of the effects of UTP may be limited by enzymatic degradation. We therefore have analyzed the metabolism of UTP and its metabolite UDP on polarized human nasal epithelium (HNE), and have compared the pharmacological activities of these two uridine nucleotides. HPLC analysis of medium bathing the mucosal surface of HNE cells revealed the presence of an ecto-nucleotidase(s) that hydrolyzed [³H]UTP and [³H]UDP with t_½ values (at 1 μM nucleotide) of 14 and 27 min, respectively. An ecto-nucleoside diphosphokinase activity also was observed, which promoted conversion of [³H]UDP into [³H]UTP in the presence of ATP. The effects of UDP on [³H]inositol phosphate accumulation, intracellular calcium levels ([Ca²⁺]_i), and Cl⁻ secretory rates (I_Cl⁻) were quantitated in HNE cells in the presence of hexokinase and glucose to ensure that no UTP (or ATP) contaminated UDP solutions during the assays. Although UDP does not activate the human P2Y₂ receptor, mucosal addition of UDP promoted [³H]inositol phosphate accumulation with an EC₅₀ of 190 nM. Mucosal addition of UTP stimulated [³H]inositol phosphate accumulation with an EC₅₀ of 280 nM. The maximal effects of mucosal UDP on [³H]inositol phosphate, [Ca²⁺]_i, and I_Cl⁻ responses were approximately one-half of those observed with mucosal UTP. Serosal application of UTP promoted a 50% greater [³H]inositol phosphate and calcium response than did mucosal application of UTP. In contrast, UDP had no effect when added to the serosal medium. Repetitive mucosal applications of UDP to HNE cells resulted in a progressive loss, i.e., desensitization, of the [Ca²⁺]_i and I_Cl⁻ response to UDP, whereas the corresponding responses to UTP remained unchanged. Our results provide evidence for the existence of a UDP receptor on HNE cells that is pharmacologically distinct from the P2Y₂ receptor. The relative stability of UDP on the airway surface and the apparent predominant mucosal expression of this putative UDP receptor make it a potential target for cystic fibrosis treatment.     

v-K-ras leads to preferential farnesylation of p21ras in FRTL-5 cells: Multiple interference with the isoprenoid pathway

The isoprenoid pathway in FRTL-5 thyroid cells was found to be deeply altered on transformation with v-K-ras. A dramatic overall reduction of protein prenylation was found in v-K-ras-transformed cells in comparison with the parent FRTL-5 cells, as shown by labeling cells with [³H]mevalonic acid. This phenomenon was accompanied by a relative increase of p21^ras farnesylation and by a decrease of the ratio between the amounts of geranylgeraniol and farnesol bound to prenylated proteins. Analysis of protein prenylation in FRTL-5 cells transformed by a temperature-sensitive mutant of the v-K-ras oncogene indicated that these variations represent an early and specific marker of active K-ras. Conversely, FRTL-5 cells transformed with Harvey-ras showed a pattern of [³H]-mevalonate (MVA)-labeled proteins similar to that of nontransformed cells. The K-ras oncogene activation also resulted in an overall decrease of [³H]-MVA incorporation into isopentenyl-tRNA together with an increase of unprocessed [³H]-MVA and no alteration in [³H]-MVA uptake. The effects of v-K-ras on protein prenylation could be mimicked in FRTL-5 cells by lowering the concentration of exogenous [³H]-MVA whereas increasing the [³H]-MVA concentration did not revert the alterations observed in transformed cells. Accordingly, v-K-ras expression was found to: (i) down-regulate mevalonate kinase; (ii) induce farnesyl-pyrophosphate synthase expression; and (iii) augment protein farnesyltransferase but not protein geranylgeranyl-transferase-I activity. Among these events, mevalonate kinase down-regulation appeared to be related strictly to differential protein prenylation. This study represents an example of how expression of the v-K-ras oncogene, through multiple interferences with the isoprenoid metabolic pathway, may result in the preferential farnesylation of the ras oncogene product p21^ras.     

Gsα-selective G protein antagonists

Suramin acts as a G protein inhibitor because it inhibits the rate-limiting step in activation of the G_α subunit, i.e., the exchange of GDP for GTP. Here, we have searched for analogues that are selective for G_sα. Two compounds have been identified: NF449 (4,4′,4",4′"-[carbonyl-bis[imino-5,1,3-benzenetriyl bis-(carbonylimino)]]tetrakis-(benzene-1,3-disulfonate) and NF503 (4,4′-[carbonylbis[imino-3,1-phenylene-(2,5-benzimidazolylene)carbonylimino]]bis-benzenesulfonate). These compounds (i) suppress the association rate of guanosine 5′-[γ-thio]triphosphate ([³⁵S]GTP[γS]) binding to G_sα-s but not to G_iα-1, (ii) inhibit stimulation of adenylyl cyclase activity in S49 cyc⁻ membranes (deficient in endogenous G_sα) by exogenously added G_sα-s, and (iii) block the coupling of β-adrenergic receptors to G_s with half-maximum effects in the low micromolar range. In contrast to suramin, which is not selective, NF503 and NF449 disrupt the interaction of the A₁-adenosine receptor with its cognate G proteins (G_i/G_o) at concentrations that are >30-fold higher than those required for uncoupling of β-adrenergic receptor/G_s tandems; similarly, the angiotensin II type-1 receptor (a prototypical G_q-coupled receptor) is barely affected by the compounds. Thus, NF503 and NF449 fulfill essential criteria for G_sα-selective antagonists. The observations demonstrate the feasibility of subtype-selective G protein inhibition.     

Formation of stable adducts and absence of depurinating DNA adducts in cells and DNA treated with the potent carcinogen dibenzo[a,l]pyrene or its diol epoxides

Polycyclic aromatic hydrocarbons (PAH) are widespread environmental contaminants, and some are potent carcinogens in rodents. Carcinogenic PAH are activated in cells to metabolites that react with DNA to form stable covalent DNA adducts. It has been proposed [Cavalieri, E. L. & Roger, E. G. (1995) Xenobiotica 25, 677–688] that unstable DNA adducts are also formed and that apurinic sites in the DNA resulting from unstable PAH adducts play a key role in the initiation of cancer. The potent carcinogen dibenzo[a,l]pyrene (DB[a,l]P) is activated in cells to (+)-syn- and (−)-anti-DB[a,l]P-11,12-diol-13,14-epoxide (DB[a,l]PDE), which have been shown to form stable adducts with DNA. To evaluate the importance of unstable PAH adducts, we compared stable adduct formation to apurinic site formation. Stable DB[a,l]PDE adducts were determined by ³³P-postlabeling and HPLC. To measure apurinic sites they were converted to strand breaks, and these were monitored by examining the integrity of a particular restriction fragment of the dihydrofolate reductase gene. The method easily detected apurinic sites resulting from methylation by treatment of cells or DNA with dimethyl sulfate or from reaction of DNA with DB[a,l]P in the presence of horseradish peroxidase. We estimate the method could detect 0.1 apurinic site in the 14-kb fragment examined. However, apurinic sites were below our limit of detection in DNA treated directly with (+)-syn- or (−)-anti-DB[a,l]PDE or in DNA from Chinese hamster ovary B11 cells so treated, although in these samples the frequency of stable adducts ranged from 3 to 10 per 14 kb. We also treated the human mammary carcinoma cell line MCF-7 with DB[a,l]P and again could not detect significant amounts of unstable adducts. These results indicate that the proportion of stable adducts formed by DB[a,l]P activated in cells and its diol epoxides is greater than 99% and suggest a predominant role for stable DNA adducts in the carcinogenic activity of DB[a,l]P.     

Cytohesin-1, a cytosolic guanine nucleotide-exchange protein for ADP-ribosylation factor

Cytohesin-1, a protein abundant in cells of the immune system, has been proposed to be a human homolog of the Saccharomyces cerevisiae Sec7 gene product, which is crucial in protein transport. More recently, the same protein has been reported to be a regulatory factor for the αLβ2 integrin in lymphocytes. Overexpression of human or yeast ADP-ribosylation factor (ARF) genes rescues yeast with Sec7 defects, restoring secretory pathway function. ARFs, 20-kDa guanine nucleotide-binding proteins initially identified by their ability to stimulate cholera toxin ADP-ribosyltransferase activity and now recognized as critical components in intracellular vesicular transport, exist in an inactive cytosolic form with GDP bound (ARF-GDP). Interaction with a guanine nucleotide-exchange protein (GEP) accelerates exchange of GDP for GTP, producing the active ARF-GTP. Both soluble and particulate GEPs have been described. To define better the interaction between ARF and Sec7-related proteins, effects of cytohesin-1, synthesized in Escherichia coli, on ARF activity were evaluated. Cytohesin-1 enhanced binding of ³⁵S-labeled guanosine 5′-[γ-thio]triphosphate [³⁵S]GTP[γS] or [³H]GDP to ARF purified from bovine brain (i.e., it appeared to function as an ARF-GEP). Addition of cytohesin-1 to ARF3 with [³⁵S]GTP[γS] bound, accelerated [³⁵S]GTP[γS] release to a similar degree in the presence of unlabeled GDP or GTP[γS] and to a lesser degree with GDP[βS]; release was negligible without added nucleotide. Cytohesin-1 also increased ARF1 binding to a Golgi fraction, but its effect was not inhibited by brefeldin A (BFA), a drug that reversibly inhibits Golgi function. In this regard, it differs from a recently reported BFA-sensitive ARF-GEP that contains a Sec7 domain.     

Evidence for two nonidentical drug-interaction sites in the human P-glycoprotein

Human P-glycoprotein (Pgp) confers multidrug resistance to cancer cells by ATP-dependent extrusion of a great many structurally dissimilar hydrophobic compounds. The manner in which Pgp recognizes these different substrates is unknown. The protein shows internal homology between its N- and C-terminal halves, each comprised of six putative transmembrane helices and a consensus ATP binding/utilization site. Photoactive derivatives of certain Pgp substrates specifically label two regions, one on each half of the protein. In this study, using [¹²⁵I]iodoarylazidoprazosin ([¹²⁵I]IAAP), a photoactive analog of prazosin, we have demonstrated the presence of two nonidentical drug-interaction sites within Pgp. Taking advantage of a highly susceptible trypsin cleavage site in the linker region of Pgp, we characterized the [¹²⁵I]IAAP binding to the N- and C-terminal halves. cis(Z)-Flupentixol, a modulator of Pgp function, preferentially increased the affinity of [¹²⁵I]IAAP for the C-terminal half of the protein (C-site) by reducing the K_d from 20 to 6 nM without changing the labeling or affinity (K_d = 42–46 nM) of the N-terminal half (N-site). Also, the concentration of vinblastine (Pgp substrate) and cyclosporin A (Pgp modulator) required for 50% inhibition of [¹²⁵I]IAAP binding to the C-site was increased 5- to 6-fold by cis(Z)-flupentixol without any effect on the N-site. In addition, [¹²⁵I]IAAP binding to the N-site was less susceptible than to C-site to inhibition by vanadate which blocks ATP hydrolysis and drug transport. These data demonstrate the presence of at least two nonidentical substrate interaction sites in Pgp.