Structural determinants in the second intracellular loop of the human cannabinoid CB1 receptor mediate selective coupling to Gs and Gi

BACKGROUND AND PURPOSE

The cannabinoid CB₁ receptor is primarily thought to be functionally coupled to the G_i form of G proteins, through which it negatively regulates cAMP accumulation. Here, we investigated the dual coupling properties of CB₁ receptors and characterized the structural determinants that mediate selective coupling to G_s and G_i.

EXPERIMENTAL APPROACH

A cAMP-response element reporter gene system was employed to quantitatively analyze cAMP change. CB₁/CB₂ receptor chimeras and site-directed mutagenesis combined with functional assays and computer modelling were used to determine the structural determinants mediating selective coupling to G_s and G_i.

KEY RESULTS

CB₁ receptors could couple to both G_s-mediated cAMP accumulation and G_i-induced activation of ERK1/2 and Ca²⁺ mobilization, whereas CB₂ receptors selectively coupled to G_i and inhibited cAMP production. Using CB₁/CB₂ chimeric receptors, the second intracellular loop (ICL2) of the CB₁ receptor was identified as primarily responsible for mediating G_s and G_i coupling specificity. Furthermore, mutation of Leu-222 in ICL2 to either Ala or Pro switched G protein coupling from G_s to G_i, while to Ile or Val led to balanced coupling of the mutant receptor with G_s and G_i.

CONCLUSIONS AND IMPLICATIONS

The ICL2 of CB₁ receptors and in particular Leu-222, which resides within a highly conserved DRY(X)₅PL motif, played a critical role in G_s and G_i protein coupling and specificity. Our studies provide new insight into the mechanisms governing the coupling of CB₁ receptors to G proteins and cannabinoid-induced tolerance.     

Chronic exposure of rat glioma C6 cells to cholera toxin induces loss of the α-subunit of the stimulatory guanine nucleotide-binding protein (Gs)

Rat glioma C6 BU1 cells were treated in tissue culture with cholera toxin. Incubation of membranes derived from these cells with fresh cholera toxin and [³²P]NAD⁺ failed to promote incorporation of radioactivity into polypeptides corresponding to forms of G_sα. This is generally assumed to reflect prior ADP ribosylation of these polypeptides in vivo using endogenouss NAD⁺ as substrate. However, immunological studies with anti-peptide antisera which identify all forms of G_sα demonstrated that concentrations of this polypeptide were now substantially reduced in the membranes. This effect was specific for G_sα as neither the α-subunits of the pertussis toxin-sensitive G-proteins G_i2 and G_i3, nor the β subunit common to the various G-proteins were lost in parallel. Pertussis toxin-catalysed ADP ribosylation did not cause the downregulation of G_sα nor of the α-subunits of G_i2 or G_i3 although it did cause ADP ribosylation of the entire complement of both G_i2 and G_i3 in the membranes. Despite the reduction in levels of immunoreactive G_sα from the membranes of cholera toxin-treated cells, no alterations in levels of MRNA corresponding to this G-protein were noted.     

Molecular modeling of salsolinol,a full Gi protein agonist of the μ‐opioid receptor,within the receptor binding site

(R/S)‐Salsolinol is a full agonist of the μ‐opioid receptor (μOR) G_i protein pathway via its (S)‐enantiomer and is functionally selective as it does not promote β‐arrestin recruitment. Compared to (S)‐salsolinol, the (R)‐enantiomer is a less potent agonist of the G_i protein pathway. We have now studied the interactions of the salsolinol enantiomers docked in the binding pocket of the μOR to determine the molecular interactions that promote enantiomeric specificity and functional selectivity of (R/S)‐salsolinol. Molecular dynamics simulations showed that (S)‐salsolinol interacted with 8 of the 11 residues of the μOR binding site, enough to stabilize the molecule. (R)‐Salsolinol showed higher mobility with fewer prevalent bonds. Hence, the methyl group bound to the (S)‐stereogenic center promoted more favorable interactions in the μOR binding site than in the (R)‐orientation. Because (S)‐salsolinol is a small molecule (179.2 Da), it did not interact with residues implicated in the binding of larger morphinan agonists that are located toward the extracellular portion of the binding pocket: W318^7.35, I322^7.39, and Y326^7.43. Our results suggest that contact with residues which (S)‐salsolinol interacts with are enough to elicit G_i protein activation, and possibly define a minimum set required by μOR ligands to promote activation of the G_i protein pathway.     

Nuances in the Calculation of Amorphous Solubility Enhancement Ratio

The theoretical amorphous solubility enhancement ratio (R_s) can be calculated based on the free energy difference between amorphous and crystalline forms (ΔG_x→a), using several experimentally determined input parameters. This work compares the various approaches for the calculation of R_s and explores the nuances associated with its calculation. The uncertainty of R_s values owing to experimental conditions (differential scanning calorimetry heating rates) used to measure the input parameters was determined for 3 drugs (indomethacin, itraconazole, and spironolactone). The calculated value of R_s was most influenced by the measurement of heat of fusion. The range in values of R_s using the various equations in the literature was within the calculated uncertainty of the theoretical R_s value. Still, all equations appear to overpredict the experimental value of R_s, sometimes by more than a factor of 5, when an experimental value is attainable. Methods for the calculation of ΔG_x→a for molecules undergoing additional phase transitions (other than glass transition and melting) were developed, employing itraconazole as a model drug. In addition, the influences of enthalpy relaxation and entropy of mixing for racemic compounds on R_s were also considered. These additional corrections improved agreement between theoretical and experimental R_s.     

G-Protein-coupled receptors in HL-60 human leukemia cells

• 1.1. HL-60 human leukemia cells are a widely employed model system for the analysis of signal transduction processes mediated via regulatory heterotrimeric guanine nucleotide-binding proteins (G-proteins). HL-60 promyelocytes are pluripotent and can be differentiated into neutrophilic or monocytic cells.
• 2.2. HL-60 cells express formyl peptide-, complement C5a-, leukotriene B₄ (LTB₄)- and platelet-activating factor receptors, receptors for purine and pyrimidine nucleotides, histamine H₁- and H₂-receptors, β₂-adrenoceptors and prostaglandin receptors.
• 3.3. The major G-proteins in HL-60 cells are pertussis toxin (PTX)-sensitive G_i-proteins (G_i2 > G_i3). G_s-proteins and G-proteins of the G_q-family (e.g., G₁₆) are expressed, too.
• 4.4. G-protein-regulated effector systems in HL-60 cells are adenylyl cyclase and phospholipase C-β₂ (PLC-β₂) and, possibly, phospholipase D (PLD), nonselective cation (NSC) channels and NADPH oxidase.
• 5.5. The expression of signal transduction pathways in HL-60 cells strongly depends on the differentiation state of cells.
• 6.6. Formyl peptides, via G_i-proteins, mediate activation of PLC, PLD, NSC channels, NADPH oxidase and azurophilic granule release and are referred to as full secretagogues. In dibutyryl cAMP (Bt₂cAMP)-differentiated HL-60 cells, C5a and LTB₄ are partial and incomplete secretagogues, respectively. There are substantial differences in the G_i-protein activations induced by formyl peptides, C5a and LTB₄.
• 7.7. In HL-60 promyelocytes, purine and pyrimidine nucleotides mediate activation of PLC and NSC channels largely via PTX-insensitive G-proteins and induce functional differentiation. In Bt₂cAMP-differentiated HL-60 cells, they additionally activate PLD, NADPH oxidase and granule release via PTX-sensitive and -insensitive pathways. ATP and UTP are partial secretagogues. Multiple types of receptors (i.e., P_2Y- and P_2U-receptors and pyrimidinocyeptors) may mediate the effects of nucleotides in HL-60 cells.
• 8.8. Bt₂cAMP- and 1α,25-dihydroxycholecalciferol-differentiated HL-60 cells express H_l-receptors coupled to G_i-proteins and PTX-insensitive G-proteins. In the former cells, histamine mediates activation of PLC and NSC channels, and in the latter, activation of NSC channels. Histamine is an incomplete secretagogue in these cells.
• 9.9. HL-60 promyelocytes express H₂-receptors coupled to adenylyl cyclase, PLC, and NSC channels. There are substantial differences in the agonist/antagonist profiles of H₂-receptor-mediated cAMP formation and rises in cytosolic Ca²⁺ concentration, indicative of the involvement of different H₂-receptor subtypes. H₂-receptors mediate functional differentiation of HL-60 cells.
• 10.10. Certain cationic-amphiphilic histamine receptor ligands (i.e., 2-substituted histamines, lipophilic guanidines, and a histamine trifluoromethyl-toluidide derivative) show stimulatory effects in HL-60 cells that are attributable to receptor-independent activation of G_i-proteins.

     

Tumor necrosis factor α up-regulates Giα and Gß proteins and adenylyl cyclase responsiveness in rat cardiomyocytes

Treatment of cultured rat cardiomyocytes in serum-free medium for 48 h with recombinant human tumor necrosis factor α (TNFα) led to a concentration-dependent increase in the level of membrane-inhibitory guanine nucleotide-binding protein (G₁) α-subunits and in pertussis toxin-catalyzed [³²P]ADP ribosylation of 40 kDa proteins. Both G_iα protein subtypes present in rat cardiac myocyte membranes, G_iα40 and G_iα41, were up-regulated by the cytokine, with the maximal increase occurring at 10 U/ml TNFα. In contrast to noradrenaline exposure, which causes a similar, but apparently exclusive, increase in α₁-subunits, treatment with TNFα in addition increased the level of membrane G protein ß₃₆-subunits. Furthermore, while noradrenaline exposure led to a decrease in receptor-dependent and -independent adenylyl cyclase activity, treatment of cardiomyocytes with TNFα caused a concentration-dependent increase in cyclase responsiveness to either forskolin, guanosine 5′-O-(3-thiotriphosphate) or isoproterenol, even though ß-adrenoceptor density was decreased by TNFα. The increase in adenylyl cyclase activity induced by TNFα was completely suppressed when the cells were cocultured with noradrenaline, a condition leading to an additive increase in G_iα level. The data indicate that the cytokine TNFα can potently modulate G protein-mediated signal transduction in rat cardiac myocytes. Although TNFα, like noradrenaline, exposure of the cells increased the level of membrane G_iα proteins, it did not decrease but rather caused an increase in adenylyl cyclase responsiveness. This hypertensitivity may be due to concomitant alterations of other components, e.g. G_ß, G_sα and/or the cyclase itself, of this multi-subunit signal transduction system following TNFα exposure.     

G12/13-dependent signaling of G-protein-coupled receptors: disease context and impact on drug discovery

G-protein-coupled receptors (GPCRs) transmit extracellular signals across the plasma membrane via intracellular activation of heterotrimeric G proteins. The signal transduction pathways of G_s, G_i and G_q protein families are widely studied, whereas signaling properties of G₁₂ proteins are only emerging. Many GPCRs were found to couple to G_12/13 proteins in addition to coupling to one or more other types of G proteins. G_12/13 proteins couple GPCRs to activation of the small monomeric GTPase RhoA. Activation of RhoA modulates various downstream effector systems relevant to diseases such as hypertension, artherosclerosis, asthma and cancer. GPCR screening assays exist for G_s-, G_i- and G_q-linked pathways, whereas a drug-screening assay for the G₁₂-Rho pathway was developed only recently. The review gives an overview of the present understanding of the G_12/13-related biology of GPCRs.     

Antinociceptive Effects of Racemic AM1241 and Its Chirally Synthesized Enantiomers: Lack of Dependence upon Opioid Receptor Activation

Cannabinoid CB₂ receptors represent a therapeutic target that circumvents unwanted central side effects (e.g., psychoactivity and/or addiction) associated with activation of CB₁ receptors. One of the primary investigative tools used to study functions of the CB₂ receptor is the aminoalkylindole (R,S)-AM1241. However, (R,S)-AM1241 has been described as an atypical CB₂ agonist which produces antinociception mediated indirectly by opioid receptors. (R,S)-AM1241 and its enantiomers, (R)-AM1241 and (S)-AM1241, were evaluated for antinociception in response to thermal (Hargreaves) and mechanical (von Frey) stimulation. Pharmacological specificity was established using antagonists for CB₁ (rimonabant [SR141716]) and CB₂ (SR144528). The opioid antagonist naloxone was administered locally in the paw or systemically to evaluate the contribution of opioid receptors to CB₂-mediated antinociception produced by (R,S)-AM1241, (R)-AM1241, and (S)-AM1241. Comparisons were made with the opioid analgesic morphine. (R,S)-AM1241, (R)-AM1241, and (S)-AM1241 (0.033–10 mg/kg i.p.) produced antinociception to thermal, but not mechanical, stimulation of the hindpaw in naive rats. Antinociception produced by (R,S)-AM1241 and (S)-AM1241 exhibited an inverted U-shaped dose response curve. (R)-AM1241 produced greater antinociception than either (S)-AM1241 or (R,S)-AM1241 at the lowest (0.033 and 0.1 mg/kg i.p.) and highest (10 mg/kg i.p.) doses. Similar levels of antinociception were observed at intermediate doses. (R,S)-AM1241, (R)-AM1241, and (S)-AM1241 each produced CB₂-mediated antinociception that was blocked by SR144528 but not by rimonabant. Local and systemic naloxone blocked morphine-induced antinociception but did not block antinociceptive effects of (R,S)-AM1241, (R)-AM1241, or (S)-AM1241. The antinociceptive effects of the CB₂-selective cannabinoid (R,S)-AM1241 and its enantiomers, (R)-AM1241 and (S)-AM1241, are not dependent upon opioid receptors.     

Lateral Diffusion of Gαs in the Plasma Membrane Is Decreased after Chronic but not Acute Antidepressant Treatment: Role of Lipid Raft and Non-Raft Membrane Microdomains

GPCR signaling is modified both in major depressive disorder and by chronic antidepressant treatment. Endogenous Gα_s redistributes from raft- to nonraft-membrane fractions after chronic antidepressant treatment. Modification of G protein anchoring may participate in this process. Regulation of Gα_s signaling by antidepressants was studied using fluorescence recovery after photobleaching (FRAP) of GFP-Gα_s. Here we find that extended antidepressant treatment both increases the half-time of maximum recovery of GFP-Gα_s and decreases the extent of recovery. Furthermore, this effect parallels the movement of Gα_s out of lipid rafts as determined by cold detergent membrane extraction with respect to both dose and duration of drug treatment. This effect was observed for several classes of compounds with antidepressant activity, whereas closely related molecules lacking antidepressant activity (eg, R-citalopram) did not produce the effect. These results are consistent with previously observed antidepressant-induced translocation of Gα_s, but also suggest an alternate membrane attachment site for this G protein. Furthermore, FRAP analysis provides the possibility of a relatively high-throughput screening tool for compounds with putative antidepressant activity.     

Different inhibition patterns of tedisamil for fast and slowly inactivating transient outward current in rat ventricular myocytes

Tedisamil has been described as a selective inhibitor of a fast inactivating transient outward current (i_to,f) in rat ventricular myocytes. Because recent reports demonstrated the existence of a second slowly inactivating transient component (i_to,s) we investigated i_to,s and differentiated the effects of tedisamil on both transient outward current components and their influence on action potential duration. Standard electrophysiological techniques were used for whole cell recordings at 24–26° C from enzymatically isolated myocytes. Inhibition of i_to,f by tedisamil was the result of an acceleration of inactivation at positive test potentials with a concentration for halfmaximal inhibition (EC₅₀) of 4–7 μmol/l, which is confirmatory to reports from other investigators. Our new results show that i_to,s is more sensitive to tedisamil with an EC₅₀ of 0.5 μmol/l. Furthermore the pattern of i_to,s inhibition is different compared with i_to,f, because inactivation of i_to,s is not accelerated by tedisamil. Instead the amplitude of the steady state inactivation curve of i_to,s is attenuated which indicates a reduction of maximally available current. I_to,s was evaluated by three different methods as time-dependently inactivating current (7.5 s test pulse duration), voltage-dependently inactivated current and tedisamil-sensitive current. All approaches yield similar inactivation curves. The potential for halfmaximal inactivation of i_to,s lies about 35 mV more negative than that for i_to,f and the slope factor (K = –23 mV) is different to that of i_to,f (K = –3 mV). Effectiveness of tedisamil-induced modulation of i_to,f and i_to,s on action potential repolarization was tested. Action potentials stimulated at 0.5 Hz were not prolonged by 1 μmol/l tedisamil (dominant i_to,s block) at a repolarization level of 0 mV but prolonged to about 120% of control at –70 mV. This indicates that i_to,f was sufficient to guarantee a regular early repolarization whereas decrease of i_to,s delayed the final repolarization. In conclusion, the observation that tedisamil inhibits i_to,f and i_to,s differently supports the hypothesis that the two i_to-components are related to two different channel populations expressed in rat ventricular myocytes. Received: 28 July 1997 / Accepted: 3 December 1997     

Marked changes in signal transduction upon heteromerization of dopamine D1 and histamine H3 receptors

Background and purpose:

Functional interactions between the G protein-coupled dopamine D₁ and histamine H₃ receptors have been described in the brain. In the present study we investigated the existence of D₁–H₃ receptor heteromers and their biochemical characteristics.

Experimental approach:

D₁–H₃ receptor heteromerization was studied in mammalian transfected cells with Bioluminescence Resonance Energy Transfer and binding assays. Furthermore, signalling through mitogen-activated protein kinase (MAPK) and adenylyl cyclase pathways was studied in co-transfected cells and compared with cells transfected with either D₁ or H₃ receptors.

Key results:

Bioluminescence Resonance Energy Transfer and binding assays confirmed that D₁ and H₃ receptors can heteromerize. Activation of histamine H₃ receptors did not lead to signalling towards the MAPK pathway unless dopamine D₁ receptors were co-expressed. Also, dopamine D₁ receptors, usually coupled to G_s proteins and leading to increases in cAMP, did not couple to G_s but to G_i in co-transfected cells. Furthermore, signalling via each receptor was blocked not only by a selective antagonist but also by an antagonist of the partner receptor.

Conclusions and implications:

D₁–H₃ receptor heteromers constitute unique devices that can direct dopaminergic and histaminergic signalling towards the MAPK pathway in a G_s-independent and G_i-dependent manner. An antagonist of one of the receptor units in the D₁–H₃ receptor heteromer can induce conformational changes in the other receptor unit and block specific signals originating in the heteromer. This gives rise to unsuspected therapeutic potentials for G protein-coupled receptor antagonists.     

Phosphorylation of G-protein α-subunits in intact adipose cells: evidence against a mediating role in insulin-dependent metabolic effects

The phosphorylation of G-protein α-subunits was studied in plasma membranes prepared from isolated, intact adipocytes equilibrated with [³²P]phosphate and subsequently incubated in the presence or absence of insulin. In iodinated or unlabeled plasma membranes, antiserum generated against a peptide corresponding to a region common to G-protein α-subunits immunoprecipitated two major proteins of 45 and 40 kDa, which were identified as G_s and G_i α-subunit, respectively, by comparison with [³²P]ADP-ribosylated G-proteins. In membranes prepared from cells equilibrated with [³²P]phosphate, the antiserum precipitated a 45 kDa phosphoprotein. Pre-immune serum failed to immunoprecipitate the phosphoprotein. Insulin stimulated [³²P]phosphate incorporation into the 45 kDa protein approximately 2-fold. Control experiments suggested that the 45 kDa phosphoprotein was not identical with Gα_s, since (1) the peptide used to raise the antiserum failed to inhibit significantly immunoprecipitation of the 45 kDa phosphoprotein with the antiserum, (2) in contrast to the G_s α-subunit, the phosphorprotein was readily removed from the immunocomplex by washing with sodium dodecyl sulfate (SDS), and (3) the subcellular localization of the phosphoprotein differed considerably from that of the G_s α-subunit. No phosphate was detected in immunoprecipitates from either basal or insulin-treated cells after the 45 kDa phosphoprotein had been removed. These data argue against a mediating role of phosphorylated G-protein α-subunits in the action of insulin.     

Antagonist interaction with the human 5-HT(7) receptor mediates the rapid and potent inhibition of non-G-protein-stimulated adenylate cyclase activity: a novel GPCR effect

BACKGROUND AND PURPOSE

The human 5-hydroxytryptamine₇ (h5-HT₇) receptor is G_s-coupled and stimulates the production of the intracellular signalling molecule cAMP. Previously, we reported a novel property of the h5-HT₇ receptor: pseudo-irreversible antagonists irreversibly inhibit forskolin-stimulated (non-receptor-mediated) cAMP production. Herein, we sought to determine if competitive antagonists also affect forskolin-stimulated activity and if this effect is common among other G_s-coupled receptors.

EXPERIMENTAL APPROACH

Recombinant cell lines expressing h5-HT₇ receptors or other receptors of interest were briefly exposed to antagonists; cAMP production was then stimulated by forskolin and quantified by an immunocompetitive assay.

KEY RESULTS

In human embryonic kidney 293 cells stably expressing h5-HT₇ receptors, all competitive antagonists inhibited nearly 100% of forskolin-stimulated cAMP production. This effect was insensitive to pertussis toxin, that is, not G_i/o-mediated. Potency to inhibit forskolin-stimulated activity strongly correlated with h5-HT₇ binding affinity (r²= 0.91), indicating that the antagonists acted through h5-HT₇ receptors to inhibit forskolin. Potency and maximal effects of clozapine, a prototypical competitive h5-HT₇ antagonist, were unaffected by varying forskolin concentration. Antagonist interaction with h5-HT₆, human β₁, β₂, and β₃ adrenoceptors did not inhibit forskolin''s activity.

CONCLUSIONS AND IMPLICATIONS

The inhibition of adenylate cyclase, as measured by forskolin''s activity, is an underlying property of antagonist interaction with h5-HT₇ receptors; however, this is not a common property of other G_s-coupled receptors. This phenomenon may be involved in the roles played by h5-HT₇ receptors in human physiology. Development of h5-HT₇ antagonists that do not elicit this effect would aid in the elucidation of its mechanisms and shed light on its possible physiological relevance.     

Crosstalk between thrombin and adenylyl cyclase-stimulating agonists in proliferating human erythroid progenitor cells

Human erythroid progenitor cells grown in a suspension culture system were used to study possible interactions between different guanine nucleotide-binding protein (G-protein)-coupled receptor-effector systems during normal cell differentiation. Agonist-stimulated adenylyl cyclase was not inhibited by any one of a panel of ligands (ADP, UTP, platelet-activating factor, thrombin, ₂-adrenoceptor agonists, interleukin 8, lysophosphatidic acid) most of which are known, in other cells, to reduce cAMP formation by a G_i-mediated, pertussis toxin-sensitive mechanism. The first four of these ligands are also known to cause transient changes in intracellular [Ca²⁺] in erythroid cells. Rather than inhibiting, thrombin (but not ADP, UTP or PAF) specifically caused a fivefold increase in the maximum adenosine- or prostaglandin El-stimulated cAMP formation, without any shift of the concentration/response curves. Thrombin did not enhance forskolin- and AlF₄ ^--stimulated cyclase activity and had only a marginal effect on isoprenaline-dependent stimulation. The effect of thrombin seemed to be unrelated to intracellular Ca²⁺ release but could be partially mimicked by phorbol ester (PMA)-induced stimulation of protein kinase C (PKC) and was inhibited by staurosporin or by inactivation of PKC after long-term incubation with PMA. The activity of thrombin was restricted to proliferating, colony-forming progenitor cells while proerythroblasts were completely unresponsive. Our results suggest that the interaction of thrombin with Gs-linked receptors requires phosphorylation of a target protein that is different from adenylyl cyclase, G_s or G_i but may be involved in the regulation of receptor desensitization.     

G Protein coupling of CGS 21680 binding sites in the rat hippocampus and cortex is different from that of adenosine A1 and striatal A2A receptors

The effect of guanine nucleotide-binding protein (G protein) modifiers on the binding of the adenosine A_2A receptor agonist 2-[4-(2-p-carboxyethyl[³H])phenyl-amino]-5’-N-ethylcarboxamidoadenosine ([³H]CGS 21680) and of the adenosine A₁ receptor agonist [³H]R-phenylisopropyladenosine ([³H]R-PIA) to rat cortical and striatal membranes was studied. Guanosine 5’-(β,γ-imido)triphosphate (1–300 μM), which uncouples all G proteins, more effectively inhibited [³H]CGS 21680 (30 nM) binding in the cortex than [³H]R-PIA (2 nM) binding to cortical or striatal membranes or [³H]CGS 21680 (30 nM) binding in the striatum. N-Ethylmaleimide (1–300 μM) or pertussis toxin (1–100 μg/ml), which uncouple G_i/G_o protein-coupled receptors, more effectively inhibited [³H]R-PIA binding to cortical or striatal membranes and [³H]CGS 21680 binding in the cortex than [³H]CGS 21680 binding in the striatum. Cholera toxin (2.5–250 μg/ml), which uncouples G_s protein-coupled receptors, more effectively inhibited [³H]CGS 21680 binding in the striatum than [³H]CGS 21680 binding in the cortex and less effectively inhibited [³H]R-PIA binding to cortical or striatal membranes. Treatment of solubilised cortical membranes with pertussis toxin (50 μg/ml) decreased [³H]CGS 21680 (30–100 nM) binding which almost fully recovered after reconstitution with G_i/G_o proteins. The K _i for displacement of [2-³H]-(4-{2-[7-amino-2-(2-furyl)(1,2,4)triazolo(2,3-a)(1,3,5)triazin-5-ylamino]ethyl}phenol) ([³H]ZM 241385, 1 nM) by CGS 21680 was 110 nM (95%CI: 98–122 nM) in non-treated, 230 (167–292) nM in pertussis toxin (25 μg/ml)-treated and 222 (150–295) nM in cholera toxin (50 μg/ml)-treated cortical membranes; in contrast, the K _i for displacement of [³H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo(4,3-e)-1,2,4-triazolo(1,5-c)pyrimidine ([³H]SCH 58261, 1 nM) by CGS 21680 was 74 (57–91) nM in non-treated, 71 (44–100) nM in pertussis toxin-treated and 147 (100–193) nM in cholera toxin-treated cortical membranes. Finally, CGS 21680 displaced monophasically the binding of the A₁ antagonist, [³H]8-cyclopentyl-1,3-dipropylxanthine (2 nM), and the A₁ agonist, [³H]R-PIA (2 nM), in 2 or 10 mM Mg²⁺-medium, either at 25°C or 37°C, in cortical or striatal membranes. These results indicate that CGS 21680 does not bind to A₁ receptors and that limbic CGS 21680 binding sites differ from striatal-like A_2A receptors since they couple to G_i/G_o proteins, as well as to G_s proteins. Received: 22 July 1998 / Accepted: 18 January 1999     

Mono- and divalent cations modulate the affinities of brain D1 and D2 receptors for dopamine by a mechanism independent of receptor coupling to guanyl nucleotide binding proteins

Summary In order to clarify the question of whether the modulatory effects of cations on dopamine receptor affinities are brought about by shifts in the equilibrium of receptor — G-protein — coupling, it was investigated whether mono- and divalent cations were still able to modulate rat striatal D1 and D2 receptor affinities after selective inactivation of the G-proteins linked to the two receptors. The G_S-protein coupled to the D1 receptor was eliminated by mild thermal inactivation, and the G_i- (or G_o-) protein associated with the D2 receptor by alkylation with a low concentration of N-ethyl-maleimide. Incubation of striatal membranes at 60°C completely abolished the specific binding of³H-GTP. Both treatments resulted in an increase of the IC₅₀-values for dopamine as a displacer of³H-SCH 23390 from D1- and of³H-spiperone from D2 receptors. Concomitantly, the formerly shallow D1 displacement curves became steeper, with their Hill coefficients increasing. This effect was less evident at D2 receptors. Guanosine triphosphate (GTP), which increased the IC₅₀'s of dopamine for both receptors approximately two-fold in control membranes, was without effect in pretreated samples, indicating an effective inactivation of the G-proteins. Na⁺ ions were still able to lower, and Ca²⁺ ions to increase the affinities of D1 and D2 receptors for dopamine after such inactivation of the respective G-proteins. It is concluded that the mechanism underlying the regulation of dopamine receptor affinities by mono- and divalent cations is independent of and superimposed upon the coupling of these receptors to guanyl nucleotide binding proteins.Abbreviations ANOVA Analysis of variance; G-proteins, guanyl nucleotide binding proteins (G_s: stimulatory, G_i: inhibitory); - GTP guanosine-5-triphosphate; Gpp(NH)p, 5-guanylylimidodiphos-phate; - NEM N-ethyl-maleimide     

Regulation of RhoGEF proteins by G12/13-coupled receptors

G protein-coupled receptors (GPCRs) represent a large family of seven transmembrane receptors, which communicate extracellular signals into the cellular lumen. The human genome contains 720–800 GPCRs, and their diverse signal characteristics are determined by their specific tissue and subcellular expression profiles, as well as their coupling profile to the various G protein families (G_s, G_i, G_q, G₁₂). The G protein coupling pattern links GPCR activation to the specific downstream effector pathways. G_12/13 signalling of GPCRs has been studied only recently in more detail, and involves activation of RhoGTPase nucleotide exchange factors (RhoGEFs). Four mammalian RhoGEFs regulated by G_12/13 proteins are known: p115-RhoGEF, PSD-95/Disc-large/ZO-1 homology-RhoGEF, leukemia-associated RhoGEF and lymphoid blast crisis-RhoGEF. These link GPCRs to activation of the small monomeric GTPase RhoA, and other downstream effectors. Misregulated G_12/13 signalling is involved in multiple pathophysiological conditions such as cancer, cardiovascular diseases, arterial and pulmonary hypertension, and bronchial asthma. Specific targeting of G_12/13 signalling-related diseases of GPCRs hence provides novel therapeutic approaches. Assays to quantitatively measure GPCR-mediated activation of G_12/13 are only emerging, and are required to understand the G_12/13-linked pharmacology. The review gives an overview of G_12/13 signalling of GPCRs with a focus on RhoGEF proteins as the immediate mediators of G_12/13 activation.     

Labelling of Ri adenosine receptors in rat fat cell membranes with (−)-[125iodo]N6-hydroxyphenylisopropyladenosine

Summary A new adenosine analogue, (–)-iodo-N⁶-p-hydroxyphenylisopropyladenosine [(–)-IHPIA], has been developed for radioligand binding studies of R_i adenosine receptors. In addition, the effects of (–)IHPIA on adenosine-mediated responses of rat fat cells have been characterized. (–)IHPIA is slightly less potent at R_i adenosine receptors than (–)N⁶-phenylisopropyladenosine [(–)PIA] as assessed by adenylate cyclase and lipolysis studies. (–)IHPIA inhibited basal adenylate cyclase activity with an IC₅₀ of 60 nmol/l compared to an IC₅₀ of 16.3 nmol/l for (–)PIA. (–)PIA and (–)IHPIA inhibited adenosine deaminase-stimulated lipolysis of intact rat fat cells with an IC₅₀ of 0.55 and 3.6 nmol/l. The potency of (–)N⁶-p-hydroxyphenylisopropyladenosine [(–)HPIA] was intermediate. (–)HPIA has been labelled with carrier-free Na[¹²⁵I] to very high specific activity (2,175 Ci/mmol) and used as agonist radioligand in binding studies of R_i adenosine receptors. The binding of (–)[¹²⁵I]HPIA was saturable, reversible and stereospecific. Saturation analysis revealed two affinity states with dissociation constants (K _D) of 0.7 and 7.6 nmol/l and maximal number of binding sites (B _max) of 0.94 and 0.95 pmol/mg protein. The rate constant of association, k ₁, was 3.7×10⁸ l×mol^–1×min^–1. Binding was slowly reversible with a t_1/2 of 88 min. In competition experiments specific binding was most potently inhibited by (–)PIA, N⁶-cyclohexyladenosine (CHA), (–)HPIA and (–)IHPIA, followed by 5-N-ethylcarboxamidoadenosine (NECA) and 2-chloroadenosine. 1,3-Diethyl-8-phenylxanthine (DPX) and 8-phenyltheophylline were the most potent adenosine antagonists with K _i-values of 67 and 83 nmol/l, whereas the methylxanthines 3-isobutyl-1-methylxanthine, theophylline and caffeine had K _i-values between 1 and 21 mol/l. Binding is highly stereospecific, as indicated by an approximately 20-fold higher K _i-value of the (+)isomer of PIA in comparison to the (–)isomer. The pharmacological profile of (–)[¹²⁵I]HPIA binding sites is consistent with an interaction at R _i adenosine receptors. (–)[¹²⁵I]HPIA appears to be a suitable agonist for radioligand binding studies at R _i adenosine receptors.     

Airway Response to Inhaled Methacholine in Normal Human Subjects

Summary: The individual airway responsiveness to inhaled, nebulized methacholine (MeCh) was estimated in normal volunteers, measuring specific airway conductance (sGAW). The dose of MeCh was increased logarithmically until a 60-65% reduction from baseline sGAW or an asymptotic approach to a maximal response was attained. The concentration of MeCh that caused a 35% reduction in sGAW (PC₃₅), the dose that caused a 62.5% reduction in sGAW, the slope of the straight, central part of the log-dose-response curve (LDRC), the slope of the straight, initial part of the dose-response curve, the maximal response attainable (E_max) and the dose causing a half-maximal response (ED₅₀) were derived. These parameters were transformed as necessary to attain normality of distribution. Relationships between them were examined by measuring the correlations between their transformed values. The ED₅₀ was taken to represent the least biased estimate of the sensitivity to MeCh. The PC₃₅ was the best practical estimate of sensitivity. The E_max was taken to represent the least biased estimate of the reactivity to MeCh. The slope of the LDRC was the best practical estimate of reactivity. The sensitivity and reactivity varied independently in these normal subjects. Each was also independent of the baseline sGAW.