Maria Rosa Mazzoni

Antagonists of the Receptor-G Protein Interface Block Gi-coupled Signal Transduction

The carboxyl terminus of heterotrimeric G protein α subunits plays an important role in receptor interaction. We demonstrate that peptides corresponding to the last 11 residues of Gαi1/2 or Gαo1 impair agonist binding to A1 adenosine receptors, whereas Gαs or Gαt peptides have no effect. Previously, by using a combinatorial library we identified a series of Gαtpeptide analogs that bind rhodopsin with high affinity (Martin, E. L., Rens-Domiano, S., Schatz, P. J., and Hamm, H. E. (1996)J. Biol. Chem. 271, 361–366). Native Gαi1/2 peptide as well as several analogs were tested for their ability to modulate agonist binding or antagonist-agonist competition using cells overexpressing human A1 adenosine receptors. Three peptide analogs decreased the K i , suggesting that they disrupt the high affinity receptor-G protein interaction and stabilize an intermediate affinity state. To study the ability of the peptides to compete with endogenous Gαiproteins and block signal transduction in a native setting, we measured activation of G protein-coupled K+ channels through A1 adenosine or γ-aminobutyric acid, type B, receptors in hippocampal CA1 pyramidal neurons. Native Gαi1/2, peptide, and certain analog peptides inhibited receptor-mediated K+ channel gating, dependent on which receptor was activated. This differential perturbation of receptor-G protein interaction suggests that receptors that act on the same G protein can be selectively disrupted.

Region-dependent effects of flibanserin and buspirone on adenylyl cyclase activity in the human brain

The mode of action of antidepressant drugs may be related to mechanisms of receptor adaptation, involving overall the serotonin 1A (5-HT1A) receptor subtype. However, so far, the clinical effectiveness of selective compounds acting at this level has proved disappointing. This could be explained by the heterogeneity of 5-HT1A receptors within the central nervous system. In animals, two 5-HT1A agonists, flibanserin and buspirone, have shown different pharmacological properties, depending on the brain region. Since no evidence supports this observation in humans, this study sought to investigate whether these two drugs exert different effects on 5-HT1A receptor activation in three different human brain areas: the prefrontal cortex, hippocampus and raphe nuclei. 5-HT1A-mediated inhibition of forskolin-stimulated adenylyl cyclase (AC) was taken as an index of 5-HT1A receptor activation. Flibanserin significantly reduced the activity of AC post-synaptically, i.e. in the prefrontal cortex [EC50 (mean +/- S.E.M.), 28 +/- 10.2 nM; Emax, 18 +/- 2.3%] and in the hippocampus (EC50, 3.5 +/- 3.1 nM; Emax, 20 +/- 4.0%), but had no effect in the raphe nuclei, i.e. at pre-synaptic level. Vice versa, buspirone was only slightly but significantly effective in the raphe (EC50, 3.0 +/- 2.8 nM; Emax, 12 +/- 1.9%). Agonist effects were sensitive to the 5-HT1A antagonists WAY-100135 and pindobind 5-HT1A in the cortex and raphe nuclei, whereas buspirone antagonized flibanserin in the hippocampus. These findings suggest a region-related action of flibanserin and buspirone on forskolin-stimulated AC activity in human brain.

Interaction of Transducin with Light-activated Rhodopsin Protects It from Proteolytic Digestion by Trypsin*

The tryptic cleavage pattern of transducin (Gt) in solution was compared with that in the presence of phospholipid vesicles, rod outer segment (ROS) membranes kept in the dark, or ROS membranes containing light-activated rhodopsin, metarhodopsin II (Rh*). When Gt was in the high affinity complex with Rh*, the αt subunit was almost completely protected from proteolysis. The protection of αt at Arg310 was complete, while Arg204 was substantially protected. The cleavage of αt at Lys18 was protected in the presence of phospholipid vesicles, ROS membranes kept in the dark, or ROS membranes containing Rh*. The cleavage of βt was slower in the presence of ROS membranes or phospholipid vesicles. When the Rh*·Gt complex was incubated with guanyl-5′-yl thiophosphate, a guanine nucleotide analog known to release the high affinity interaction between Gt and Rh*, the protection at Arg310 and Arg204 was diminished. From our results, we propose that Rh* either physically blocks access of trypsin to Arg204 and Arg310 or maintains the heterotrimer in such a conformation that these cleavage sites are not available. Since Arg204 is involved in the switch interface with βγt (Lambright, D. G., Sondek, J., Bohm, A., Skiba, N. P., Hamm, H. E., and Sigler, P. B. (1996) Nature 379, 311-319), it may be that βγt is implicated in protecting this cleavage site in the receptor-bound, stabilized heterotrimer. Arg310 is not near the βγt subunit, thus we believe that the high affinity binding of Gt to Rh* physically or sterically blocks access of trypsin to this site. Thus, Arg310, only a few angstroms away from the carboxyl terminus of αt, which is known to directly bind to Rh*, is likely to also be a part of the Rh* binding site. This is in agreement with other studies and has implications for the mechanism by which receptors catalyze GDP release from G proteins. The protection of Lys18 in the presence of phospholipid vesicles suggests that the amino-terminal region is in contact with the membrane, consistent with the crystal structure of the heterotrimer (Lambright, D. G., Sondek, J., Bohm, A., Skiba, N. P., Hamm, H. E., and Sigler, P. B. (1996) Nature 379, 311-319).

Increased Inhibitory Activity of Protein Kinase C on the Serotonin Transporter in OCD

Different observations show a reduced functionality of the serotonin (5-HT) transporter in obsessive-compulsive disorder (OCD) that might be due to a disturbance of its regulation at intracellular level. Protein kinase C (PKC) has been reported to provoke a decrease in the number of the 5-HT transporter proteins. Therefore, we investigated whether OCD patients differed from control subjects in the effect of PKC upon the 5-HT transporter, after stimulation of this enzyme with 4β-12-tetradecanoylphorbol 13-acetate (β-TPA). Fifteen patients affected by OCD, according to DSM-IV criteria, were compared with a similar group of healthy subjects. The determination of 5-HT uptake was carried out according to the method of Arora and Meltzer with slight modifications. At baseline, OCD patients showed a significant decrease in the maximal velocity (Vmax) of 5-HT uptake, as compared with control subjects, with no change in the Michaelis-Menten constant (Km). The activation of PKC with β-TPA provoked a significant decrease in Vmax values in both groups, but the effect was significantly more robust in OCD patients who, in turn, also showed also an increase in Km values. These findings could indicate the presence of hyperactivity of PKC in OCD that could be the result of increased activity of the phosphatidylinositol pathway. In addition, this suggests new potential therapeutic targets in OCD.

Regulation of agonist binding to A2A adenosine receptors: Effects of guanine nucleotides (GDP[S] and GTP[S]) and Mg2+ ion

Adenosine acts as a neuromodulator through at least two receptor subtypes, A1 and A2. A2 receptors have been further divided into A2A (high agonist affinity) and A2B (low agonist affinity) receptors. Both A1 and A2 receptors belong to the superfamily of guanine nucleotide-binding regulatory protein (G protein)-coupled receptors. A Gs protein couples the A2A receptor to the activation of adenylyl cyclase. In order to elucidate the mechanism of coupling between the A2A receptor and Gs, we studied the modulation by guanine nucleotides and divalent cations of agonist binding to the A2A receptor in rat striatal membranes, using [3H]CGS 21680 as a selective high-affinity agonist. We demonstrated that in rat striatal membranes agonist binding to A2A receptors was modulated by guanine nucleotides. Both GDP and GTP inhibited [3H]CGS 21680 binding to rat striatal membranes with about equal potency. The nonhydrolyzable analogs, GDP[S] and GTP[S], were equipotent inhibitors and approx. 100-times more potent than GDP and GTP. Data from competition studies with labeled and unlabeled CGS 21680 when analyzed by nonlinear regression demonstrated the presence of two binding sites in rat striatal membranes with mean values for KD of 5.6 and 343 nM and Bmax of 200 and 942 fmol/mg protein. The high-affinity binding site has the characteristics of the A2A receptor. In the presence both of (0.1 mM) GDP[S] and GTP[S], the KD values for the high-affinity site were increased severalfold, whereas the low-affinity site was no longer detected in filtration assays. Dissociation studies revealed monophasic dissociation curves both in the absence and presence of 0.1 mM GDP[S]. However the K-1 value increased in the presence of guanine nucleotide. We also showed that in bovine striatal membranes agonist binding to A2A receptors was modestly modulated by guanine nucleotides, suggesting differences of receptor Gs-protein-coupling a mechanism in different species. Divalent cations often increase agonist binding to different receptors, whereas Mg2+ ions play a role in regulating the initial steps of G-protein activation. We investigated the effects of divalent cations on [3H]CGS 21680 binding to the A2A receptor and determined the requirement of these cations to obtain the modulation of binding by guanine nucleotides. We found that millimolar concentrations of divalent cations were required to obtain an effective interaction between the A2A receptor and Gs. The high-affinity binding of [3H]CGS 21680 to the A2A receptor in rat striatal membranes was dependent on the presence of Mg2+ ions.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of the platelet serotonin transporter by protein kinase C in the young and elderly.

BACKGROUND Some data show that different factors may influence the serotonin (5-HT) uptake rate. Our study aimed at evaluating the possible role of a protein kinase C (PKC) activator, i.e., 4-beta-12-tetradecanoylphorbol-13-acetate (beta-TPA) on the platelet 5-HT uptake of young and elderly subjects, through the measurement of the 5-HT uptake itself and 3H-paroxetine ([3H]PAR) binding sites, which correspond to the transporter protein. METHODS Human platelets and 5-HT uptake were evaluated according to the method of Arora and Meltzer, while [3H]PAR binding was performed following the Marazziti et al method. RESULTS The results showed that beta-TPA reduced significantly the maximal velocity (Vmax) of 5-HT uptake, with no change in the Michaelis constant or in [3H]PAR binding parameters, in platelets of both young and elderly subjects. Although this last group of subjects had a significantly lower Vmax than the other, the degree of inhibition was almost the same (75%) in both. CONCLUSIONS These findings indicate that PKC decreases the 5-HT uptake rate by modifying the phosphorylation state of the transporter and with no change in the number of [3H]PAR binding sites. The responsiveness of this pathway is identical in both young and elderly subjects.

Effects of postmortem delay on serotonin and (+)8-OH-DPAT-mediated inhibition of adenylyl cyclase activity in rat and human brain tissues

The reproducibility of serotonin (5-HT) and (+)8-OH-DPAT-mediated inhibition of adenylyl cyclase activity was assessed in membranes, stimulated by forskolin, of rat frontal cortex postmortem as well as of human fronto-cortical, hippocampal and dorsal raphe tissues obtained from autopsy brains. The results revealed that differences between basal and forskolin-stimulated enzyme activities were still significant after 48 h postmortem in rat cortex and in all human brain regions up to 46 h after death. However, a decrease of about 17 and 26% in forskolin-stimulated adenylyl cyclase activity was observed at 24 and 48 h, respectively, in rat cortex. 5-HT and the 5-HT1A receptor agonist, (+)8-hydroxy-2(di-N-propylamino)tetraline (8-OH-DPAT), were able to inhibit forskolin-stimulated adenylyl cyclase activity in a dose-dependent manner for 48 h after death in rat and human brain. In rat cortex, both 5-HT and (+)8-OH-DPAT potencies (EC50, nM) and efficacies (percent of maximum inhibition capacity, %) varied significantly with postmortem delay. Conversely, in human tissues, postmortem delay and subject age did not modify agonist potencies and efficacies. Furthermore, a regionality of 5-HT potency and efficacy was revealed in the human brain. 5-HT was equally potent in cortex and raphe nuclei, while being more potent but less effective in hippocampus. (+)8-OH-DPAT was more active in hippocampus and raphe nuclei than in cortex. (+)8-OH-DPAT behaved as an agonist in all areas, as its efficacy was similar or greater than those obtained with 5-HT. The (+)8-OH-DPAT dose-response curve was completely reversed by 5-HT1A receptor antagonists in rat cortex and all human brain areas. In conclusion, we suggest here that differences between rat and human brain might exist at the level of postmortem degradation of 5-HT-sensitive adenylyl cyclase activity. In human brain, 5-HT1A receptor-mediated inhibition of adenylyl cyclase seems to be reproducible, suggesting that reliable experiments can be carried out on postmortem specimens from patients with neuropsychiatric disorders.

Tryptophan207 is involved in the GTP-dependent conformational switch in the α subunit of the G protein transducin: Chymotryptic digestion patterns of the GTPγS and GDP-bound forms

The limited proteolytic pattern of transducin,Gt, and its purified subunits with chymotrypsin were analyzed and the cleavage sites on the αt subunit were identified. The αt subunit in the GTPγS bound form was cleaved into a major 38 kD fragment, whereas αt-GDP was progressively digested into 38, 23, 21, and 15 kD fragments. The βγt subunit was not very sensitive to proteolytic digestion with chymotrypsin. The γt subunit was not cleaved and only a small portion of βt was digested into several fragments. In order to determine which proteolytic fragment of αt still contained the carboxyl terminal region, chymotrypsinization was carried out usingGt previously32P-labeled at Cys347 by petrussis toxin-catalyzed ADP-ribosylation. The32P-label was mainly associated with the αt subunit and a 15 kD fragment. The 23 and 21 kD fragments were not32P-labeled. Analysis of amino terminal sequences of 38, 21, and 15 kD proteolytic bands allowed the identification of the major cleavage sites. Chymotrypsin had two cleavage sites in the amino terminal region of αt, at Leu15 and Leu19. Chymotrypsin removed 15–19 amino acid residues from the amino terminus of αt, generating two peptides (38 kD) which comigrates in gel electrophoresis. Chymotrypsin also cleaved at Trp207 in a conformation-dependent manner. Trp207 of αt-GTPγS was resistant to proteolysis but αt-GDP and the 38 kD fragments of αt-GDP produced the 23 and 21 kD fragments, respectively, and a 15 kD fragment containing the carboxyl terminus. This proves that the environment of Trp207 changes when GTP or GTPγS is bound, leading to its inaccessibility to chymotrypsin.

erythro- and threo-2-Hydroxynonyl substituted 2-phenyladenines and 2-phenyl-8-azaadenines: ligands for A1 adenosine receptors and adenosine deaminase

erythro-2-Phenyl-9-(2-hydroxy-3-nonyl)adenine and its 8-aza analog were prepared and showed a very high inhibitory activity towards adenosine deaminase (ADA), with Ki 0.55 and 1.67 nM, respectively, and high affinity for A1 adenosine receptors, with Ki 28 and 2.8 nM, respectively. To increase affinity for A1 receptors we introduced a substituent on the N6 position such as alkyl or cycloalkyl groups, which are present in effective agonists or antagonists. Furthermore, for some compounds, we prepared the two diastereoisomers erythro and threo to verify whether the binding with A1 receptors is stereoselective, as in ADA. Results show that some of the synthesised compounds are good inhibitors for ADA and good ligands for A1, and the erythro diastereoisomers are more active than the threo ones. The experimental evidence allows us to hypothesise some similarity in the three dimensional structures of the binding site of the two proteins, ADA and A1 adenosine receptor, in spite of lacking any homologies in the amino acid sequences.

Toward the rational development of peptidomimetic analogs of the C-terminal endothelin hexapeptide: development of a theoretical model

In an early report on the structure-activity relationship of endothelin (ET) peptides, it was reported that the C-terminal hexapeptide ET(16-21), His-Leu-Asp-Ile-Ile-Trp, is the minimum ET fragment which maintains biological activity in some, but not all the tissues responding to ETs. Subsequently, other authors described a series of analogs of this peptide, in which the His 16 residue was replaced by non-natural amino acids, characterized by bulky aromatic side chains. Among them, two well-characterized non-selective ETA/ETB antagonists were PD 142893 and PD 145065; interest in these potent ET antagonists was, however, reduced by their peptidic structure which was likely to lead to undesirable properties such as poor bioavailability and short duration of action. On the basis of these premises, our previous studies led to the development of a peptidomimetic ligand of ET receptors (compound 3), based on the replacement of the His 16 residue of ET(16-21) with an (E)-N-(benzyloxy)iminoacyl moiety; compound 3 proved to possess a certain affinity for ET receptors, albeit lower than that shown by PD 142893 and PD 145065. We report here on ETA/ETB binding affinity of compounds 4-12, designed as a new series of ET(16-21) analogs. Compounds 4 and 5 were practically devoid of any affinity; derivatives 6-12 exhibited appreciable affinity indices for ETB receptors higher than that shown by 3, even if still lower than that obtained for PD 145065. This paper also describes the development of a pharmacophoric model able to explain the ET receptor binding properties of our hexapeptide analogs compared with those of PD 142893 and PD 145065 and IRL2500, recently reported as a potent ETB selective endothelin antagonist.