Regulation of Functional Activity of Adenylyl Cyclase Signal System by Synthetic Coil-Forming Peptides in Mouse Fibroblast Culture of Line L (ISM Subline)

The signal transduction process via adenylyl cyclase system (ACS) requires coordinated functioning of signal proteins—components of ACS. It is suggested that functional coupling between them, together with other molecular mechanisms, is based on coiled-coil interactions. To study role of these interactions in functioning of ACS, we synthesized cationic coiled-forming peptides with a regular structure Ac–Ala–His– (Ala)₂–His–Ala–NH₂ (I), Ac–Ala–His–(Ala)₃–His– (Ala)₂–His–Ala–NH₂ (II), and Ac–(Pro(₂–His– (Ala)₂–His– (Ala)₂–His– (Ala)₂–His–Ala–NH₂ (III). Using circular dichroism (CD) spectroscopy, a portion of -helix conformation in their secondary structure was determined, and effects of these peptides on basal adenylyl cyclase (AC) activity as well as on the activity stimulated by non-hormonal (NaF and Gpp[NH]p) and hormonal (serotonin) agents was studied in homogenate of mouse fibroblasts, line L (subline LSM). The synthetic peptides were shown to inhibit in a dose-dependent manner both basal and induced AC activity, which indicates their uncoupling action on ACS. The biological effect of these peptides correlated with their length (I < II < III), but not with coiled-coil structure, which was 20, 7, and 21%, respectively, according to data of circular dichroism spectroscopy in 3-fluoroethanol. However, there are reasons to believe that the coiled-coil structure of peptides, first place extended ones, increases at interaction with plasma membrane and signal proteins, which affects the degree of their effect on functional ACS activity. At micromolar concentrations, peptides II and III were established to markedly stimulate the basal AC activity, thereby mimicking G-protein-binding sites of cytoplasmic receptor loops. The data obtained indicate participation of the coiled-coil interactions in functional coupling of ACS components, and the methodology itself of the use of model peptides with different coiled-coil structure and distribution of charged amino acids is an efficient approach for studying molecular bases for functioning of signal systems.     

Inhibitory action of polycationic peptides on hormonal regulation of adenylyl cyclase of the ciliate Dileptus anser

To analyse molecular mechanisms of regulatory action of different hormones on the activity of the adenylyl cyclase signaling system (ACS) of the ciliate Dileptus anser, we studied the influence on this process of six synthetic polycationic peptides and peptides, corresponding to C-terminal regions of mammalian G-protein 385-394 alphas- and 346-355 alphai2-subunits. As we reported earlier, these peptides block hormonal signal transduction in tissues of the higher eukaryotes. Now it has been found that both polycationic peptides, containing hydrophobic C to-radicals, and branched peptides decrease regulatory effects of peptide hormones (insulin, relaxin) and biogenic amines (serotonin, adrenaline) on adenylyl cyclase (AC) activity and GTP-binding. In regard to the following peptides Cys-epsilonAhx-Trp-Lys-Lys(C10)-Lys2-Lys(C10)-Lys3-Lys(C10)-Tyr-Lys-Lys(C10)-Lys-Lys-amide and [(Gly-Arg-Gly-Asp-Ser-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro- Pro-Gly)2-Lys-EAhx-Cys]2 (epsilonAhx - E-aminocaproyl, C10 - caprinoyl group) their dose-dependent inhibitory action is shown. In cell culture of D. anser with a lower basal AC activity, both hydrophobic and branched peptides stimulated AC and GTP-binding without hormones. The data give evidence that these peptides can activate ACS of ciliates in a receptor-independent manner. No influence of peptides 385-394 alphas and 346-355 alphai2 on hormonal signal transduction in D. anser was observed, due, presumably, to some structural differences of G-proteins of the lower and higher eukaryotes. A conclusion was made about an important role of polycationic regions for functional coupling of hormone-activated receptor and G-proteins in the ciliate D. anser.     

Molecular mechanisms of action of dendrons, containing 48-60 sequence of HIV-1 TAT-protein, on the functional activity of the adenylyl cyclase signaling systems

For the aims of studying molecular mechanisms of functioning of adenylyl cyclase signaling systems (ACS), we investigated the influence of synthetic polycationic peptides of the star-like structure (dendrons), containing 48-60 sequence of HIV-1 TAT-protein, on the functional activity of ACS components in smooth muscles of the mollusc Anodonta cygnea and in rat skeletal muscles. It has been shown that the following peptides (Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln)2-Lys-epsilonAhx(= epsilon-aminohexanoic acid)-Cys(Acm), referred to as peptide I, (Gly-Arg-Gly-Asp-Ser-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln)2-Lys-epsilonAhx-Cys(Acm) (peptide II), [(Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln)2-Lys-epsilonAhx-Cys]2 (peptide III), and [(Gly-Arg-Gly-Asp-Ser-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln)2-Lys-epsilonAhx-Cys]2 (peptide IV) inhibit in a dose-dependent manner the adenylyl cyclase (AC) activity stimulated by both nonhormanal agents (GppNHp and forskolin) and hormones, such as serotonin (mollusc) and isoproterenol (rat). Peptides III and IV (tetrameric dendrons) were most effective in comparison with peptides I and II (dimeric dendrons). The AC activity stimulated by hormones and forskolin was most sensitive to the action of dendrons. All dendrons stimulated GTP-binding activity of G-proteins: dimeric dendrons were most effective at 10(-5) M concentration, whereas tetrameric dendrons at 10(-6) M. In the presence of dendrons, the affinity of beta-antagonist [3H]-dihydroalprenolol to P-adrenergic receptor in rat muscle mem- branes was unchanged. At the same time, the affinity of beta-agonist isoproterenol to the receptor decreased, and no shift to the right was observed on the curve of isoproterenol-induced [3H]-dihydroalprenolol displacement in the presence of GTP. The obtained data show the disturbance of the coupling between the receptor and G-protein, which is the main reason of dendron inhibitory action on AC stimulation by hormones. Besides, these data demonstrated that hormones could disturb the functional activity of AC, i.e. a catalytic component of ACS.     

Involvement of SH-groups in the regulation of adenylate cyclase signal system by isoproterenol and serotonin in cultured murine fibroblasts L (subline LSM)

The adenylyl cyclase system (ACS) plays a key role in transduction of a hormonal signal into eukaryotic cells. The functional activity of the system depends on SH-groups of proteins involved in the ACS: receptor, G-protein, and enzyme adenylyl cyclase (AC). We studied the influence of thiols and SH-blockers on the regulation of AC activity by nonhormonal (NaF and Gpp[NH]p) and hormonal (biogenic amines isoproterenol and serotonin) agents in homogenates of cultured murine fibroblasts of line L (subline LSM). In the presence of thiols 2-mercaptoethanol (5 mM) and dithiothreitol (1 mM) the basal AC activity somewhat increased, whereas the stimulating effects of NaF, Gpp[NH]p, and hormones decreased. No potentiating action of Gpp[NH]p on hormonal effect in this case was found. The SH-blockers 25 mkM p-chloromercuribenzoic acid (CMBA) and 0.2 mM N-ethylmaleimide significantly inhibited both the basal AC activity and that stimulated by different agents. Thiols partially restored CMBA inhibited AC activity (in the case of N-ethylmaleimide restoring effects of thiols were insignificant). This, the ACS of murine fibroblasts of subline LSM is SH-sensitive. The forms of SH-groups in proteins involved in the ACS determine their functional activities and a possibility of transduction of the hormonal signal on the effector systems.     

Dissociation action of cationic peptides with hydrophobic radicals on functional coupling between serpentine type receptors and GTP-binding proteins

The coupling of hormone-activated receptor and heterotrimeric G protein is an important step of the signal transduction through adenylyl cyclase signal system (ACS). The numerous literature data and own results show that G protein-interacting regions, that are localized in cytoplasmic loops of receptors, have considerable positive charge, can form amphiphilic alpha-helices and are tightly associated with the membrane. We studied the influence of model cationic peptides on both basal and stimulated by hormones and nonhormonal agents adenylyl cyclase (AC) activity and on GTP binding activity of heterotrimeric G proteins in skeletal muscles of rats and smooth muscles of mollusc Anodonta cygnea. Peptides with hydrophobic radicals of caprinoyl acid (C10): Lys(C10)-His-Glu-Lys-Lys-(C10)-His-Glu-Lys-Lys(C10)-His-Glu-Lys-Lys(C10)- His-Glu-Lys-Ala-amide (peptide I), Cys-Lys(C10)-X-Tyr-Lys-Ala-Lys7-Trp-Lys-amide (II), Cys-X-Trp-Lys-Lys(C10)-Lys2-Lys(C10)-Lys3-Lys(C10)-Tyr-Lys-Lys(C10)-Lys-Lys- amide (III), where X--epsilon-aminocaproyl acid residue, were synthesized by solid-phase methodology. IC50 values for inhibiting the influence of peptides on serotonin-(molluscs) and isoproterenol-stimulated (rats) AC activity were: for peptide I--56 and 70 mkM, for peptide II--32 and 47 mkM, for peptide III--22 and 28 mkM, respectively. At the same time the peptides weakly decreased AC activity stimulated by nonhormonal agents (NaF, Gpp[NH]p, forskolin). Peptides I--III stimulated basal activity of the enzyme in both investigated tissues. The maximum stimulating effects (28--52%) of the peptides were observed at their concentration 10 mkM. Peptides (10--100 mkM) increased Gpp[NH]p binding in plasma membranes of mollusc and rat muscles and strongly decreased the influence of the hormones on the binding. Based on the obtained data we supposed that cationic peptides with hydrophobic radicals mimic G protein-binding regions of the receptors and can be involved in the regulation of functional coupling between the receptors and G proteins.     

Role of Sulfhydryl Groups in Functioning of Adenylyl Cyclase Signaling System

This review considers the literature data and author's own results on the role of SH-groups in functioning of the hormone-sensitive adenylyl cyclase system (ACS). It has been shown that the state of SH-groups affects crucially all main stages of the hormonal signal transudation: the ligand-binding properties of receptor and its coupling to G-proteins, interaction of G-proteins with adenylyl cyclase (AC) and its catalytic activity. It is noted that for the receptors, coupled to AC by a stimulating mode, the central aspect of the SH-dependent regulation of ACS is shifted to the receptor, while for the receptors coupled to AC by an inhibiting mode, it coincides with G-protein of the inhibiting type, which is sensitive to the SH-group state. Based on the performed comparative analysis of primary structures of signalling proteins—ACS components and of literature data, there are revealed the cysteine residues determining the functional activity of these proteins in the process of the hormonal signal transudation. The conclusion is made that the SH-group state (the ratio of free SH-groups and disulfide bonds) is the main factor determining the ACS reactivity to hormonal effects and selectivity of process of the signal transudation.     

Comparative study of molecular mechanisms of natural and synthetic polycationic peptides action on the activity of the adenylyl cyclase signaling system

The molecular mechanisms of action of natural and synthetic polycationic peptides, forming amphiphilic helices, on the heterotrimeric G-proteins and enzyme adenylyl cyclase (AC), components of hormone-sensitive AC system, were studied. It is shown that synthetic peptides C-epsilonAhx-WKK(C10)-KKK(C10)-KKKK(C10)-YKK(C10)-KK (peptide I) and (GRGDSGRKKRRQRRRPPQ)2-K-epsilonAhx-C(Acm)(peptide II) in dose-dependent manner stimulate the basal AC activity, inhibit forskolin-stimulated AC activity and decrease both stimulating and inhibiting AC effects of the hormones in the tissues (brain striatum, heart muscle) of rat and in smooth muscles of the mollusc Anodonta cygnea. AC effects of these peptides are decreased after membrane treatment by cholera and pertussis toxins and are inhibited in the presence of the peptides, corresponding to C-terminal regions 385-394 alphas- and 346-355 alphai2-subunits of G-proteins. These data give evidence that the peptides I and II act on the signaling pathways which are realized through Gs- and Gi-proteins. At the same time, natural polycationic peptide mastoparan acts on AC system through Gi-proteins and blocks hormonal signals mediated via Gi-proteins only. Consequently, the action of mastoparan on G-proteins is selective and differs from the action of the synthetic peptides. It is also shown that peptide II, with branched structure, directly interacts not only with G-proteins (less effective in comparison with peptide I with hydrophobic radicals and mastoparan), but also with enzyme AC, the catalytic component of AC system. On the basis of data obtained the following conclusions were made: 1) the formation of amphiphilic helices is not enough for selective activation of G-protein by polycationic peptides, and 2) the primary structure of the peptides, the distribution of positive charged amino acids and hydrophobic radicals in them are very important for selective interaction between polycationic peptides and G-proteins.     

Molecular causes of changes in sensitivity of adenylyl cyclase signaling system to biogenic amines in the heart muscle during experimental streptozotocin diabetes

Changes in hormonal sensitivity of the adenylyl cyclase signaling system (ACS) and their possible molecular causes in the heart muscle of rats with experimental streptozotocin diabetes (type I diabetes) are investigated. An increase in stimulating effects of noradrenaline and isoproterenol on adenylyl cyclase (AC) activity have been shown. In the case of noradrenaline, this increase is due to suppression of Gi-protein function and Gi-coupled inhibitory AC signaling pathway. Meanwhile, in diabetic rats the influence of C-terminal peptide 346-355 of alphai2-subunit on hormonal activation of AC and GTP-binding is diminished. In the case of isoproterenol, along with its stimulating effect, at micromolar concentrations this hormone exerts inhibitory action, realized, presu- mably, through beta3-adrenergic receptors. Effect of isoproterenol on AC and GTP-binding in the heart of diabetic animals is modified by peptide 385-394 alphas, blocking Gs-coupled signaling pathways, and by peptide 346-355 alphai2, blocking transduction of inhibitory signals. In addition, a decrease in serotonin stimulating effect on components of ACS in diabetic animals was shown. The data obtained provide evidence for changes in ACS function in diabetes, which can be detected mainly at the G-protein level. The proposed peptide strategy is a new and perspective approach for studying molecular causes of functional violations in hormonal signaling systems arising at endocrine pathology.     

Regulatory Action of Synthetic Cationic Peptides Containing Residues of Glutamic Acid on Functional Activity of Components of the Adenylyl Cyclase Signal System

One of the most important stages of hormonal signal transduction in cells through the hormone-sensitive adenylyl cyclase signal system (ACS) is functional coupling of receptor of the serpentine type to heterotrimeric GTP-binding protein (G-protein). The main role in realization of such coupling is played by spiralized regions of the receptor cytoplasmic loops proximal in relation to membrane, most of them carrying positive charge. To study molecular mechanisms of interaction of the receptor with G-protein, we compared effects of synthetic cationic peptides containing residues of glutamic acid on the process of regulation of ACS by hormones (biogenic amines) and non-hormonal agents in smooth muscles of the freshwater bivalve mollusc Anodonta cygnea and skeletal muscles of rat. All peptides had the clearly expressed ability to form -helices. Peptides H-(Leu-His-Glu-Lys)₄-Leu-NH₂ (I), H-(Leu-His-Glu-Lys)₃-Lys-His-Glu-Lys-Leu-NH₂ (II), H-(Leu-Lys-Glu-Lys)₄-Leu-NH₂ (III), and H-(Ile-His-Glu-Lys)₄-Ala-NH₂ (IV) at concentrations of 10^–6–10^–3 M reduced dose-dependently the value of stimulating effects of serotonin (in mollusc muscles) and isoproterenol (in rat muscles) on the adenylyl cyclase (AC) and protein kinase A (PKA) activities. Values of concentration of these peptide causing a 50% decrease of the hormone-stimulating effect (IC₅₀) vary from 150 to 750 µM. According to the degree of this inhibitory action on stimulating effects of hormones, they may be arranged in the following series: III II > IV I. The peptides I–IV were more effective than the peptide H-(Glu-Lys)₈-Ala-NH₂ (V) with the charge close to zero, but much less effective than the studied earlier cationic peptides containing only positively charged amino acid residues. The inhibitory effect of the peptides I-IV on stimulation of AC by non-hormonal agents, NaF, Gpp[NH]p, and forskolin, was essentially less pronounced and was marked only at 10^–4–10^–3 M concentrations. Thus, the inclusion of negatively charged amino acid residues in the primary structure of polycationic peptides leads to a decrease in their ability to inhibit hormonal stimulation of AC and PKA, which indicates importance both of the total positive charge of peptides and of distribution of the charged amino acids in the formed helices for realization of the uncoupling action on the ACS components—the receptor and G-protein.     

The signal transfer regions of G alpha(s)

The crystal structure of soluble functional fragments of adenylyl cyclase complexed with G alpha(s) and forskolin, shows three regions of G alpha(s) in direct contact with adenylyl cyclase. The functions of these three regions are not known. We tested synthetic peptides encoding these regions of G alpha(s) on the activities of full-length adenylyl cyclases 2 and 6. A peptide encoding the Switch II region (amino acids 222-247) stimulated both adenylyl cyclases 2- to 3-fold. Forskolin synergized the stimulation. Addition of peptides in the presence of activated G alpha(s) partially inhibited G alpha(s) stimulation. Corresponding Switch II region peptides from G alpha(q) and G alpha(i) did not stimulate adenylyl cyclase. A peptide encoding the Switch I region (amino acids 199-216) also stimulated AC2 and AC6. The stimulatory effects of the two peptides at saturating concentrations were non-additive. A peptide encoding the third contact region (amino acids 268-286) located in the alpha 3-beta 5 region, inhibits basal, forskolin, and G alpha(s)-stimulated enzymatic activities. Since this region in G alpha(s) interacts with both the central cytoplasmic loop and C-terminal tail of adenylyl cyclases this peptide may be involved in blocking interactions between these two domains. These functional data in conjunction with the available structural information suggest that G alpha(s) activation of adenylyl cyclase is a complex event where the alpha 3-beta 5 loop of G alpha(s) may bring together the central cytoplasmic loop and C-terminal tail of adenylyl cyclase thus allowing the Switch I and Switch II regions to function as signal transfer regions to activate adenylyl cyclase.     

Regulation of Adenylyl Cyclase Signaling System in Cell Cultures of Infusoria Dileptus anser and Tetrahymena pyriformis by Peptides of Insulin Superfamily

Hormone-sensitive adenylyl cyclase signaling system (ACS) provides transduction of a wide spectrum of hormonal signals in cells of the higher eucaryotes. At the same time, ACS in the lower eucaryotes at present is practically not studied. We studied regulatory effects on ACS of the infusoria Dileptus anser and Tetrahymena pyriformis of peptide hormones of the higher eukaryotes—insulin, IGF-1, and relaxin, whose action on ACS of the higher eucaryotes was the subject of our earlier studies. The action of these hormones at concentrations of 10^–10–10^–8 M on the AC activity in infusoria had clearly stimulating character, the dose–effect curves being of a bell-shaped form with a maximum of the stimulating effect of the hormones at concentrations of 10^–9–10^–8 M. the shape of the curves and the value of the stimulating effect of the peptide hormones depended substantially on the level of the AC basal activity in homogenates of infusorian cell cultures. All the hormones (10^–8 M) stimulated GTP-binding activity of G-proteins. It was shown by the example of relaxin that its stimulating effect on GTP-binding in infusorian cells was dose-dependent and increased in the range of hormone concentrations from 10^–10 to 10^–8 M to reach its maximum at concentrations of 10^–8–10^–7 M. In the presence of suramin, an inhibitor of heterotrimeric G-proteins, the stimulating effects of the hormones on the GTP-binding and the AC activity decreased essentially or were absent completely. This indicates that the heterotrimeric G-proteins are ones of components of the signaling cascade that mediates regulatory effects of the hormones of the insulin group on the AC activity in infusorian cell cultures. Based on the obtained data, it is suggested that the basic molecular mechanisms of regulation of ACS by insulin and the related peptides that are similar to those found in the higher vertebrates already begin to be formed as early as at the level of the lower eucaryotes.     

Structural and functional characteristics of the adenylyl cyclase signaling system regulated by biogenic amines and peptide hormones in the muscle of a worm Lumbricus terrestris

It has been shown for the first time that biogenic amines (catecholamines and tryptophane derivatives) stimulate dose-dependently activity of adenylyl cyclase (AC) and GTP-binding of G-proteins in muscle of the cutaneous-muscle bag of the earthworm Lumbricus terrestris. By efficiency of their stimulating action on the AC activity, biogenic amines can be arranged in the following sequence: octopamine > tyramine > tryptamine = serotonin > dopamine > isoproterenol = adrenalin. The sequence of efficiency of their action on GTP-binding is somewhat different: serotonin > tryptamine > octopamine > dopamine = tyramine > adrenaline > isoproterenol. Sensitivity of AC and G-proteins in the worm muscle to biogenic amines is similar with that in smooth muscle of the molluse Anodonta cygnea (invertebrates), but differs markedly by this parameter from the rat myocardium (vertebrates). It has also been revealed that AC in the worm muscle is regulated by peptide hormones relaxin and somatostatin whose action is comparable with that in the mollusk muscle, but much weaker that the action of these hormones on the rat myocardium AC activity. Use of C-terminal peptides of alpha-subunits of G-proteins of the stimulatory (385-394 Galpha(s)) and inhibitory (346-355 Galpha(i2)) types that disrupt selectively the hormonal signal transduction realized via G(s)- and G(i)-proteins, respectively, allowed establishing that the AC-stimulating effects of relaxin, octopamine, tyramine, and dopamine in the worm muscle are realized via the receptors coupled functionally with G(s)-protein; the AC-inhibiting effect of somatostatin is realized via the receptor coupled with G(i)-protein, whereas serotonin and tryptamine activate both types of G-proteins.     

Peptides derived from the third cytoplasmatic loop of serotonin 1B receptor subtype selectively inhibit serotonin signal transduction via a homologous receptor

The third intracellular loops of hormonal receptors play the main role in the interaction of majority of the serpentine type receptors with heterotrimeric G-proteins. In recent years, it was shown that synthetic peptides corresponding to membrane-proximal regions of these loops could be selectively influenced with hormonal signal transduction via the receptors homologous to them and trigger signalling cascade in absence of the hormone. For the first time, we succeeded in synthesizing the peptides derived from C-terminal region of the third intracellular loop of the IB-subtype serotonin receptor and studied their influence on serotonin-sensitive adenylyl cyclase system in the rat brain. The peptides 300-316 and 306-316 (the numbers correspond to amino acid positions in the rat IB-subtype serotonin receptor) at micromolar concentrations in absence of hormone-stimulated GTP-binding of Gi,-proteins coupled with the IB-subtype serotonin receptors and inhibited forskolin-stimulated adenylyl cyclase activity. Using selective agonists and antagonists of serotonin receptors it was shown that the peptides 300-316 and 306--316 inhibited serotonin signal transduction via homologous to them receptor and weakly influenced other types of serotonin receptors. The peptide 300-316 is more active compared with its shorter analogue 306-316 in the selectivity and efficiency of action on adenylyl cyclase signalling system regulated via the IB-subtype serotonin receptors. These findings indicate that the regions 300-316 of the IB-subtype serotonin receptor are involved in interaction with Grproteins and consist of the main molecular determinants responsible for serotonin signal transduction to adenylyl cyclase.     

Role of cysteine sulfhydryl groups in activation of adenylyl cyclase signaling system by biogenic amines in molluscan and rat tissues

It has been shown that in smooth muscles of the freshwater bivalve molluscAnodonta cygnea as well as in skeletal muscles and brain striatum of rats a blocker of SH-groups,para-chlormercury benzoate (ChMB), and an alkylating agent,N-ethylmaleimide, inhibit both the basal adenylyl cyclase (AC) activity and the activity of the enzyme stimulated by non-hormonal agents (NaF, Gpp[NH]p) and by hormonal agents such as serotonin (mollusc muscles, rat brain) or isoproterenol (rat muscles and rat brain). The inhibitory effects of ChMB andN-ethylmaleimide on AC are partly eliminated by an SH-group containing reagent, β-mercaptoethanol (ME, 5 mM). Restoration of the basal and of the stimulated enzyme activity inhibited by ME is better in the case of the ChMB than of theN-ethylmaleimide action. It has also been found that ME stimulates both the basal and the stimulated by non-hormonal agents AC activity. In the presence of ME the hormonal stimulating effects on the enzyme are also preserved, except for the effect of isoproterenol on AC in rat skeletal muscles; this effect is inhibited by ME. Potentiation of the stimulating effect of the hormones on AC by Gpp[NH]p is only preserved in the molluscan smooth muscles (the effect of serotonin—90%). The data obtained indicate that cysteine sulfhydryl groups play a key role in hormonal regulation of the functional activity of the components of the hormone-sensitive adenylyl cyclase signaling system.     

Enhanced expression of Gialpha protein and adenylyl cyclase signaling in aortas from 1 kidney 1 clip hypertensive rats

We have previously shown the augmented levels of Gialpha-2 and Gialpha-3 proteins (isoforms of inhibitory guanine nucleotide regulatory protein (G-protein)), and not of Gsalpha, in the hearts and aortas of spontaneously and experimentally induced hypertensive rats. The increased expression of Gialpha and blood pressure was restored toward WKY levels by captopril treatment, suggesting a role for angiotensin (Ang) II in the enhanced expression of Gialpha protein and blood pressure. This study was undertaken to investigate whether 1 kidney 1 clip (1K-1C) hypertensive rats that exhibit enhanced levels of Ang II also express enhanced levels of Gialpha proteins. Aortas from 1K-1C hypertensive rats were used. The expression of G-proteins was determined at protein levels with immunoblotting techniques, using specific antibodies for different isoforms of G-proteins. The levels of Gialpha-2 and Gialpha-3 proteins were significantly higher in aortas from 1K-1C hypertensive rats than in control rats; Gsalpha levels were unchanged. The inhibitory effect of low concentrations of guanosine 5'-[gamma-thio]triphosphate (GTPgammaS) on forskolin (FSK)-stimulated adenylyl cyclase (AC) activity was significantly enhanced in aortas from 1K-1C hypertensive rats; the inhibitory effect of C-ANP(4-23), which specifically interacts with the atrial natriuretic peptide (ANP)-C receptor, and Ang II on AC was attenuated. GTPgammaS, isoproterenol, glucagon, NaF, and FSK stimulated the AC activity in aortas from control and hypertensive rats to varying degrees; however, the stimulations were significantly lower in hypertensive rats than in control rats. These data suggest that aortas from 1K-1C hypertensive rats exhibit enhanced expression of Gialpha proteins and associated functions.     

Cytoplasmic domain of natriuretic peptide receptor C constitutes Gi activator sequences that inhibit adenylyl cyclase activity

We have recently demonstrated that a 37-amino acid peptide corresponding to the cytoplasmic domain of the natriuretic peptide receptor C (NPR-C) inhibited adenylyl cyclase activity via pertussis toxin (PT)-sensitive G(i) protein. In the present studies, we have used seven different peptide fragments of the cytoplasmic domain of the NPR-C receptor with complete, partial, or no G(i) activator sequence to examine their effects on adenylyl cyclase activity. The peptides used were KKYRITIERRNH (peptide 1), RRNHQEESNIGK (peptide 2), HRELREDSIRSH (peptide 3), RRNHQEESNIGKHRELR (peptide 4), QEESNIGK (peptide X), ITIERRNH (peptide Y), and ITIYKKRRNHRE (peptide Z). Peptides 1, 3, and 4 have complete G(i) activator sequences, whereas peptides 2 and Y have partial G(i) activator sequences with truncated carboxyl or amino terminus, respectively. Peptide X has no structural specificity, whereas peptide Z is the scrambled peptide control for peptide 1. Peptides 1, 3, and 4 inhibited adenylyl cyclase activity in a concentration-dependent manner with apparent K(i) between 0.1 and 1 nm; however, peptide 2 inhibited adenylyl cyclase activity with a higher K(i) of about 10 nm, and peptides X, Y, and Z were unable to inhibit adenylyl cyclase activity. The maximal inhibitions observed were between 30 and 40%. The inhibition of adenylyl cyclase activity by peptides 1-4 was absolutely dependent on the presence of guanine nucleotides and was completely attenuated by PT treatment. In addition, the stimulatory effects of isoproterenol, glucagon, and forskolin on adenylyl cyclase activity were inhibited to different degrees by these peptides. These results suggest that the small peptide fragments of the cytoplasmic domain of the NPR-C receptor containing 12 or 17 amino acids were sufficient to inhibit adenylyl cyclase activity through a PT-sensitive G(i) protein. The peptides having complete structural specificity of G(i) activator sequences at both amino and carboxyl termini were more potent to inhibit adenylyl cyclase activity as compared with the peptides having a truncated carboxyl terminus, whereas the truncation of the amino-terminal motif completely attenuates adenylyl cyclase inhibition.     

The hormone-sensitive adenylyl cyclase system of the ciliate Dileptus anser

The hormone-sensitive adenylyl cyclase system (AC system) was found and characterized for unicellular eukaryotes--the ciliatae Dileptus anser. It has been first shown that hormones of higher eukaryotes--biogenic amines (adrenalin, isoproterenol and serotonin) and peptide glucagon--stimulate in dose-dependent manner the activity of adenylyl cyclase (AC) of D. anser. The enzymatic activity was stimulated also by guanine nucleotides--GTP and their non-hydrolysable analogue Gpp[NH]p. Stimulating effects of hormones and guanine nucleotides strongly depend on the level of AC basal activity, which is relatively easy to reach (1430 to 3900 pmol cAMP/min per 1 mg of protein). The sensitivity of D. anser AC system to hormones and guanine nucleotides shows the presence of receptor or receptor-related molecules, capable of interacting with the hormone and activating AC through heterotrimeric G-proteins, in ciliatae. On the base of obtained data, a conclusion is made about the similarity of the structural-functional organization of AC systems of D. anser and higher eukaryotes.     

The secondary structure of peptides derived from the third intracellular loop of the serpentine-type receptors and its interrelation with their biological activity

We and other authors have shown that synthetic peptides corresponding to regions of the third cytoplasmic loop (CL-3) of receptors of the serpentine type are capable of activating G-protein signaling cascades and triggering them in the absence of a hormone. To create selective regulators of hormonal signaling systems on the basis of these peptides, the relationship between their biological activity and secondary structure is studied. It is suggested that the most suitable is the helical conformation, which allows the peptide to effectively interact with signaling proteins. The goal of this study was to test the biological activity and secondary structure of linear peptides that we synthesized and their dimeric and palmitoylated analogs corresponding to the C-terminal region of CL-3 of luteinizing hormone receptor (LHR) and 5-hydroxytryptamine (serotonin) receptor of type 6 (Ser₆R). It is shown that LHR peptides at micromolar concentrations stimulate the basal activity of adenylyl cyclase (AC) and the GTP-binding of G-proteins in plasma membranes of rat testes, while Ser₆R peptides activate AC and G-proteins in synaptosomal membranes of rat brain. The action of peptides is tissue-specific and observed in tissues where there are homologous receptors. The most effective were palmitoylated peptides. LHR peptide reduced the AC stimulatory effect of human chorionic gonadotropin, while Ser₆R peptides, the effect of Ser₆R-agonist, EMD-386088, and the action of the peptides was not found in the case of nonhomologous receptors. Using circular dichroism spectroscopy, it is shown that in the neutral (pH 7) and acidic (pH 2) medium, all the peptides exist predominantly in the antiparallel β-sheet (37–42%) and disordered conformations (33–35%). In the alkaline medium (pH 10) in the case of palmitoylated peptides the increase of the contribution of the helical conformation to 12–27% was observed. In the presence of trifluoroethanol (10–80%), a helix-forming solvent, the contribution of helical conformation for the majority of peptides was slightly increased (for palmitoylated analogs by 14%); however, in this case, the antiparallel β-sheet and disordered conformation prevailed. The conclusion was drawn that the lack of a clearly expressed ability to form helices in peptides derived from CL-3 of receptors did not significantly affect their activity. This is consistent with the proposed mechanism of peptide action, whereby peptide interacts with the complementary regions of homologous receptor that does not require helix formation.     

The role of sulfhydryl groups in functioning of components of adenylate cyclase signal system in smooth muscles of the mollusk Anodonta cygnea (effect of N-ethylmaleimide and p-chloromercuribenzoic acid)]

The alkylating agent N-ethylameimide and the sulfhydryl group blocker p-chloromercuribenzoic acid (CPMA) inhibited in dose-dependent manner both basal activity of adenylyl cyclase (AC) and its activity stimulated by non-hormonal substances (forskolin, sodium fluoride, guanylilimidodiphosphate) in smooth muscles of the freshwater bivalve mollusk Anodonta cygnea. The double increase (from 30 to 60 min) in the time of preincubation of a sarcolemmal membrane fraction with ethylmaleimide and CPMA led to an essential increase in enzyme inhibition (especially for CPMA). 50 mM SH-containing reagent beta-mercaptoethanol (ME) partially restored the AC activity, inhibited by N-ethylmaleimide and CPMA, except when these two latter reagents were in high concentrations (1-10 and 0.5 mM, respectively). The data obtained point to the key role of cysteine SH-groups in regulation of the functional activity of proteins, components of the adenylyl cyclase system--AC and heterotrimeric G-proteins.     

A decrease in the sensitivity of adenylyl cyclase and heterotrimeric g proteins to chorionic gonadotrophin and peptide hormones action in the tissues of reproductive system of rats with experimental type 2 diabetes

Patients with different forms of the diabetes, particularly with insulin-independent type 2 diabetes, have a wide spectrum of the disturbances of the functions of reproductive system. It is suggested that the main reason of these disturbances is altered sensitivity of reproductive system tissues to the regulatory action of hormones. The aim of this study was the identification of the changes in functioning of adenylyl cyclase system (ACS) sensitive to human chorionic gonadotrophin (hCG) and the peptide hormones in the ovary, testes and uterus of rats with neonatal streptozotocin (STZ) diabetes that is similar to the type 2 diabetes in humans. The effects of hCG, PACAP-38 and relaxin, realizing their effects via stimulatory G proteins (G_s), and somatostatin, acting via the inhibitory G protein (G_i), on adenylyl cyclase (AC) activity and GTP binding to the G proteins were studied. In rats with STZ type 2 diabetes the regulatory effects of hCG and PACAP-38 decreased in the ovary and testes, while the effects of somatostatin decreased in all investigated tissues (especially in the uterus). This caused attenuation of the hormonal effects, stimulating (hCG and PACAP-38) or inhibiting (somatostatin) AC activity, and in the decrease of their stimulatory effect on the GTP binding. At the same time a significant decrease of ACS sensitivity to relaxin in the tissues of diabetic rats was not found. Data obtained suggest that one of the key reasons for impairments of reproductive functions in experimental type 2 diabetes is the decrease of ACS sensitivity to the hormones, hCG, PACAP-38 and somatostatin, which play an important role in the reproductive system functioning.