G. P. Vlasov

Studies of the molecular mechanisms of action of relaxin on the adenylyl cyclase signaling system using synthetic peptides derived from the LGR7 relaxin receptor.

The peptide hormone relaxin produces dose-dependent stimulation of adenylyl cyclase activity in rat tissues (striatum, cardiac and skeletal muscle) and the muscle tissues of invertebrates, i.e., the bivalve mollusk Anodonta cygnea and the earthworm Lumbricus terrestris, adenylyl cyclase stimulation being more marked in the rat striatum and cardiac muscle. Our studies of the type of relaxin receptor involved in mediating these actions of relaxin involved the first synthesis of peptides 619–629, 619–629-Lys(Palm), and 615–629, which are derivatives of the primary structure of the C-terminal part of the third cytoplasmic loop of the type 1 relaxin receptor (LGR7). Peptides 619–629-Lys(Palm) and 615–629 showed competitive inhibition of adenylyl cyclase stimulation by relaxin in rat striatum and cardiac muscle but had no effect on the action of relaxin in rat skeletal muscle or invertebrate muscle, which is evidence for the tissue and species specificity of their actions. On the one hand, this indicates involvement of the LGR7 receptor in mediating the adenylyl cyclase-stimulating action of relaxin in rat striatum and cardiac muscle and, on the other, demonstrates the existence of other adenylyl cyclase signal mechanisms for the actions of relaxin in rat skeletal muscle and invertebrate muscle, not involving LGR7 receptors. The adenylyl cyclase-stimulating effect of relaxin in the striatum and cardiac muscles was found to be decreased in the presence of C-terminal peptide 385–394 of the αs subunit of the mammalian G protein and to be blocked by treatment of membranes with cholera toxin. These data provide evidence that in the striatum and cardiac muscle, relaxin stimulates adenylyl cyclase via the LGR7 receptor, this being functionally linked with Gs protein. It is also demonstrated that linkage of relaxin-activated LGR7 receptor with the Gs protein is mediated by interaction of the C-terminal half of the third cytoplasmic loop of the receptor with the C-terminal segment of the αs subunit of the G protein.

Receptor of the serpentine-type and heterotrimeric G protein as targets of action of polylysine dendrimers

Molecular processes of the action of polycationic peptides that represent polylysine homo- and heterodendrimers on the functional activity of the biogenic amine- and peptide hormone-sensitive adenylyl cyclase signaling system (AC system) in rat myocardium and brains were studied. An intended use of these peptides is that of highly effective polymer carriers for biologically active substances. The polylysine homodendrimers of the third [(NH2)16(Lys)8(Lys)4(Lys)2Lys-Ala-NH2] (I), fourth [(NH2)32(Lys)16(Lys)8(Lys)4(Lys)2Lys-Ala-NH2] (II), and fifth [(NH2)64(Lys)32(Lys)16(Lys)8(Lys)4(Lys)2Lys-Ala-NH2] (III) generations, as well as polylysine heterodendrimers of the fifth generation, [(NH2)64(Lys-Glu)32(Lys-Glu)16(Lys-Glu)8(Lys-Glu)4(Lys-Glu)2Lys-Ala-Ala-Lys(ClAc)-Ala-NH2] (IV), [(NH2)64(Lys-Ala)32(Lys-Ala)16(Lys-Ala)8(Lys-Ala)4(Lys-Ala)2Lys-Ala-Lys(ClAc)-Ala-Ala-NH2] (V) and [(NH2)64(Lys-Gly-Gly)32(Lys-Gly-Gly)16(Lys-Gly-Gly)8(Lys-Gly-Gly)4(Lys-Gly-Gly)2Lys-Gly-Gly-Lys(ClAc)-Ala-Ala-NH2] (VI), interact with the C-terminal regions of α subunits of the heterotrimeric G proteins, preferably of the inhibitor type, and stimulate its activity in respector-independent manner. The most effective G-protein activators were homodendrimers II and III and heterodendrimer V. The polylysine dendrimers disturbed the functional coupling of receptors of biogenic amines and peptide hormones with Gi proteins and, to a lesser extent, Gs proteins. This was manifested as a decrease in the regulatory effects of the hormones on AC activity and the GTP binding of the G protein, as well as by a decrease in the affinity of receptors to agonists in the presence of polylysine dendrimers, which is a consequence of the dissociation of the receptor-G protein complex. It has also been shown that, based on their molecular mechanisms and selectivity of action on G proteins, polylysine dendrimers are similar to mastoparan and melittin, which are natural toxins of insect venom.

Effects of polycationic peptides of different natures on the functional state of the serotonin-regulated adenylate cyclase system in the rat brain

In animals of different phylogenetic levels, polycationic peptides are one of the most important regulators of the functional activities of hormonal signaling systems, including the adenylate cyclase signaling system (AC system). We compared the functional state of the AC system in the rat brain after treatment with three synthetic polycationic peptides (C-ɛAhx-YKAKKKKKKKWK (I), CK(C10)-ɛAhx-YKAKKKKKKKWK (II), and (NH2)64(K)32(K)16(K)8(K)4(K)2KA (III)) and the peptide toxin mastoparan, which do not have homology to signaling proteins, and the synthetic polycationic peptides ARERKATKTL255–264K (IV) and HSRKALKASL306–315K (V), which are derivatives of the C-terminal domains of the third cytoplasmic loop of the 1B and 6 serotonin receptors (SR). We found that the peptides I, II, and III stimulated heterotrimeric G-proteins of both the stimulating (Gs) and inhibiting (Gi) type, peptide IV and, mastoparan predominantly affected Gi-proteins, and peptide V mainly activated Gs-proteins. Peptides II and V stimulated basal AC activity, while mastoparan and peptides III and IV inhibited the forskolin-stimulated activity of the enzyme. The serotonin-induced stimulation of AC strongly decreased in the presence of peptide V, a derivative of SR of the sixth type coupled with Gs-proteins (the efficacy of the inhibitory effect of the peptides was V > II > III > IV ≅ mastoparan ≅ I). Inhibition of the AC system by serotonin decreased in the presence of peptide IV, a derivative of 1B SR coupled with Gi-proteins (the efficacy of inhibition was IV > III ≅ mastoparan > II ≥ I ≅ V). Inhibition of AC by the D2-agonist bromocriptine strongly decreased in the presence of mastoparan and peptide III, and peptide IV was less active (mastoparan ≥ III > IV > II > I ≅ V). Among the polycationic peptides studied peptides IV and V and SR derivatives caused the strongest inhibition of serotonin-stimulated GTP binding and specific binding of the hormone with membranes, and their effect was observed at a concentration of 10−6 M. Thus, in contrast to polycationic peptides that have no homology with signal proteins, the polycationic peptides and SR derivatives selectively affected the functional state of the AC system regulated by serotonin in the rat brain and transmission of hormonal signals.

Molecular mechanisms of interaction of polycationic peptides with G proteins.

The key stage in the transduction of a hormonal signal into a cell is the interaction of serpentine-type receptors with heterotrimeric G proteins. At this stage, as a result of activation of G proteins of different types, the signal path way is selected, which eventually determines cell response to the hormonal effect. In the past years, we and other authors obtained data indicating that G proteins may be activated via a receptor-independent pathway as a result of their interaction with natural polycationic peptides [1, 2]; with peptides that are derivatives of the cytoplasmic loops of receptors (for details, see [3]); and with polycationic compounds of an artificial origin that represent synthetic peptides [4–8], derivatives of positively charged amino acids [9, 10], or biopolymers containing amino groups [11]. Studying peptide and nonpeptide activators of G proteins mimicking the effect of receptors is a promising approach for the search and development of new-generation drugs that are able to regulate the activity of hormonal signal systems at the stage of functional coupling between receptor and G proteins. However, before the use of such activators in practice, it is necessary to understand the molecular mechanisms of their action on the G-protein molecules and the complex of G protein and receptor. This study is devoted to the determination of these mechanisms, poorly studied thus far. Earlier, we showed that the effectiveness with which synthetic peptides interact regulate the functional activity of proteins—the components of the adenylate cyclase signal system (ACSS)—is largely determined by their structural characteristics, such as the number and distribution of positively charged amino acid residues, the ability to form amphipathic helices, the presence in the peptide molecule of hydrophobic radicals, their number, and relative orientation [6–8]. The goal of this study was to investigate the molecular mechanisms of action of two polycationic peptides synthesized earlier [7, 8] on the receptor and coupling (G protein) components of ACSS sensitive to biogenic amines. For this purpose, we studied the effect of these peptides on the binding characteristics of receptors of biogenic amines and the GTP-binding activity of G proteins. Peptide I ( Cε Ahx-WKK(C 10 )-KKK(C 10 )KKKK(C 10 )-YKK(C 10 )-KK , where ε Ahx is the residue of ε -aminohexanoic acid and C 10 is the residue of capric acid) contained hydrophobic C 10 radicals, whereas peptide II ( [(GRGDSGRKKRRQRRRPPQ) 2 -Kε Ahx-C] 2 ) had a branched structure and represented a tetramer created on the basis of fragment 48–60 of HIV-1 TAT protein.

Peptides Derived from the Third Cytoplasmic Loop of the Serotonin Subtype 1B Receptor Selectively Inhibit Transmission of Serotoninergic Signals via Their Homologous Receptors

A leading role in the interactions of most serotonin-type hormone receptors with heterotrimeric G proteins is played by their third cytoplasmic loops. Studies in recent years have shown that synthetic peptides corresponding to the membrane-proximal parts of these loops may have selective influences on the transmission of hormone signals via their homologous receptors and trigger the signal cascade in the absence of hormone. We report the first synthesis of peptides derived from the C-terminal region of the third cytoplasmic loop of type 1B serotonin receptors, along with studies of their influences on the serotonin sensitivity of the adenylate cyclase system in the rat brain. Peptides 300–316 and 306–316 (numbered from amino acid positions in the rat serotonin subtype 1B receptor molecule) at micromolar concentrations, in the absence of hormone, stimulated GTP-binding Gi-proteins coupled with subtype 1B serotonin receptors and inhibited forskolin-stimulated adenylate cyclase activity. Studies using selective serotonin receptor agonists and antagonists showed that peptides 300–316 and 306–316 inhibit the transmission of the serotonin signal via the homologous receptor but have weak effects on other types of serotonin receptor. Peptide 300–316 were markedly more active than its shortened analog 306–316 in terms of the selectivity and efficacy of actions on the adenylate cyclase signaling system, which is regulated via subtype 1B serotonin receptors. These data provide evidence that region 300–316 of subtype 1B serotonin receptors is involved in the interaction with Gi proteins and includes the main molecular determinants responsible for transmission of the serotonin signal to adenylate cyclase.

Molecular mechanisms of interaction of polycationic peptides with serpentine type receptors and heterotrimeric G-proteins in rat tissues

The key step in the hormonal signal transduction into cell is interaction of receptors with heterotrimeric G-proteins. We and other authors have shown that G-proteins may be activated as a result of their direct interaction with polycationic peptides. The goal of this work was to study molecular mechanisms of effect of hydrophobic peptide I, C-εAhx-WKK(C10)-KKK(C10)-KKKK(C10)-YKK(C10)-KK, and branched peptide II, [(GRGDSGRKKRRQRRRPPQ)2-K-εAhx-C]2 including the 48–60 fragment of the HIV-1 TAT-protein, on receptor and G-protein. These two peptides (10−6−10−4 M) produced a dose-dependent simulation of the GTP-binding activity of G-proteins in plasma membrane fractions of the brain striatum and cardiac muscle in rats. The effect of peptide I was more pronounced and decreased to a considerable degree in the presence of the C-terminal 385–394 peptide of the G-protein αs-subunit that selectively disrupts interaction of receptors with Gs-protein. Peptide I reduced markedly affinity of serotonin (agonist) to the serotonin striatum receptors, whereas peptide II inhibited to the significant extent the binding of dihydroalprenolol (antagonist) to β-adrenergic receptors in cardiac muscle. Peptide I, unlike peptide II, decreased essentially the high affinity binding of β-agonist isoproterenol. The obtained data indicate the ability of polycationic peptides to activate G1-proteins, to disturb their coupling with receptor, and to affect binding properties of the receptor. There are differences in molecular mechanisms of action of peptides with different structures on G-proteins and receptors.

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 α-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.

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 α-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.