E. A. Shpakova

The Peptides Mimicking the Third Intracellular Loop of 5-Hydroxytryptamine Receptors of the Types 1B and 6 Selectively Activate G Proteins and Receptor-Specifically Inhibit Serotonin Signaling via the Adenylyl Cyclase System

The third intracellular loop (ICL3) of G protein-coupled receptors has, as a rule, a key role in their interaction with heterotrimeric G proteins. We synthesized peptides corresponding to the C-terminal region of the ICL3 (C-ICL3) of 5-hydroxytryptamine receptors of the type 1B (5-HT1BR) and 6 (5-HT6R) and studied their influence on the functional activity of adenylyl cyclase signaling system (ACSS) in synaptosomal membranes isolated from the rat brain. The 5-HT1BR-peptide ARERKATKTL307–316K-amide mimicking agonist-activated 5-HT1BR reduced forskolin-stimulated adenylyl cyclase (AC) activity and activated pertussis toxin-sensitive G proteins. It lowered inhibitory effects of serotonin and 5-HT1BR-agonists on forskolin-stimulated AC activity and their stimulating effects on GTP binding. This was not the case in the presence of 5-HT1BR-antagonists. The 5-HT6R-peptides mimicking 5-HT6R activated both the basal AC activity and GTP binding of cholera toxin-sensitive G proteins. They lowered the stimulating effect of serotonin and 5-HT6R-agonists on AC and Gs proteins, but in the presence of 5-HT6R-antagonists their action was blocked. Of all the 5-HT6R-peptides with linear and dimeric structure we studied the palmitoylated peptide KHSRKALKASL258–268K(Pal)A-amide had a most pronounced effect both on the basal and 5-HT6R-agonist-stimulated ACSS. The data was obtained indicating that the peptides corresponding to C-ICL3 of 5-HT1BR and 5-HT6R selectively activate Gi and Gs proteins, respectively, and in a receptor-specific manner reduce signal transduction via serotonin-sensitive ACSS in the rat brain. The results of the study give strong evidence in favor of active participation of C-ICL3 of these 5-HTRs in their coupling with the G proteins.

Receptor and tissue specificity of the effects of peptides corresponding to intracellular regions of the serpentine type receptors

Proximal regions of the third intracellular loop (ICL-3) are responsible for the interaction with heterotrimeric G proteins in most of the serpentine type receptors. The peptides corresponding to these regions are able to activate G proteins in the absence of hormone and to alter the transduction of hormonal signal via the respective homologous receptor. However, the molecular mechanisms of action of the peptides, their specificity to receptors and target tissues are currently not well understood. The goal of this work was to study the receptor and tissue specificity of peptides-derivatives of C-terminal regions of the ICL-3 of luteinizing hormone receptor (LHR), type 1 relaxin receptor (RXFP1), somatostatin receptors of types 1 and 2 (Som1R and Som2R), and 5-hydroxytryptamine receptors of subtype 1B and type 6 (5-HT1BR and 5-HT6R) on the functional activity of adenylyl cyclase (AC) and GppNHp-binding of G proteins in the brain, myocardium, and testis of rats. It was shown that the influence of peptides on AC and G proteins is well detected in tissues enriched in homologous receptors. The effects stimulating AC and GppNHp-binding were most pronounced in the testes for LHR peptide, in the brain for peptide 5-HT6R, and in all of the tested tissues (but mainly in the myocardium) for the RXFP1 peptide. The AC-inhibiting effects of peptides Som1R, Som2R and 5-HT1BR, as well as the stimulation of GppNHp binding induced by these peptides, were most pronounced in the brain. In the presence of the peptides, the AC effects of hormones acting via homologous receptors were significantly attenuated, while the AC effects of other hormones changed insignificantly. The findings suggest that biological activity of the peptides depends on their interaction with complementary regions of homologous receptors, which should be taken into account when developing highly selective regulators of hormonal signaling systems on the basis of these peptides.

Biological activity in vitro and in vivo of peptides corresponding to the third intracellular loop of thyrotropin receptor

64 In recent years, one of the rapidly developing directions in molecular endocrinology and nanobioo technology is the peptide strategy, which has become widely used for studying the molecular mechanisms of transduction of hormonal signals into the cell and for designing highly selective and highly effective regg ulators of hormonal signaling systems [1, 2]. It is based on the search and development of peptides corr responding in structure to the functionally important parts of signaling proteins—receptors, heterotrimeric GGproteins, and enzymes that generate second mess sengers. The most promising direction in the peptide strategy is the synthesis and study of peptides derived from serpentineetype receptors, because at the level of these receptors not only the specific recognition of an external signal but also the choice of the pathway of its transduction into the cell takes place and, therefore, the intracellular targets of the hormone are deterr mined. We and other authors demonstrated that pepp tides corresponding to the cytoplasmic loops (CLs) of serpentineetype receptors selectively interact with G proteins, trigger signaling cascades in the absence of hormone, affect the transduction of the signal generr ated by them through a homologous receptor, thus functioning as regulators of intracellular signaling [3– 9]. One of the urgent problems of modern endocrinol ogy is the search for an effective regulator of the thyy roid gland and the entire hypothalamiccpituitaryythyy roid axis, which act at the stage of activation of the thyroiddstimulating hormone (TSH) receptor by the hormone. Disturbances that occur at this signal transs duction stage lead to a wide spectrum of thyroid gland diseases, including autoimmune thyroiditis and thyy roid cancer. The purpose of the study was to develop selective regulators of the TSHHsensitive signaling pathways on the basis of the peptides derived from the CCterminal region 612–627 of the third intracellular loop (CCICLL3) of the TSH receptor. We also studied the activity of the peptides in vitro by their effects on the basal and hormoneestimulated level of GTPPbindd ing of heterotrimeric G s and G q proteins, which are components of these cascades, and in vivo by the effect of intranasal administration of peptides on the level of thyroid hormones in the blood plasma of experimental animals. It should be noted that mutations in the CCICLL3 of the TSH receptor disturb its interaction with G s proteins and lead to loss of the ability of the mutant receptor to stimulate adenylate cyclase activity …

Peptide 612–627 of thyrotropin receptor and its modified analogs as regulators of adenylyl cyclase in rat thyroid gland

The specific activity of the thyroid gland is regulated by thyroid-stimulating hormone (TSH) via TSH receptor (TSHR). This receptor is coupled with various types of G proteins, including Gs proteins, through which TSH stimulates activity of enzyme adenylyl cyclase (AC). Since the use of TSH in medicine is restricted, selective regulators of TSHR with activity of agonists and antagonists are being developed. One approach to their creation is development of peptides corresponding to the functionally important TSHR regions that are located in its cytoplasmic loops and are involved in binding and activation of G proteins. We synthesized peptide corresponding to the C-terminal region 612–627 of the third cytoplasmic loop of TSHR and its derivatives modified by residues of palmitic acid (from the N- or C-termini) or by polylysine dendrimer (from the N-terminus) and we studied their effect on the basal and TSH-stimulated AC activity in membrane fractions isolated from the rat thyroid gland. The most active was peptide 612–627-K(Pal)A modified by palmitate from the C-terminus, where the hydrophobic transmembrane region is located in TSHR. At the micromolar concentrations, it increased AC activity and reduced the AC-stimulating TSH effect. The action of 612–627-K(Pal)A was directed to its homologous TSHR, which is indicated by the following facts: the ingibition of Gs proteins, the transductory component of the AC system, by treatment of the membranes with cholera toxin blocked the AC effect of peptide and this effect was not revealed in the tissues where TSHR are absent, the peptide did not influence the stimulating AC effects of hormones acting via other receptors. The unmodified peptide and the peptide with N-terminal dendrimer had a much lower ability to activate AC in the thyroid gland than 612–627-K(Pal)A, whereas the peptide modified by palmitate from the N-terminus was inactive. At the same time, the peptide modified by dendrimer was comparable with 612–627-K(Pal)A by the ability to inhibit the AC action of TSH, but it also decreased (although to the lesser degree) the AC effects of other hormones, which indicates its low receptor specificity. Thereby, the obtained data indicate the high efficiency of the peptide 612–627-K(Pal)A as a regulator of TSHR and the possibilities of creation of drugs on its basis for regulation of thyroid-gland functions in the case of pathology.

Regulation of adenylyl cyclase activity in rat testes by acylated derivatives of peptide 562–572 of a luteinizing hormone receptor

One area of the search for hormonal signaling systems regulators is development of peptides that correspond to the cytoplasmic regions of G protein-coupled receptors (GPCR). Modification of such peptides with hydrophobic radicals increases their efficiency and selectivity. However, at present it has not been studied how the activity of the peptide depends on the localization of hydrophobic radicals, their number, and chemical nature. The aim of this work consisted in synthesis of peptide 562–572 derivatives modified by fatty-acid radicals and corresponding to the C-terminal region of the luteinizing hormone receptor (LHR) and in the study of regulatory effects of the acylated LHR peptides on the basal and hormone-stimulated activity of adenylyl cyclase (AC) in rat tissues. To elucidate the effects of localization of hydrophobic radicals and of their number, modifications of peptide 562–572 were carried out only at the N-or at the C-terminus or at both ends. To study the effect of hydrophobicity, residues of palmitic (Pal) and decanoic (Dec) acids were chosen. Using a solid-phase strategy synthesis was performed of the unmodified peptide NKDTKIAKK-Nle-A562-572-KA (1) and five of its acylated analogues, N[K(Dec)]DTKIAKK-Nle-A562-572-KA (2), NKDTKIAKK-Nle-A562-572-[K(Dec)]A (3), N[K(Dec)]DTKIAKK-Nle-A562-572-[K(Dec)]A (4), N[K(Pal)]DTKIAKK-Nle-A562-572-KA (5), and NKDTKIAKK-Nle-A562-572-[K(Pal)]A (6). Peptide 6 modified with palmitate at the C-terminus to a large extent increased the basal AC activity and reduced the AC stimulating effect of human chorionic gonadotropin (hCG) in testes of rats; peptides 3 and 4 modified with decanoate at the C-terminus were less effective, but exceeded in activity the unmodified peptide 1; and peptides 2 and 5 acylated at the N-terminus were little active. The action of peptides was characterized by tissue and the receptor specificity. Thus, modification of the LHR peptide 562–572 with fatty-acid radicals at the C-terminus enhances its regulatory effect on the functional activity of the adenylyl cyclase system in rat testes, which indicates a promising modification of GPCR peptides with hydrophobic radicals. These data confirm the hypothesis that the hydrophobic radical is to be localized in the locus of GPCR peptide, where a transmembrane domain is located in the receptor.

The functional activity of the adenylate cyclase system in the brains of rats with metabolic syndrome induced by immunization with peptide 11–25 of the type 4 melanocortin receptor

The melanocortin system of the brain, which includes melanocortin receptors of the fourth type (M4R), plays a key role in the regulation of energy homeostasis and controls functions of the nervous system. Inhibition of M4R results in obesity and the metabolic syndrome, which presumably occur due to changes in the neuromediator systems of the brain. To examine this hypothesis, we examined the effect of long-term immunization of rats with the BSA-conjugated K-[TSLHLWNRSSHGLHG11–25]-A peptide (K-[11–25]-A), which corresponds to the extracellular N-terminal domain of M4R, on the activity of the hormone-sensitive adenylate cyclase signaling system (ACSS) of the brain. In rats that were immunized numerous times with the BSA conjugate (the I group), we observed an increase in body weight, impaired glucose tolerance, insulin resistance, and dyslipidemia. At 13 months after the beginning of the experiment, we evaluated the ACSS activity in synaptosomal membranes from the brain. The basal activity of AC and its regulation by GppNHp and forskolin did not differ from the control. In the I group both the AC-stimulating effects of the α-melanocyte-stimulating hormone (α-MSH), M4R-agonist THIQ, dopamine, and pituitary AC-activating polypeptide and the AC-inhibiting effects of serotonin and 5-nonyloxytryptamine, an agonist of 5-hydroxytryptamine receptor (5-HTR) of the 1B/1D-subtype, decreased. The affinity of M4R to agonists did not change. The AC-stimulating effect of the M3R agonist γ-MSH was enhanced, which is a compensation for the weakening of M4R functions. The AC-stimulating effects of serotonin, EMD-386088, an agonist of 6 type 5-HTR relaxin, and noradrenaline, as well as the inhibitory effects of noradrenaline, the D2-agonist bromocriptine, and somatostatin in the I group did not change. Thus, inhibition of M4R as a result of immunization with the BSA conjugate of the K-[11–25]-A peptide alters the sensitivity of the ACSS of the rat brain to peptides of melanocortin family and other neurohormones. These alterations are characterized by hormonal and receptor specificity and may be one of the causes of insulin resistance, metabolic syndrome, and functional disturbances in the CNS and at the periphery under conditions of M4R deficit.

The metabolic changes in rats immunized with BSA conjugate of peptides derived from the N-terminal region of type 4 melanocortin receptor

163 To study the etiology and pathogenesis of diseases associated with metabolic disorders, their experimenn tal models are required. A promising approach for cree ating them is to induce changes in hormonal signaling systems of the brain and peripheral tissues responsible for the regulation and control of metabolic processes. It was found that disturbances in the functional activv ity of the type 4 melanocortin receptor (М 4 R) in the brain lead to changes in eating behavior, obesity, and metabolic disorders, which are determined by М 4 R dependent central mechanisms [1, 2]. These disorders can be caused by inactivating mutations in М 4 R, longg term treatment with synthetic М 4 R antagonists, or increased production of the agoutiilike peptide, an endogenous М 4 R antagonist [3, 4]. In this work, to simulate and study the metabolic disorders caused by М 4 R inhibition, we developed the strategy of repeated immunization of experimental animals (rats) with a synthetic peptide that corresponds to the extracellular М 4 R region responsible for recognizing and binding М 4 R agonists. In developing this strategy, we proceeded from the fact that the antibodies generated against the extracellular regions of other serpentine receptors spee cifically inhibit the binding of agonists to these recepp tors and remove the latter from signal transduction, leading to the development of autoimmune diseases [5–7]. For immunization, we used the previously synn thesized peptide KK[TSLHLWNRSSHGLHG 11–25 ]] Aamide (K[11–25]A), which structurally corree sponds to the extracellular NNterminal region of rat М 4 R. To improve the immunization efficiency, the peptide was conjugated to bovine serum albumin (BSA) [8, 9]. The goal of this work was to examine the effect of longgterm immunization of male rats with the BSA conjugate of peptide KK[11–25]]A on the body weight, metabolic parameters (blood glucose and insulin levels, glucose tolerance, and lipid status), and the level of thyroid hormones regulating the metabolic processes in the CNS and peripheral tissues. Immunii zation was started at an age of 1.5 months and was perr formed several times during the year. Peptide KK[11–25]]A was obtained by soliddphase synthesis on paramethylbenzhydrylamine resin (capacity, 1.16 mmol/g) using protected Nαtert butyloxycarbonyl amino groups as described previi ously [10]. To form the peptide bond, 11oxybenzotriaa zolyl esters of amino acids were used, which were obtained using N,Ndiisopropyl carbodiimide. Pepp tide KK[11–25]]A was conjugated with BSA by conn densation with glutaraldehyde. For this …

Biological activity of lipophilic derivatives of peptide 562-572 of rat luteinizing hormone receptor.

248 One of the directions in the research of highly active regulators of hormonal signaling systems is the design of peptides whose primary structure corree sponds to the structure of cytoplasmic loops of the serr pentineetype receptors, which are able to modulate and regulate the functional activity of homologous receptors [1–4]. Earlier, we and other authors showed that modification of such peptides with lipophilic radd icals leads to an increased potency and selectivity of their action compared to the unmodified analogues [5–10]. For example, when acylated at the C terminus with the palmitic acid residue, peptide 612–627, which corresponds to the third cytoplasmic loop of the thyroid stimulating hormone receptor, had a higher biological activity in vitro compared to the unmodified analogues and stimulated the secretion of thyroid horr mones in the thyroid gland in vivo [10]. However, it is still obscure how the biological activity of this peptide depends on the location of lipophilic residues, their number, and their chemical nature. It was assumed that the hydrophobic radical mimics the transmemm brane domain of the receptor, which is located adjaa cent to the cytoplasmic part corresponding to the pepp tide [3, 4]. As a result, the hydrophobic radical should be located in the peptide segment proximal to the membrane and should be comparable in size to the transmembrane domain, thus ensuring the proper orii entation and anchoring of the modified peptide in the membrane. To test this hypothesis, we synthesized the lipoo philic derivatives of peptide 562–572, corresponding to the CCterminal region of the rat luteinizing horr mone receptor (LHR), and performed a comparative study of their effect on the basal activity of adenylyl cyclase (AC), the catalytic component of adenylyl cyclase signaling system (ACSS), and the regulation of AC by human chorionic gonadotropin (hCG) in rat testicular membranes. Human chorionic gonadotroo pin is a structural and functional homologue of LH; it binds specifically to LHR and activates AC through the heterotrimeric stimulatory G proteins [11]. It should be noted that LH plays a key role in regulating the synthesis and secretion of steroid hormones by reproductive tissue and controls spermatogenesis, foll liculogenesis, and oogenesis [12, 13]. To determine the effect of localization of hydrophobic radicals and their numbers on the activity of peptide 562–572, we implemented its modification only at the N or C terr minus or at both termini. To study the effect of radical hydrophobicity, we …

Palmitoylated Peptide 562-572 of Luteinizing Hormone Receptor Increases Testosterone Level in Male Rats

We studied the effect of intraperitoneal and intratesticular administration of NKDTKIAKKNle-A562-572 peptide and its palmotoylated analog NKDTKIAKK-Nle-A562-572-K(Palm)A to male rats on adenylate cyclase activity in the testicular membranes in vitro and on plasma testosterone levels. Peptide NKDTKIAKK-Nle-A562-572-K(Palm)A stimulated basal adenylate cyclase activity and reduced activity of the enzyme in testicular membranes stimulated by chorionic gonadotropin. After intratesticular administration in a dose of 200 μg/kg, it significantly increased testosterone so that 1, 3 and 5 h after administration its level was increased by 74, 44 and 35%, respectively. Administered intraperitoneally in a lower dose (50 μg/kg), the peptide had little effect on testosterone level. Unmodified peptide was inactive.

Effect of Peptides Corresponding to Extracellular Domains of Serotonin 1B/1D Receptors and Melanocortin 3 and 4 Receptors on Hormonal Regulation of Adenylate Cyclase in Rat Brain

The ligand-recognizing part of G protein-coupled receptors consists of their extracellular loops and N-terminal domain. Identifi cation of these sites is essential for receptor mapping and for the development and testing of new hormone system regulators. The peptides corresponding by their structure to extracellular loop 2 of serotonin 1B/1D receptor (peptide 1), extracellular loop 3 of melanocortin 3 receptor (peptide 2), and N-terminal domain of melanocortin 4 (peptide 3) were synthesized by the solid-phase method. In synaptosomal membranes isolated from rat brain, peptide 1 (10–5-10–4 M) attenuated the effects of 5-nonyloxytryptamine (selective agonist of serotonin 1B/1D receptor) and to a lesser extent serotonin and 5-methoxy-N,N-dimethyltryptamine acting on all the subtypes of serotonin receptor 1. Peptide 2 (10–5-10–4 M) signifi cantly reduced the adenylate cyclase-stimulating effect of γ-melanocyte-stimulating hormone (agonist of melanocortin receptor 3), but had no effect on the adenylate cyclase effect of THIQ (agonist melanocortin receptor 4). Peptide 3 reduced the adenylate cyclase-stimulating effects of THIQ and α-melanocyte-stimulating hormone (non-selective agonist of melanocortin receptors 3 and 4), but did not modulate the effect of γ-melanocyte-stimulating hormone. The effect of peptide 3 was weaker: it was observed at peptide 3 concentration of 10–4 M. Peptides 1-3 did no change the adenylate cyclase-modulating effects of hormones acting through non-homologous receptors. Thus, the synthesized peptides specifi cally inhibited the regulatory effects of hormones acting through homologous receptors. This suggests that the corresponding extracellular domains are involved in ligand recognition and binding and determine functional activity of the receptor.