Design and synthesis of fluorescent probes for GPR54

Kisspeptins are neuropeptides that induce the secretion of gonadotropin-releasing hormone via the activation of the cognate receptor, G-protein coupled receptor 54 (GPR54). The kisspeptin–GPR54 axis is associated with the onset of puberty and the maintenance of the reproductive system. In this study, several fluorescent probes have been designed and synthesized for rat GPR54 through the modification of the N-terminus of rat kisspeptins to allow for the visualization of the expression and localization of kisspeptin receptor(s) in living cells and native tissues. The tetramethylrhodamine (TMR) and rhodamine green (RG)-labeled kisspeptins exhibited good binding and agonistic activities towards GPR54, and the results of the application studies demonstrated that these fluorescent probes could be used effectively for the detection of GPR54 receptors in flow cytometry and confocal microscopy experiments.     

Kisspeptins and the control of gonadotropin secretion in male and female rodents

Kisspeptins, the products of KiSS-1 gene acting via G protein-coupled receptor 54 (GPR54), have recently emerged as fundamental gatekeepers of gonadal function by virtue of their ability to stimulate gonadotropin secretion. Indeed, since the original disclosure of the reproductive facet of the KiSS-1/GPR54 system, an ever-growing number of studies have substantiated the extraordinary potency of kisspeptins to elicit gonadotropin secretion in different mammalian species, under different physiologic and experimental conditions, and through different routes of administration. In this context, studies conducted in laboratory rodents have been enormously instrumental to characterize: (i) the primary mechanisms of action of kisspeptins in the control of gonadotropin secretion; (ii) the pharmacological consequences of acute vs. continuous activation of GPR54; (iii) the roles of specific populations of kisspeptin-producing neurons at the hypothalamus in mediating the feedback effects of sex steroids; (v) the function of kisspeptins in the generation of the pre-ovulatory surge of gonadotropins; and (iv) the influence of sex steroids on GnRH/gonadotropin responsiveness to kisspeptins. While some of those aspects of kisspeptin function will be covered elsewhere in this Special Issue, we summarize herein the most salient data, obtained in laboratory rodents, that have helped to define the physiologic roles and putative pharmacological implications of kisspeptins in the control of male and female gonadotropic axis.     

Expression of a functional g protein-coupled receptor 54-kisspeptin autoregulatory system in hypothalamic gonadotropin-releasing hormone neurons

The G protein-coupled receptor 54 (GPR54) and its endogenous ligand, kisspeptin, are essential for activation and regulation of the hypothalamic-pituitary-gonadal axis. Analysis of RNA extracts from individually identified hypothalamic GnRH neurons with primers for GnRH, kisspeptin-1, and GPR54 revealed expression of all three gene products. Also, constitutive and GnRH agonist-induced bioluminescence resonance energy transfer between Renilla luciferase-tagged GnRH receptor and GPR54 tagged with green fluorescent protein, expressed in human embryonic kidney 293 cells, revealed heterooligomerization of the two receptors. Whole cell patch-clamp recordings from identified GnRH neurons showed initial depolarizing effects of kisspeptin on membrane potential, followed by increased action potential firing. In perifusion studies, treatment of GT1-7 neuronal cells with kisspeptin-10 increased GnRH peak amplitude and duration. The production and secretion of kisspeptin in cultured hypothalamic neurons and GT1-7 cells were detected by a specific RIA and was significantly reduced by treatment with GnRH. The expression of kisspeptin and GPR54 mRNAs in identified hypothalamic GnRH neurons, as well as kisspeptin secretion, indicate that kisspeptins may act as paracrine and/or autocrine regulators of the GnRH neuron. Stimulation of GnRH secretion by kisspeptin and the opposing effects of GnRH on kisspeptin secretion indicate that GnRH receptor/GnRH and GPR54/kisspeptin autoregulatory systems are integrated by negative feedback to regulate GnRH and kisspeptin secretion from GnRH neurons.     

Physical association of GPR54 C-terminal with protein phosphatase 2A

KiSS1 was discovered as a metastasis suppressor gene and subsequently found to encode kisspeptins (KP), ligands for a G protein coupled receptor (GPCR), GPR54. This ligand-receptor pair was later shown to play a critical role in the neuro-endocrine regulation of puberty. The C-terminal cytoplasmic (C-ter) domain of GPR54 contains a segment rich in proline and arginine residues that corresponds to the primary structure of four overlapping SH3 binding motifs. Yeast two hybrid experiments identified the catalytic subunit of protein phosphatase 2A (PP2A-C) as an interacting protein. Pull-down experiments with GST fusion proteins containing the GPR54 C-ter confirmed binding to PP2A-C in cell lysates and these complexes contained phosphatase activity. The proline arginine rich segment is necessary for these interactions. The GPR54 C-ter bound directly to purified recombinant PP2A-C, indicating the GPR54 C-ter may form complexes involving the catalytic subunit of PP2A that regulate phosphorylation of critical signaling intermediates.     

The metastasis suppressor gene KiSS-1 encodes kisspeptins, the natural ligands of the orphan G protein-coupled receptor GPR54.

Natural peptides displaying agonist activity on the orphan G protein-coupled receptor GPR54 were isolated from human placenta. These 54-, 14,- and 13-amino acid peptides, with a common RF-amide C terminus, derive from the product of KiSS-1, a metastasis suppressor gene for melanoma cells, and were therefore designated kisspeptins. They bound with low nanomolar affinities to rat and human GPR54 expressed in Chinese hamster ovary K1 cells and stimulated PIP(2) hydrolysis, Ca(2+) mobilization, arachidonic acid release, ERK1/2 and p38 MAP kinase phosphorylation, and stress fiber formation but inhibited cell proliferation. Human GPR54 was highly expressed in placenta, pituitary, pancreas, and spinal cord, suggesting a role in the regulation of endocrine function. Stimulation of oxytocin secretion after kisspeptin administration to rats confirmed this hypothesis.     

KiSS-1 and GPR54 at the pituitary level: overview and recent insights

Since the stimulatory effect of kisspeptin on gonadotropin secretion is blocked by a GnRH antagonist, it has been suggested that the effect of kisspeptin is manifest exclusively at the level of hypothalamic GnRH secretion. However, kisspeptins are present in ovine hypophysial portal blood suggesting that the pituitary gland may be a target of kisspeptin. Dual fluorescence labeling with a specific mouse monoclonal antibody against LHbeta demonstrates that KiSS-1 and GPR54 are expressed by the gonadotrophs. Different paradigms were designed in animals and in humans in vivo to elucidate its role. However, in vitro studies assessing the direct stimulatory effects of kisspeptins on gonadotropin secretion in the pituitary have given conflicting results, depending on the hormonal (GnRH and/or estradiol) environment of the cells. Kisspeptins alone seem unable to induce the LH surge. It is therefore likely that kisspeptin has a synergic effect with GnRH and estradiol, at both hypothalamic and pituitary levels. However, kisspeptin may also play another role, distinct from that restricted to the reproductive axis. In this paper, we shall also review data on the potential role of kisspeptin in the control of other pituitary functions, e.g. somatotroph and lactotroph. Finally, kisspeptins could act as endocrine/autocrine/paracrine signals in modulating hormonal secretions of the anterior pituitary.     

Molecular cloning of the bullfrog kisspeptin receptor GPR54 with high sensitivity to Xenopus kisspeptin

Kisspeptin and its receptor, GPR54, play important roles in mammalian reproduction and cancer development. However, little is known about their function in nonmammalian species. In the present study, we have isolated the cDNA encoding the kisspeptin receptor, GPR54, from the bullfrog, Rana catesbeiana. The bullfrog GPR54 (bfGPR54) cDNA encodes a 379-amino acid heptahelical G protein-coupled receptor. bfGPR54 exhibits 45-46% amino acid identity with mammalian GPR54s and 70-74% identity with fish GPR54s. RT-PCR analysis showed that bfGPR54 mRNA is highly expressed in the forebrain, hypothalamus and pituitary. Upon stimulation by synthetic human kisspeptin-10 with Phe-amide residue at the C-terminus (h-Kiss-10F), bfGPR54 induces SRE-luc activity, a PKC-specific reporter, evidencing the PKC-linked signaling pathway of bfGPR54. Using a blast search, we found a gene encoding a kisspeptin-like peptide in Xenopus. The C-terminal decapeptide of Xenopus kisspeptin shows higher amino acid sequence identity to fish Kiss-10s than mammalian Kiss-10s. A synthetic Xenopus kisspeptin peptide (x-Kiss-12Y) showed a higher potency than mammalian Kiss-10s in the activation of bfGPR54. This study expands our understanding of the physiological roles and molecular evolution of kisspeptins and their receptors.     

Structure-activity relationship study on small peptidic GPR54 agonists

Metastin (kisspeptin-54) is an endogenous ligand that modulates gonadotropin-releasing hormone (GnRH) secretion through the interaction with a G protein-coupled receptor (GPCR), GPR54. The short-chain C-terminal decapeptide amide, metastin (45-54) (kisspeptin-10), exerts the identical bioactivities to metastin, such as metastasis suppression of cancer cells and inhibition of trophoblast migration and invasion. In order to understand the structural requirement for GPR54 agonistic activity, structure-activity relationship (SAR) study on pentapeptide-based C-terminal metastin analogues was carried out. As a result, H-Amb-Nal(2)-Gly-Leu-Arg-Trp-NH2 34 was identified as a novel GPR54 agonist that possessed the most potent GPR54 agonistic activity reported so far.     

Structure-activity relationship study and NMR analysis of fluorobenzoyl pentapeptide GPR54 agonists

GPR54 is a Gq-protein coupled receptor involved in cancer metastasis and regulation of the endocrine system. GPR54 activation by endogenous ligands attenuates the mobility of carcinomas and stimulates the secretion of gonadotropin-releasing hormone. GPR54 agonists are, therefore, potential therapeutic candidates for cancer metastasis and hormonal diseases. Pentapeptide derivatives of kisspeptin C-terminus were identified as potent GPR54 agonists in our previous studies. In the present study, we investigated the structure-activity relationship of a variety of pentapeptides having various fluorine-substituted benzoyl groups at the N-terminus. Among these, a 4-fluorobenzoyl derivative was the most potent agonist. On the other hand, the derivatives having multiple fluoro-substituting groups showed less binding affinity. NMR analysis of these peptides and their N-terminal partial structures suggested that fluorine substituents affect the benzoyl conformation. o-Monofluorobenzoyl is likely to be in a coplanar conformation due to the intramolecular CF--HN hydrogen bonding between o-fluorine and amide hydrogen; the o,o-difluorobenzoyl moiety exists in a distorted conformation probably due to the steric hindrance and/or electrostatic repulsion between two o-fluorine atoms and carbonyl oxygen.     

GPR54 (KISS1R) transactivates EGFR to promote breast cancer cell invasiveness

Kisspeptins (Kp), peptide products of the Kisspeptin-1 (KISS1) gene are endogenous ligands for a G protein-coupled receptor 54 (GPR54). Previous findings have shown that KISS1 acts as a metastasis suppressor in numerous cancers in humans. However, recent studies have demonstrated that an increase in KISS1 and GPR54 expression in human breast tumors correlates with higher tumor grade and metastatic potential. At present, whether or not Kp signaling promotes breast cancer cell invasiveness, required for metastasis and the underlying mechanisms, is unknown. We have found that kisspeptin-10 (Kp-10), the most potent Kp, stimulates the invasion of human breast cancer MDA-MB-231 and Hs578T cells using Matrigel-coated Transwell chamber assays and induces the formation of invasive stellate structures in three-dimensional invasion assays. Furthermore, Kp-10 stimulated an increase in matrix metalloprotease (MMP)-9 activity. We also found that Kp-10 induced the transactivation of epidermal growth factor receptor (EGFR). Knockdown of the GPCR scaffolding protein, β-arrestin 2, inhibited Kp-10-induced EGFR transactivation as well as Kp-10 induced invasion of breast cancer cells via modulation of MMP-9 secretion and activity. Finally, we found that the two receptors associate with each other under basal conditions, and FRET analysis revealed that GPR54 interacts directly with EGFR. The stability of the receptor complex formation was increased upon treatment of cells by Kp-10. Taken together, our findings suggest a novel mechanism by which Kp signaling via GPR54 stimulates breast cancer cell invasiveness.     

The neuroanatomy of the kisspeptin system in the mammalian brain

The kisspeptin precursor is the protein transcribed from the Kiss-1 gene and the kisspeptins are the peptides that are posttranslationally processed from the precursor. The kisspeptins activate the G-protein coupled receptor GPR54 and are strongly implicated in puberty onset and in regulation of the hypothalamo-pituitary gonadal axis in mammals. Physiological studies have indicated that these effects occur via a direct activation of the GnRH neurons, and at an unknown site in the median eminence or directly on the gonadotropes. Paradoxically, while the function of kisspeptin is relatively well understood, little data are available about the localization of kisspeptin neurons in the brain, and in particular the projection patterns of kisspeptin containing axons implicated in regulation of the hypothalamo-pituitary gonadal axis. This review covers the current information about the localization of kisspeptin neurons in the mammalian brain and discusses the facts and artifacts of the methods of their detection. The available data suggest that kisspeptins are synthesized in neurons in the anteroventral periventricular nucleus and the arcuate nucleus. Both populations are considered to be involved in control of gonadotropes. In addition, kisspeptin nerve terminals and receptors are found in other hypothalamic area suggesting that kisspeptins are involved in regulation of other yet unknown homeostatic or neuroendocrine functions.     

Identification of the GPR55 agonist binding site using a novel set of high-potency GPR55 selective ligands

Marijuana is the most widely abused illegal drug, and its spectrum of effects suggests that several receptors are responsible for the activity. Two cannabinoid receptor subtypes, CB1 and CB2, have been identified, but the complex pharmacological properties of exogenous cannabinoids and endocannabinoids are not fully explained by their signaling. The orphan receptor GPR55 binds a subset of CB1 and CB2 ligands and has been proposed as a cannabinoid receptor. This designation, however, is controversial as a result of recent studies in which lysophosphatidylinositol (LPI) was identified as a GPR55 agonist. Defining a biological role for GPR55 requires GPR55 selective ligands that have been unavailable. From a β-arrestin, high-throughput, high-content screen of 300000 compounds run in collaboration with the Molecular Libraries Probe Production Centers Network initiative (PubChem AID1965), we identified potent GPR55 selective agonists. By modeling of the GPR55 activated state, we compared the GPR55 binding conformations of three of the novel agonists obtained from the screen, CID1792197, CID1172084, and CID2440433 (PubChem Compound IDs), with that of LPI. Our modeling indicates the molecular shapes and electrostatic potential distributions of these agonists mimic those of LPI; the GPR55 binding site accommodates ligands that have inverted-L or T shapes with long, thin profiles that can fit vertically deep in the receptor binding pocket while their broad head regions occupy a horizontal binding pocket near the GPR55 extracellular loops. Our results will allow the optimization and design of second-generation GPR55 ligands and provide a means for distinguishing GPR55 selective ligands from those interacting with cannabinoid receptors.     

Lipid derivatives activate GPR119 and trigger GLP-1 secretion in primary murine L-cells

Aims/hypothesisGlucagon-like peptide-1 (GLP-1) is an incretin hormone derived from proglucagon, which is released from intestinal L-cells and increases insulin secretion in a glucose dependent manner. GPR119 is a lipid derivative receptor present in L-cells, believed to play a role in the detection of dietary fat. This study aimed to characterize the responses of primary murine L-cells to GPR119 agonism and assess the importance of GPR119 for the detection of ingested lipid.MethodsGLP-1 secretion was measured from murine primary cell cultures stimulated with a panel of GPR119 ligands. Plasma GLP-1 levels were measured in mice lacking GPR119 in proglucagon-expressing cells and controls after lipid gavage. Intracellular cAMP responses to GPR119 agonists were measured in single primary L-cells using transgenic mice expressing a cAMP FRET sensor driven by the proglucagon promoter.ResultsL-cell specific knockout of GPR119 dramatically decreased plasma GLP-1 levels after a lipid gavage. GPR119 ligands triggered GLP-1 secretion in a GPR119 dependent manner in primary epithelial cultures from the colon, but were less effective in the upper small intestine. GPR119 agonists elevated cAMP in ∼70% of colonic L-cells and 50% of small intestinal L-cells.Conclusions/interpretationGPR119 ligands strongly enhanced GLP-1 release from colonic cultures, reflecting the high proportion of colonic L-cells that exhibited cAMP responses to GPR119 agonists. Less GPR119-dependence could be demonstrated in the upper small intestine. In vivo, GPR119 in L-cells plays a key role in oral lipid-triggered GLP-1 secretion.     

Photoaffinity labeling of insect nicotinic acetylcholine receptors with a novel [(3)H]azidoneonicotinoid

The nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel in the insect CNS and a target for major insecticides. Here we use photoaffinity labeling to approach the functional architecture of insect nAChRs. Two candidate 5-azido-6-chloropyridin-3-yl photoaffinity probes are evaluated for their receptor potencies: azidoneonicotinoid (AzNN) with an acyclic nitroguanidine moiety; azidodehydrothiacloprid. Compared to their non-azido parents, both probes are of decreased potencies at Drosophila (fruit fly) and Musca (housefly) receptors but AzNN retains full potency at the Myzus (aphid) receptor. [(3)H]AzNN was therefore radiosynthesized at high specific activity (84 Ci/mmol) as a novel photoaffinity probe. [(3)H]AzNN binds to a single high-affinity site in Myzus that is competitively inhibited by imidacloprid and nicotine and further characterized as to its pharmacological profile with various nicotinic ligands. [(3)H]AzNN photoaffinity labeling of Myzus and Homalodisca (leafhopper) detects a single radiolabeled peak in each case displaceable with imidacloprid and nicotine and with molecular masses corresponding to approximately 45 and approximately 56 kDa, respectively. The photoaffinity-labeled receptor in both Drosophila and Musca has imidacloprid- and nicotine-sensitive profiles and migrates at approximately 66 kDa. These photoaffinity-labeled polypeptides are considered to be the insecticide-binding subunits of native insect nAChRs.     

Development of bifunctional photoactivatable benzophenone probes and their application to glycoside substrates

Photoaffinity labeling is used to covalently attach ligands to macromolecules to determine their spatial arrangement and structure. Benzophenone (BP) groups are widely used for covalent photoaffinity labeling and for probing protein interactions. We developed bifunctional BP photoactivatable derivatives using three different general chemical approaches. In addition to the photoaffinity reactivity of the BP, these derivatives contain an additional group: A radioactive tracer for biological studies, or an N-ethylmaleimide group as an additional crosslinker, or a biotin group to be used during purification and characterization of probe-protein complexes using the high-affinity biotin-avidin interaction. A model series of photoaffinity labeling probes was synthesized based on the arbutin ligand. These compounds can be used as probes to study the arbutin binding site of microbial beta-glucoside transporters by photolabeling residues in its vicinity. The second functionality provides additional options for studying proteins and binding sites. The probes were developed using different methodologies: (i) a diazotation reaction; (ii) protecting group methodology; and (iii) solid-phase synthesis. These procedures are general and provide a simple and versatile approach for synthesizing bifunctional BP ligands, as demonstrated here on arbutin.     

Phosphatidylinositol 3,4,5-trisphosphate activity probes for the labeling and proteomic characterization of protein binding partners

Phosphatidylinositol polyphosphate lipids, such as phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P?], regulate critical biological processes, many of which are aberrant in disease. These lipids often act as site-specific ligands in interactions that enforce membrane association of protein binding partners. Herein, we describe the development of bifunctional activity probes corresponding to the headgroup of PI(3,4,5)P? that are effective for identifying and characterizing protein binding partners from complex samples, namely cancer cell extracts. These probes contain both a photoaffinity tag for covalent labeling of target proteins and a secondary handle for subsequent detection or manipulation of labeled proteins. Probes bearing different secondary tags were exploited, either by direct attachment of a fluorescent dye for optical detection or by using an alkyne that can be derivatized after protein labeling via click chemistry. First, we describe the design and modular synthetic strategy used to generate multiple probes with different reporter tags of use for characterizing probe-labeled proteins. Next, we report initial labeling studies using purified protein, the PH domain of Akt, in which probes were found to label this target, as judged by in-gel detection. Furthermore, protein labeling was abrogated by controls including competition with an unlabeled PI(3,4,5)P? headgroup analogue as well as through protein denaturation, indicating specific labeling. In addition, probes featuring linkers of different lengths between the PI(3,4,5)P? headgroup and photoaffinity tag led to variations in protein labeling, indicating that a shorter linker was more effective in this case. Finally, proteomic labeling studies were performed using cell extracts; labeled proteins were observed by in-gel detection and characterized using postlabeling with biotin, affinity chromatography, and identification via tandem mass spectrometry. These studies yielded a total of 265 proteins, including both known and novel candidate PI(3,4,5)P?-binding proteins.     

Chronic subcutaneous administration of kisspeptin-54 causes testicular degeneration in adult male rats

The kisspeptins are KiSS-1 gene-derived peptides that signal through the G protein-coupled receptor-54 (GPR54) and have recently been shown to be critical regulators of reproduction. Acute intracerebroventricular or peripheral administration of kisspeptin stimulates the hypothalamic-pituitary-gonadal (HPG) axis. This effect is thought to be mediated via the hypothalamic gonadotropin-releasing hormone (GnRH) system. Chronic administration of GnRH agonists paradoxically suppresses the HPG axis after an initial agonistic stimulation. We investigated the effects of continuous peripheral kisspeptin administration in male rats by use of Alzet minipumps. Initially we compared the effects of acute subcutaneous administration of kisspeptin-10, -14, and -54 on the HPG axis. Kisspeptin-54 produced the greatest increase in plasma LH and total testosterone at 60 min postinjection and was used in the subsequent continuous administration experiments. Chronic subcutaneous long-term administration of 50 nmol kisspeptin-54/day for 13 days decreased testicular weight. Histological examination showed degeneration of the seminiferous tubules associated with a significant decrease in the circulating levels of the testes-derived hormone, inhibin B. Plasma free and total testosterone were also lower, although these changes did not reach statistical significance. Further studies examined the effects of shorter periods of continuous kisspeptin administration. Subcutaneous administration of 50 nmol kisspeptin-54 for 1 day increased plasma LH and testosterone. This effect was lost after 2 days of administration, suggesting a downregulation of the HPG axis response to kisspeptin following continuous administration. These findings indicate that kisspeptin may provide a novel tool for the manipulation of the HPG axis and spermatogenesis.     

Photoaffinity labeling of specific muscarinic antagonist binding sites of brain: I. Preliminary studies using two p-azidophenylacetate esters of tropine

Possible photoaffinity probes for muscarinic acetylcholine receptors have been explored for the first time: Specific [³H]-quinuclidinyl benzylate binding sites of several fractions from rat brain can be irreversibly inactivated by photoaffinity labeling with two p-azidophenylacetate esters of tropine. Inactivation of these sites depends on formation of a reversible complex with the azides prior to their photolytic conversion to the highly reactive nitrenes; it is dependent on ligand concentration and length of photolysis. Atropine and oxotremorine, but not d-tubocurarine, afford protection against photoinactivation.These findings suggest the utility of these and related azido derivatives as potent, selective photoaffinity ligands directed against binding sites for muscarinic antagonists.