Jae Young Seong

Ligand binding pocket formed by evolutionarily conserved residues in the glucagon-like peptide-1 (GLP-1) receptor core domain

Background: Little is known about the interaction between GLP-1 and the heptahelical core domain of GLP1R. Results: GLP-1 Asp9 and Gly4 interact with the evolutionarily conserved residues in extracellular loop 3. Conclusion: Ligand binding pocket formed by evolutionarily conserved residues in the GLP1R core domain. Significance: This study highlights the mechanism underlying high affinity interaction between GLP-1 and the binding pocket of the receptor. Glucagon-like peptide-1 (GLP-1) plays a pivotal role in glucose homeostasis through its receptor GLP1R. Due to its multiple beneficial effects, GLP-1 has gained great attention for treatment of type 2 diabetes and obesity. However, little is known about the molecular mechanism underlying the interaction of GLP-1 with the heptahelical core domain of GLP1R conferring high affinity ligand binding and ligand-induced receptor activation. Here, using chimeric and point-mutated GLP1R, we determined that the evolutionarily conserved amino acid residue Arg380 flanked by hydrophobic Leu379 and Phe381 in extracellular loop 3 (ECL3) may have an interaction with Asp9 and Gly4 of the GLP-1 peptide. The molecular modeling study showed that Ile196 at transmembrane helix 2, Met233 at ECL1, and Asn302 at ECL2 of GLP1R have contacts with His1 and Thr7 of GLP-1. This study may shed light on the mechanism underlying high affinity interaction between the ligand and the binding pocket that is formed by these conserved residues in the GLP1R core domain.

Molecular Evolution of Multiple Forms of Kisspeptins and GPR54 Receptors in Vertebrates

Kisspeptin and its receptor GPR54 play important roles in mammalian reproduction and cancer metastasis. Because the KiSS and GPR54 genes have been identified in a limited number of vertebrate species, mainly in mammals, the evolutionary history of these genes is poorly understood. In the present study, we have cloned multiple forms of kisspeptin and GPR54 cDNAs from a variety of vertebrate species. We found that fish have two forms of kisspeptin genes, KiSS-1 and KiSS-2, whereas Xenopus possesses three forms of kisspeptin genes, KiSS-1a, KiSS-1b, and KiSS-2. The nonmammalian KiSS-1 gene was found to be the ortholog of the mammalian KiSS-1 gene, whereas the KiSS-2 gene is a novel form, encoding a C-terminally amidated dodecapeptide in the Xenopus brain. This study is the first to identify a mature form of KiSS-2 product in the brain of any vertebrate. Likewise, fish possess two receptors, GPR54-1 and GPR54-2, whereas Xenopus carry three receptors, GPR54-1a, GPR54-1b, and GPR54-2. Sequence identity and genome synteny analyses indicate that Xenopus GPR54-1a is a human GPR54 ortholog, whereas Xenopus GPR54-1b is a fish GPR54-1 ortholog. Both kisspeptins and GPR54s were abundantly expressed in the Xenopus brain, notably in the hypothalamus, suggesting that these ligand-receptor pairs have neuroendocrine and neuromodulatory roles. Synthetic KiSS-1 and KiSS-2 peptides activated GPR54s expressed in CV-1 cells with different potencies, indicating differential ligand selectivity. These data shed new light on the molecular evolution of the kisspeptin-GPR54 system in vertebrates.

Discovery and Evolutionary History of Gonadotrophin―Inhibitory Hormone and Kisspeptin: New Key Neuropeptides Controlling Reproduction

Gonadotrophin‐releasing hormone (GnRH) is the primary hypothalamic factor responsible for the control of gonadotrophin secretion in vertebrates. However, within the last decade, two other hypothalamic neuropeptides have been found to play key roles in the control of reproductive functions: gonadotrophin‐inhibitory hormone (GnIH) and kisspeptin. In 2000, we discovered GnIH in the quail hypothalamus. GnIH inhibits gonadotrophin synthesis and release in birds through actions on GnRH neurones and gonadotrophs, mediated via GPR147. Subsequently, GnIH orthologues were identified in other vertebrate species from fish to humans. As in birds, mammalian and fish GnIH orthologues inhibit gonadotrophin release, indicating a conserved role for this neuropeptide in the control of the hypothalamic‐pituitary‐gonadal axis across species. Subsequent to the discovery of GnIH, kisspeptin, encoded by the KiSS‐1 gene, was discovered in mammals. By contrast to GnIH, kisspeptin has a direct stimulatory effect on GnRH neurones via GPR54. GPR54 is also expressed in pituitary cells, but whether gonadotrophs are targets for kisspeptin remains unresolved. The KiSS‐1 gene is also highly conserved and has been identified in mammals, amphibians and fish. We have recently found a second isoform of KiSS‐1, designated KiSS‐2, in several vertebrates, but not birds, rodents or primates. In this review, we highlight the discovery, mechanisms of action, and functional significance of these two chief regulators of the reproductive axis.

Identification of Farnesyl Pyrophosphate and N-Arachidonylglycine as Endogenous Ligands for GPR92

A series of small compounds acting at the orphan G protein-coupled receptor GPR92 were screened using a signaling pathway-specific reporter assay system. Lipid-derived molecules including farnesyl pyrophosphate (FPP), N-arachidonylglycine (NAG), and lysophosphatidic acid were found to activate GPR92. FPP and lysophosphatidic acid were able to activate both Gq/11- and Gs-mediated signaling pathways, whereas NAG activated only the Gq/11-mediated signaling pathway. Computer-simulated modeling combined with site-directed mutagenesis of GPR92 indicated that Thr97, Gly98, Phe101, and Arg267 of GPR92 are responsible for the interaction of GPR92 with FPP and NAG. Reverse transcription-PCR analysis revealed that GPR92 mRNA is highly expressed in the dorsal root ganglia (DRG) but faint in other brain regions. Peripheral tissues including, spleen, stomach, small intestine, and kidney also expressed GPR92 mRNA. Immunohistochemical analysis revealed that GPR92 is largely co-localized with TRPV1, a nonspecific cation channel that responds to noxious heat, in mouse and human DRG. FPP and NAG increased intracellular Ca2+ levels in cultured DRG neurons. These results suggest that FPP and NAG play a role in the sensory nervous system through activation of GPR92.

Suppression of NF-κB signaling by KEAP1 regulation of IKKβ activity through autophagic degradation and inhibition of phosphorylation

IkappaB kinase beta (IKKbeta) plays a crucial role in biological processes, including immune response, stress response, and tumor development by mediating the activation of various signaling molecules such as NF-kappaB. Extensive studies on the mechanisms underlying IKK activation have led to the identification of new activators and have facilitated an understanding of the cellular responses related to NF-kappaB and other target molecules. However, the molecular processes that modulate IKK activity are still unknown. In this study, we show that KEAP1 is a new IKK binding partner, which is responsible for the down-regulation of TNFalpha-stimulated NF-kappaB activation. The E(T/S)GE motif, which is found only in the IKKbeta subunit of the IKK complex, is essential for interaction with the C-terminal Kelch domain of KEAP1. Reduction of KEAP1 expression by small interfering RNA enhanced NF-kappaB activity, and up-regulated the expression of NF-kappaB target genes. Ectopic expression of KEAP1 decreased the expression of IKKbeta, which was restored by an autophagy inhibitor. IKK phosphorylation stimulated by TNFalpha was blocked by KEAP1. Our data demonstrate that KEAP1 is involved in the negative regulation of NF-kappaB signaling through the inhibition of IKKbeta phosphorylation and the mediation of autophagy-dependent IKKbeta degradation.

Revisiting the evolution of gonadotropin-releasing hormones and their receptors in vertebrates: secrets hidden in genomes.

Gonadotropin-releasing hormone (GnRH) and its G protein-coupled receptor, GnRHR, play a pivotal role in the control of reproduction in vertebrates. To date, many GnRH and GnRHR genes have been identified in a large variety of vertebrate species using conventional biochemical and molecular biological tools in combination with bioinformatic tools. Phylogenetic approaches, primarily based on amino acid sequence identity, make it possible to classify these multiple GnRHs and GnRHRs into several lineages. Four vertebrate GnRH lineages GnRH1, GnRH2, GnRH3, and GnRH4 (for lamprey) are well established. Four vertebrate GnRHR lineages have also been proposed-three for nonmammalian GnRHRs and mammalian GnRHR2 as well as one for mammalian GnRHR1. However, these phylogenetic analyses cannot fully explain the evolutionary origins of each lineage and the relationships among the lineages. Rapid and vast accumulation of genome sequence information for many vertebrate species, together with advances in bioinformatic tools, has allowed large-scale genome comparison to explore the origin and relationship of gene families of interest. The present review discusses the evolutionary mechanism of vertebrate GnRHs and GnRHRs based on extensive genome comparison. In this article, we focus only on vertebrate genomes because of the difficulty in comparing invertebrate and vertebrate genomes due to their marked divergence.

Effect of ascorbic acid supplementation on testicular steroidogenesis and germ cell death in cadmium-treated male rats.

Cadmium (Cd) is one of the environmental pollutants affecting various tissues and organs including testis. Harmful effect of Cd in testis is known to be germ cell degeneration and impairment of testicular steroidogenesis. Animals treated with high doses of Cd (0.2 and 0.3 mg/100g BW) showed a significant decrease in serum testosterone (T) level, but a significant induction of testicular lipid peroxidation levels. TUNEL assay showed that low doses of Cd (0.13 and 0.15 mg/100g BW) exhibited typical characteristics of apoptosis while high doses of Cd caused more necrosis than apoptosis. In contrast, supplementation with ascorbic acid reduced testicular lipid peroxidation levels. Ascorbic acid supplementation restored testicular 3beta-hydroxysteroiddehydrogenase (HSD) and 17beta-HSD enzyme activities, 3beta-HSD and cytochrome P450 side chain cleavage (P450(scc)) mRNA levels and serum T concentration to normal in Cd-administered rats. Moreover, administration of ascorbic acid prevented germ cell apoptosis as demonstrated by the reduced number of TUNEL-positive cells in germinal epithelium and inhibited Cd-induced necrosis. These results indicate that ascorbic acid have protective roles in vivo on the Cd-induced overall testicular damage including impaired steroidogenesis and germ cell death possibly through scavenging the reactive oxygen species generated by Cd administration.

Molecular coevolution of kisspeptins and their receptors from fish to mammals

Kisspeptin and its receptor, GPR54, play a pivotal role in vertebrate reproduction. Recent advances in bioinformatic tools combined with comparative genomics have led to the identification of a large number of kisspeptin and GPR54 genes in a variety of vertebrate species. Genome duplications may have produced at least two isoforms of both ligand (KiSS1 and KiSS2) and receptor (GPR54‐1 and GPR54‐2). Additional isoforms of kisspeptin (KiSS1b) and GPR54 (GPR54‐1b) have been found in an amphibian species, Xenopus (Silurana) tropicalis. Here, we describe the evolutionary lineages of these kisspeptin and GPR54 isoforms using genome synteny and phylogenetic analyses, and possible molecular interactions between kisspeptin and GPR54 subtypes based on ligand‐receptor selectivity. Together, kisspeptin and GPR54 provide an excellent model for understanding molecular coevolution of the peptide ligand and GPCR pairs.

Cellular and Molecular Biology of Orphan G Protein-Coupled Receptors

The superfamily of G protein-coupled receptors (GPCRs) is the largest and most diverse group of membrane-spanning proteins. It plays a variety of roles in pathophysiological processes by transmitting extracellular signals to cells via heterotrimeric G proteins. Completion of the human genome project revealed the presence of approximately 168 genes encoding established nonsensory GPCRs, as well as 207 genes predicted to encode novel GPCRs for which the natural ligands remained to be identified, the so-called orphan GPCRs. Eighty-six of these orphans have now been paired to novel or previously known molecules, and 121 remain to be deorphaned. A better understanding of the GPCR structures and classification; knowledge of the receptor activation mechanism, either dependent on or independent of an agonist; increased understanding of the control of GPCR-mediated signal transduction; and development of appropriate ligand screening systems may improve the probability of discovering novel ligands for the remaining orphan GPCRs.

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.