Mi Jin Moon

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.

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.

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.

Molecular Coevolution of Neuropeptides Gonadotropin-Releasing Hormone and Kisspeptin with their Cognate G Protein-Coupled Receptors

The neuropeptides gonadotropin-releasing hormone (GnRH) and kisspeptin (KiSS), and their receptors gonadotropin-releasing hormone receptor (GnRHR) and kisspeptin receptor (KiSSR) play key roles in vertebrate reproduction. Multiple paralogous isoforms of these genes have been identified in various vertebrate species. Two rounds of genome duplication in early vertebrates likely contributed to the generation of these paralogous genes. Genome synteny and phylogenetic analyses in a variety of vertebrate species have provided insights into the evolutionary origin of and relationship between paralogous genes. The paralogous forms of these neuropeptides and their receptors have coevolved to retain high selectivity of the ligand–receptor interaction. These paralogous forms have become subfunctionalized, neofunctionalized, or dysfunctionalized during evolution. This article reviews the evolutionary mechanism of GnRH/GnRHR and KiSS/KiSSR, and the fate of the duplicated paralogs in vertebrates.

Expansion of Secretin-Like G Protein-Coupled Receptors and Their Peptide Ligands via Local Duplications Before and After Two Rounds of Whole-Genome Duplication

In humans, the secretin-like G protein-coupled receptor (GPCR) family comprises 15 members with 18 corresponding peptide ligand genes. Although members have been identified in a large variety of vertebrate and nonvertebrate species, the origin and relationship of these proteins remain unresolved. To address this issue, we employed large-scale genome comparisons to identify genome fragments with conserved synteny and matched these fragments to linkage groups in reconstructed early gnathostome ancestral chromosomes (GAC). This genome comparison revealed that most receptor and peptide genes were clustered in three GAC linkage groups and suggested that the ancestral forms of five peptide subfamilies (corticotropin-releasing hormone-like, calcitonin-like, parathyroid hormone-like, glucagon-like, and growth hormone-releasing hormone-like) and their cognate receptor families emerged through tandem local gene duplications before two rounds (2R) of whole-genome duplication. These subfamily genes have, then, been amplified by 2R whole-genome duplication, followed by additional local duplications and gene loss prior to the divergence of land vertebrates and teleosts. This study delineates a possible evolutionary scenario for whole secretin-like peptide and receptor family members and may shed light on evolutionary mechanisms for expansion of a gene family with a large number of paralogs.

Tyr1 and Ile7 of glucose-dependent insulinotropic polypeptide (GIP) confer differential ligand selectivity toward GIP and glucagon-like peptide-1 receptors

Glucagon like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are incretin hormones released in response to food intake and potentiate insulin secretion from pancreatic β cells through their distinct yet related G protein-coupled receptors, GLP1R and GIPR. While GLP-1 and GIP exhibit similarity in their N-terminal sequence and overall α-helical structure, GLP-1 does not bind to GIPR and vice versa. To determine which amino acid residues of these peptide ligands are responsible for specific interaction with their respective receptors, we generated mutant GIP in which several GLP-1-specific amino acid residues were substituted for the original amino acids. The potency of the mutant ligands was examined using HEK293 cells transfected with GLP1R or GIPR expression plasmids together with a cAMP-responsive element-driven luciferase (CRE-luc) reporter plasmid. A mutated GIP peptide in which Tyr1, Ile7, Asp15, and His18 were replaced by His, Thr, Glu, and Ala, respectively, was able to activate both GLP1R and GIPR with moderate potency. Replacing the original Tyr1 and/or Ile7 in the N-terminal moiety of this mutant peptide allowed full activation of GIPR but not of GLP1R. However, reintroducing Asp15 and/or His18 in the central α-helical region did not significantly alter the ligand potency. These results suggest that Tyr/His1 and Ile/Thr7 of GIP/GLP-1 peptides confer differential ligand selectivity toward GIPR and GLP1R.

Evolutionarily conserved residues at glucagon-like peptide-1 (GLP-1) receptor core confer ligand-induced receptor activation.

Background: Little is known regarding the molecular interaction between GLP-1 and its receptor. Results: His1 of GLP-1 interacts with Asn302 of GLP1R, and Thr7 of GLP-1 has close contact with Ile196, Leu232, and Met233 of GLP1R. Conclusion: Evolutionarily conserved residues at the GLP1R core confer ligand-induced receptor activation. Significance: This study may provide critical clues for development of peptide and/or nonpeptide agonists acting at GLP1R. Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) play important roles in insulin secretion through their receptors, GLP1R and GIPR. Although GLP-1 and GIP are attractive candidates for treatment of type 2 diabetes and obesity, little is known regarding the molecular interaction of these peptides with the heptahelical core domain of their receptors. These core domains are important not only for specific ligand binding but also for ligand-induced receptor activation. Here, using chimeric and point-mutated GLP1R/GIPR, we determined that evolutionarily conserved amino acid residues such as Ile196 at transmembrane helix 2, Leu232 and Met233 at extracellular loop 1, and Asn302 at extracellular loop 2 of GLP1R are responsible for interaction with ligand and receptor activation. Application of chimeric GLP-1/GIP peptides together with molecular modeling suggests that His1 of GLP-1 interacts with Asn302 of GLP1R and that Thr7 of GLP-1 has close contact with a binding pocket formed by Ile196, Leu232, and Met233 of GLP1R. This study may provide critical clues for the development of peptide and/or nonpeptide agonists acting at GLP1R.

A Novel Glucagon-Related Peptide (GCRP) and Its Receptor GCRPR Account for Coevolution of Their Family Members in Vertebrates

The glucagon (GCG) peptide family consists of GCG, glucagon-like peptide 1 (GLP1), and GLP2, which are derived from a common GCG precursor, and the glucose-dependent insulinotropic polypeptide (GIP). These peptides interact with cognate receptors, GCGR, GLP1R, GLP2R, and GIPR, which belong to the secretin-like G protein-coupled receptor (GPCR) family. We used bioinformatics to identify genes encoding a novel GCG-related peptide (GCRP) and its cognate receptor, GCRPR. The GCRP and GCRPR genes were found in representative tetrapod taxa such as anole lizard, chicken, and Xenopus, and in teleosts including medaka, fugu, tetraodon, and stickleback. However, they were not present in mammals and zebrafish. Phylogenetic and genome synteny analyses showed that GCRP emerged through two rounds of whole genome duplication (2R) during early vertebrate evolution. GCRPR appears to have arisen by local tandem gene duplications from a common ancestor of GCRPR, GCGR, and GLP2R after 2R. Biochemical ligand-receptor interaction analyses revealed that GCRP had the highest affinity for GCRPR in comparison to other GCGR family members. Stimulation of chicken, Xenopus, and medaka GCRPRs activated Gαs-mediated signaling. In contrast to chicken and Xenopus GCRPRs, medaka GCRPR also induced Gαq/11-mediated signaling. Chimeric peptides and receptors showed that the K16M17K18 and G16Q17A18 motifs in GCRP and GLP1, respectively, may at least in part contribute to specific recognition of their cognate receptors through interaction with the receptor core domain. In conclusion, we present novel data demonstrating that GCRP and GCRPR evolved through gene/genome duplications followed by specific modifications that conferred selective recognition to this ligand-receptor pair.

MOLECULAR EVOLUTION OF GPCRS: GLP1/GLP1 receptors

Glucagon-like peptide 1 (GLP1) is an intestinal incretin that regulates glucose homeostasis through stimulation of insulin secretion from pancreatic β-cells and inhibits appetite by acting on the brain. Thus, it is a promising therapeutic agent for the treatment of type 2 diabetes mellitus and obesity. Studies using synteny and reconstructed ancestral chromosomes suggest that families for GLP1 and its receptor (GLP1R) have emerged through two rounds (2R) of whole genome duplication and local gene duplications before and after 2R. Exon duplications have also contributed to the expansion of the peptide family members. Specific changes in the amino acid sequence following exon/gene/genome duplications have established distinct yet related peptide and receptor families. These specific changes also confer selective interactions between GLP1 and GLP1R. In this review, we present a possible macro (genome level)- and micro (gene/exon level)-evolution mechanisms of GLP1 and GLP1R, which allows them to acquire selective interactions between this ligand-receptor pair. This information may provide critical insight for the development of potent therapeutic agents targeting GLP1R.