Evolution of new hormone function: loss and gain of a receptor

The vertebrate proglucagon gene encodes three glucagon-like sequences (glucagon, glucagon-like peptide-1 [GLP-1], and glucagon-like peptide 2 [GLP-2]) that have distinct functions in regulating metabolism in mammals. In contrast, glucagon and GLP-1 have similar physiological actions in fish, that of mammalian glucagon. We have identified sequences similar to receptors for proglucagon-derived peptides from the genomes of two fish (pufferfish and zebrafish), a frog (Xenopus tropicalis), and a bird (chicken). Phylogenetic analysis of the receptor sequences suggested an explanation for the divergent function of GLP-1 in fish and mammals. The phylogeny of our predicted and characterized receptors for proglucagon-derived peptides demonstrate that receptors for glucagon, GLP-1, and GLP-2 have an origin before the divergence of fish and mammals; however, fish have lost the gene encoding the GLP-1 class of receptors, and likely the incretin action of GLP-1. Receptors that bind GLP-1, but yield glucagon-like action, have been characterized in goldfish and zebrafish, and these sequences are most closely related to glucagon receptors. Both pufferfish and zebrafish have a second glucagon receptor-like gene that is most closely related to the characterized goldfish glucagon receptor. The phylogeny of glucagon receptor-like genes in fish indicates that a duplication of the glucagon receptor gene occurred on the ancestral fish lineage, and could explain the shared action of glucagon and GLP-1. We suggest that the binding specificity of one of the duplicated glucagon receptors has diverged, yielding receptors for GLP-1 and glucagon, but that ancestral downstream signaling has been maintained, resulting in both receptors retaining glucagon-stimulated downstream effects.     

Lamprey proglucagon and the origin of glucagon-like peptides.

We characterized two proglucagon cDNAs from the intestine of the sea lamprey Petromyzon marinus. As in other vertebrates, sea lamprey proglucagon genes encode three glucagon-like sequences, glucagon, and glucagon-like peptides 1 and 2 (GLP-1 and GLP-2). This observation indicates that all three glucagon-like sequences encoded by the proglucagon gene originated prior to the divergence of jawed and jawless vertebrates. Estimates of the rates of evolution for the glucagon-like sequences suggest that glucagon originated first, about 1 billion years ago, while GLP-1 and GLP-2 diverged from each other about 700 MYA. The two sea lamprey intestinal proglucagon cDNAs have differing coding potential. Proglucagon I cDNA encodes the previously characterized glucagon and the glucagon-like peptide GLP-1, while proglucagon II cDNA encodes a predicted GLP-2 and, possibly, a glucagon. The existence of two proglucagon cDNAs which differ with regard to their potential to encode glucagon-like peptides suggests that the lamprey may use differential gene expression as a third mechanism, in addition to alternative proteolytic processing and mRNA splicing, to regulate the production of proglucagon-derived peptides.     

Molecular evolution of proglucagon

The vertebrate proglucagon gene encodes glucagon, and the two glucagon-like peptides GLP-1 and GLP-2. To better understand the origin and diversification of the distinct hormonal roles of the three glucagon-like sequences encoded by the proglucagon gene, we have examined the evolution of this gene. The structure of proglucagon has been largely maintained within vertebrates. Duplication of the proglucagon gene or duplications of sequences within the proglucagon gene are rare. All proglucagon gene duplications are likely to be the result of genome duplication events. Examination of the rates of amino acid sequence evolution of each hormone reveals that they have not evolved in a uniform manner. Each hormone has evolved in an episodic fashion, suggesting that the selective constraints acting upon the sequence vary between, and within, vertebrate classes. Changes in selection on a sequence often reflect changes in the function of the sequence, such as the change in function of GLP-1 from a glucagon-like hormone in fish to an incretin in mammals. We found that the GLP-2 sequence underwent rapid sequence evolution in the early mammal lineage, therefore we have concluded that mammalian GLP-2 has acquired a new biological function that is not found in other vertebrates. Comparisons of the hormone sequences show that many amino acid residues that are functionally important in mammalian hormones are not conserved through vertebrate evolution. This observation suggests that the sequences involved in hormone action change through evolution.     

Comparative genomics of duplicate γ-glutamyl transferase genes in teleosts: medaka (Oryzias latipes), stickleback (Gasterosteus aculeatus), green spotted pufferfish (Tetraodon nigroviridis), fugu (Takifugu rubripes), and zebrafish (Danio rerio)

The availability of multiple teleost (bony fish) genomes is providing unprecedented opportunities to understand the diversity and function of gene duplication events using comparative genomics. Here we examine multiple paralogous genes of γ-glutamyl transferase (GGT) in several distantly related teleost species including medaka, stickleback, green spotted pufferfish, fugu, and zebrafish. Through mining genome databases, we have identified multiple GGT orthologs. Duplicate (paralogous) GGT sequences for GGT1 (GGT1 a and b), GGTL1 (GGTL1 a and b), and GGTL3 (GGTL3 a and b) were identified for each species. Phylogenetic analysis suggests that GGTs are ancient proteins conserved across most metazoan phyla and those paralogous GGTs in teleosts likely arose from the serial 3R genome duplication events. A third GGTL1 gene (GGTL1c) was found in green spotted pufferfish; however, this gene is not present in medaka, stickleback, or fugu. Similarly, one or both paralogs of GGTL3 appear to have been lost in green spotted pufferfish, fugu, and zebrafish. Syntenic relationships were highly maintained between duplicated teleost chromosomes, among teleosts and across ray-finned (Actinopterygii) and lobe-finned (Sarcopterygii) species. To assess subfunction partitioning, six medaka GGT genes were cloned and assessed for developmental and tissue-specific expression. On the basis of these data, we propose a modification of the "duplication-degeneration-complementation" model of subfunction partitioning where quantitative differences rather than absolute differences in gene expression are observed between gene paralogs. Our results demonstrate that multiple GGT genes have been retained within teleost genomes. Questions remain, however, regarding the functional roles of multiple GGTs in these species.     

Two Dipolar α-Helices within Hormone-encoding Regions of Proglucagon Are Sorting Signals to the Regulated Secretory Pathway

Proglucagon is expressed in pancreatic α cells, intestinal L cells, and some hypothalamic and brainstem neurons. Tissue-specific processing of proglucagon yields three major peptide hormones as follows: glucagon in the α cells and glucagon-like peptides (GLP)-1 and -2 in the L cells and neurons. Efficient sorting and packaging into the secretory granules of the regulated secretory pathway in each cell type are required for nutrient-regulated secretion of these proglucagon-derived peptides. Our previous work suggested that proglucagon is directed into granules by intrinsic sorting signals after initial processing to glicentin and major proglucagon fragment (McGirr, R., Guizzetti, L., and Dhanvantari, S. (2013) J. Endocrinol. 217, 229–240), leading to the hypothesis that sorting signals may be present in multiple domains. In the present study, we show that the α-helices within glucagon and GLP-1, but not GLP-2, act as sorting signals by efficiently directing a heterologous secretory protein to the regulated secretory pathway. Biophysical characterization of these peptides revealed that glucagon and GLP-1 each encode a nonamphipathic, dipolar α-helix, whereas the helix in GLP-2 is not dipolar. Surprisingly, glicentin and major proglucagon fragment were sorted with different efficiencies, thus providing evidence that proglucagon is first sorted to granules prior to processing. In contrast to many other prohormones in which sorting is directed by ordered prodomains, the sorting determinants of proglucagon lie within the ordered hormone domains of glucagon and GLP-1, illustrating that each prohormone has its own sorting “signature.”     

Evolution of receptors for proglucagon-derived peptides: isolation of frog glucagon receptors

The mammalian proglucagon gene encodes three glucagon-like sequences, glucagon, glucagon-like peptide 1 (GLP-1) and glucagon-like peptide 2 (GLP-2). Each of these three functionally distinct proglucagon-derived peptides has a unique, but related, receptor. To better understand the origin of the unique physiological functions of each proglucagon-derived glucagon-like sequence we have cloned glucagon-like receptors from two species of frogs, Xenopus laevis and Rana pipiens. The cloned glucagon-like receptor sequences were found to be most closely related to glucagon receptors. To determine whether the evolutionary history of the receptors for proglucagon-derived peptides was the same as that inferred for the peptide hormones, we conducted a phylogenetic analysis using both parsimony and distance methods. We show that the evolutionary history of the receptors for glucagon-like sequences differ from the history of the glucagon-like sequences. The phylogeny of receptors for proglucagon-derived peptides is not monophyletic (i.e. they are not each other's closest relatives), as the receptor for the hormone glucose-dependent insulinotropic peptide (GIP) is more closely related to the glucagon receptor than either the GLP-1 or GLP-2 receptors. In contrast to the evolutionary origin of glucagon-like sequences, where glucagon is of most ancient origin, we found that the GLP-2 receptor has the most ancient origin. These observations suggest that the diversification of the glucagon-like sequences encoded by the proglucagon gene and of the receptors for these peptides occurred independently, and that either these hormones or their receptors have been recruited for new functions.     

Localization of glucagon-like peptide (GLP) immunoreactants in human gut and pancreas using light and electron microscopic immunocytochemistry

The distribution of peptide immunoreactivities predicted from the sequence of the human preproglucagon gene in enteroglucagon (EG; glicentin-like immunoreactant-containing) cells of the human gut and A cells of the pancreas has been determined by light and electron microscopic immunocytochemistry. At light microscopy the application of peroxidase-antiperoxidase and immunogold-silver staining methods has revealed that glucagon-like peptide (GLP-1 and GLP-2) immunoreactivities coexist with a glicentin-related immunodeterminant in human colorectal EG cells and pancreatic A cells. Using single and double colloidal gold probe electron immunocytochemistry, we have been able to show the coexistence of glicentin, GLP-1, and GLP-2 immunoreactivities within single EG cell secretory granules. No morphologic segregation of the proglucagon immunoreactants was observed in EG cells of the colonic mucosa. In pancreatic A cells we have localized GLP-1, GLP-2, and glucagon-[16-29] immunoreactivities solely to the electron-dense core of the secretory granules, whereas glicentin-related immunoreactivity was restricted to the electron-lucent halo. The results obtained in the present study have shown that the peptide immunoreactivities predicted from cDNA sequencing of the human preproglucagon gene are indeed expressed in colorectal EG and pancreatic A cells. The topographical segregation of immunoreactivities in the A cell secretory granule shows that antigenic determinants derived from the C-terminal portion of proglucagon are stored with glucagon in the core of the secretory granule.     

Comparative genomics provides evidence for an ancient genome duplication event in fish

There are approximately 25 000 species in the division Teleostei and most are believed to have arisen during a relatively short period of time ca. 200 Myr ago. The discovery of 'extra' Hox gene clusters in zebrafish (Danio rerio), medaka (Oryzias latipes), and pufferfish (Fugu rubripes), has led to the hypothesis that genome duplication provided the genetic raw material necessary for the teleost radiation. We identified 27 groups of orthologous genes which included one gene from man, mouse and chicken, one or two genes from tetraploid Xenopus and two genes from zebrafish. A genome duplication in the ancestor of teleost fishes is the most parsimonious explanation for the observations that for 15 of these genes, the two zebrafish orthologues are sister sequences in phylogenies that otherwise match the expected organismal tree, the zebrafish gene pairs appear to have been formed at approximately the same time, and are unlinked. Phylogenies of nine genes differ a little from the tree predicted by the fish-specific genome duplication hypothesis: one tree shows a sister sequence relationship for the zebrafish genes but differs slightly from the expected organismal tree and in eight trees, one zebrafish gene is the sister sequence to a clade which includes the second zebrafish gene and orthologues from Xenopus, chicken, mouse and man. For these nine gene trees, deviations from the predictions of the fish-specific genome duplication hypothesis are poorly supported. The two zebrafish orthologues for each of the three remaining genes are tightly linked and are, therefore, unlikely to have been formed during a genome duplication event. We estimated that the unlinked duplicated zebrafish genes are between 300 and 450 Myr. Thus, genome duplication could have provided the genetic raw material for teleost radiation. Alternatively, the loss of different duplicates in different populations (i.e. 'divergent resolution') may have promoted speciation in ancient teleost populations.     

Complete sequences of glucagon-like peptide-1 from human and pig small intestine

In the small intestine, proglucagon is processed into the previously characterized peptide "glicentin" (proglucagon (PG) 1-69) and two smaller peptides showing about 50% homology with glucagon: glucagon-like peptide-1 and -2. It was assumed that the sites of post-translational cleavage in the small intestine of the proglucagon precursor were determined by pairs of basic amino acid residues flanking the two peptides. Earlier studies have shown that synthetic glucagon-like peptide-1 (GLP-1) synthesized according to the proposed structure (proglucagon 71-108 or because residue 108 is Gly, 72-107 amide) had no physiological effects, whereas a truncated from of GLP-1, corresponding to proglucagon 78-107 amide, strongly stimulated insulin secretion and depressed glucagon secretion. To determine the amino acid sequence of the naturally occurring peptide we isolated GLP-1 from human small intestine by hydrophobic, gel permeation, and reverse-phase high performance liquid chromatography. By analysis of composition and sequence it was determined that the peptide corresponded to PG 78-107. By mass spectrometry the molecular mass was determined to be 3295, corresponding to PG 78-107 amide. Furthermore, mass spectrometry of the methyl-esterified peptide showed an increase in mass compatible with the presence of alpha-carboxyl amidation. Thus, the gut-derived insulinotrophic hormone GLP-1 is shown to be PG 78-107 amide.     

The discovery of glucagon-like peptide 1

The discovery of glucagon-like peptide 1 (GLP-1) began more than two decades ago with the observations that anglerfish islet proglucagon messenger RNAs (mRNAs) contained coding sequences for two glucagon-related peptides arranged in tandem. Subsequent analyses revealed that mammalian proglucagon mRNAs encoded a precursor containing the sequence of pancreatic glucagon, intestinal glicentin and two glucagon-related peptides termed GLP-1 and GLP-2. Multidisciplinary approaches were then required to define the structure of biologically active GLP-1 7-36 amide and its role as an incretin, satiety hormone and, most recently, a neuroprotective peptide. This historial perspective outlines the use of traditional recombinant DNA approaches to derive the GLP-1 sequence and highlights the challenges and combination of clinical and basic science approaches required to define the physiology and pathophysiology of bioactive peptides discovered through genomics.     

Phylogenetic Timing of the Fish-Specific Genome Duplication Correlates with the Diversification of Teleost Fish

For many genes, ray-finned fish (Actinopterygii) have two paralogous copies, where only one ortholog is present in tetrapods. The discovery of an additional, almost-complete set of Hox clusters in teleosts (zebrafish, pufferfish, medaka, and cichlid) but not in basal actinopterygian lineages (Polypterus) led to the formulation of the fish-specific genome duplication hypothesis. The phylogenetic timing of this genome duplication during the evolution of ray-finned fish is unknown, since only a few species of basal fish lineages have been investigated so far. In this study, three nuclear genes (fzd8, sox11, tyrosinase) were sequenced from sturgeons (Acipenseriformes), gars (Semionotiformes), bony tongues (Osteoglossomorpha), and a tenpounder (Elopomorpha). For these three genes, two copies have been described previously teleosts (e.g., zebrafish, pufferfish), but only one orthologous copy is found in tetrapods. Individual gene trees for these three genes and a concatenated dataset support the hypothesis that the fish-specific genome duplication event took place after the split of the Acipenseriformes and the Semionotiformes from the lineage leading to teleost fish but before the divergence of Osteoglossiformes. If these three genes were duplicated during the proposed fish-specific genome duplication event, then this event separates the species-poor early-branching lineages from the species-rich teleost lineage. The additional number of genes resulting from this event might have facilitated the evolutionary radiation and the phenotypic diversification of the teleost fish.[Reviewing Editor: Martin Kreitman]     

Glucagon-like immunoreactivity in hypothalamic neurons of the rat

Summary Antisera specific for three different regions of pancreatic proglucagon were used to examine the distribution of such immunoreactivity in rat hypothalamus. Neurons in the supraoptic and paraventricular nuclei were immunoreactive with an antiserum against glucagon, but not with antisera directed towards the aminoterminal region of proglucagon (glicentin) or the glucagon-like peptide I sequence in the carboxyl-terminal region of proglucagon. These findings confirm a previous report of glucagon-like immunoreactivity in the supraoptic and paraventricular nuclei, but indicate that, while this material is immunochemically related to glucagon, it is not derived from a proglucagon-like precursor.     

The evolutionary origin of nodal-related genes in teleosts

Because of an extra whole-genome duplication, zebrafish and other teleosts have two copies of genes that are present in a single copy in tetrapod genomes. Some zebrafish genes, however, are present in triplicate. For example, the nodal-related genes encode secreted proteins of the transforming growth factor beta superfamily that are required in all vertebrates to induce the mesoderm and endoderm, pattern all three germ layers, and establish the left-right axis. Zebrafish have three nodal-related genes, called ndr1/squint, ndr2/cyclops, and ndr3/southpaw. As part of an analysis of enhancer elements controlling zebrafish nodal-related gene expression, we analyzed the nodal loci in the sequenced genomes of five teleost species and four tetrapod species. Each teleost genome contains three nodal-related genes, indicating that squint, cyclops, and southpaw orthologues were present early in the teleost lineage. The genes flanking the nodal-related genes are also conserved, demonstrating a high degree of conserved synteny. Although we found little homology outside of the coding sequences in this region, pufferfish enhancer sequences work in zebrafish embryos to drive reporter gene expression in the squint expression pattern. This indicates a high degree of functional conservation of enhancer elements within the teleosts. We conclude that the ancestral squint and cyclops genes arose during the teleost-specific whole-genome duplication event and that southpaw emerged from a subsequent duplication event involving ancestral squint.     

Comparative analysis of teleost fish genomes reveals preservation of different ancient clock duplicates in different fishes

Clock (Circadian locomotor output cycle kaput) was the first vertebrate circadian clock gene identified in a mouse forward genetics mutagenesis screen. It encodes a bHLH-PAS protein that is highly conserved throughout evolution. Tetrapods also have the second Clock gene, Clock2 or Npas2 (Neuronal PAS domain protein 2). Conversely, the fruit fly, an invertebrate, has only one clock gene. Interrogation of the five teleost fish genome databases revealed that the zebrafish and the Japanese pufferfish (fugu) each have three clock genes, whereas the green spotted pufferfish (tetraodon), the Japanese medaka fish and the three-spine stickleback each have two clock genes. Phylogenetic and splice site analyses indicated that zebrafish and fugu each have two clock1 genes, clock1a and clock1b and one clock2; tetraodon also have clock1a and clock1b but do not have clock2; and medaka and stickleback each have clock1b and one clock2. Genome neighborhood analysis further showed that clock1a/clock1b in zebrafish, fugu and tetraodon is an ancient duplicate. While the dN/dS ratios of these three fish clock duplicates are all <1, indicating that purifying selection has acted upon them; the Tajima relative rate test showed that all three fish clock duplicates have asymmetric evolutionary rates, implicating that one of these duplicates have been under positive selection or relaxed functional constraint. These results support the view that teleost fish clock genes were generated from an ancient genome-wide duplication, and differential gene loss after the duplication resulted in retention of different ancient duplicates in different teleost fishes, which could have contributed to the evolution of the distinct fish circadian clock mechanisms.     

More genes in fish?

Certain species of fish have recently become important model systems in comparative genomics and in developmental biology, in certain instances because of their small genome sizes (e.g., in the pufferfish) and, in other cases, because of the opportunity they provide to combine an easily accessible and experimentally manipulable embryology with the power of genetic approaches (e.g., in the zebrafish). The resulting accumulation of genomic information indicates that, surprisingly, many gene families of fish consist of more members than in mammals. Most modern fish, including the zebrafish and medakka, are diploid organisms; however, the greater number of genes in fish was possibly caused by additional ancient genome duplications which happened in the lineage leading to modern ray-finned fishes but not along the lineage leading to tetrapods. Since these two lineages shared their last common ancestor (in the Devonian about 360 million years ago) individual duplicated members of gene families were later lost in fish. Interestingly, comparative data indicate that, in some cases, genes in mammals even serve somewhat different functions than their homologues in fish, highlighting that the degree of evolutionary relatedness of genes is not always a reliable predictor of their evolutionary conservation and their similarity of function. Since fish are phenotypically probably not more complex than mammals, it is possible that evolution took alternative paths to the “economics of genomics” through alternative solutions to gene regulation. It is suggested that the more complex genomic architecture of fish permitted them to adapt and speciate quickly in response to changing selective regimes. BioEssays 20 :511–515, 1998. © 1998 John Wiley & Sons, Inc.     

Phylogenetic and chromosomal analyses of multiple gene families syntenic with vertebrate Hox clusters

Background  

Ever since the theory about two rounds of genome duplication (2R) in the vertebrate lineage was proposed, the Hox gene clusters have served as the prime example of quadruplicate paralogy in mammalian genomes. In teleost fishes, the observation of additional Hox clusters absent in other vertebrate lineages suggested a third tetraploidization (3R). Because the Hox clusters occupy a quite limited part of each chromosome, and are special in having position-dependent regulation within the multi-gene cluster, studies of syntenic gene families are needed to determine the extent of the duplicated chromosome segments. We have analyzed in detail 14 gene families that are syntenic with the Hox clusters to see if their phylogenies are compatible with the Hox duplications and the 2R/3R scenario. Our starting point was the gene family for the NPY family of peptides located near the Hox clusters in the pufferfish Takifugu rubripes, the zebrafish Danio rerio, and human.     

Evolution of the vertebrate glucose-dependent insulinotropic polypeptide (GIP) gene

The glucose-dependent insulinotropic polypeptide (GIP) gene is believed to have originated from a gene duplication event very early in vertebrate evolution that also produced the proglucagon gene, yet so far GIP has only been described within mammals. Here we report the identification of GIP genes in chicken, frogs, and zebrafish. The chicken and frog genes are organized in a similar fashion to mammalian GIP genes and contain 6 exons and 5 introns in homologous locations. These genes can also potentially be proteolytically processed in identical patterns as observed in the mammalian sequences that would yield a GIP hormone that is only one amino shorter than the mammalian sequences due to the removal of an extra basic residue by carboxypeptidase E. The zebrafish GIP gene and precursor protein is shorter than other vertebrate GIP genes and is missing exon 5. The predicted zebrafish GIP hormone is also shorter, being only 31 amino acids in length. The zebrafish GIP hormone is similar in length to the proglucagon-derived peptide hormones, peptides encoded from the gene most closely related to GIP. We suggest that the structure of zebrafish GIP is more similar to the ancestral gene, and that tetrapod GIP has been extended. The mammalian GIP hormone has also undergone a period of rapid sequence evolution early in mammalian evolution. The discovery of a conserved GIP in diverse vertebrate suggests that it has an essential role in physiology in diverse vertebrates, although it may have only recently evolved a role as an incretin hormone.