TABLE of CONTENTS
G PROTEIN COUPLED RECEPTORS
RFAMIDE NEUROPEPTIDE FAMILY AND 26-AMINO ACID RESIDUE RFAMIDE PEPTIDE (26RFA/QRFP)
PYROGLUTAMYLATED RFAMIDE PEPTIDE RECEPTOR
SURPRISES FROM THE FASCINATING RESEARCH ON QRFP AND ITS RECEPTORS
CONCLUSIONS AND FUTURE DIRECTIONS
G protein-coupled receptors (GPCRs) are members of a large protein family that share common structural motifs, including seven transmembrane helices, and play pivotal roles in cell-to-cell communications and in the regulation of cell functions. A large number of GPCRs still remain as “orphan receptors” whose cognate ligands have yet to be identified. Identification of ligands for orphan GPCRs provides a basis for understanding the physiological roles of those GPCRs and their ligands, which can involve the central nervous, endocrine, reproductive, cardiovascular, immune, inflammatory, digestive, and metabolic systems. GPR103 is an orphan GPCR that shows similarities with orexin, neuropeptide FF, and cholecystokinin receptors. In humans, 26RFa/QRFP has been found to be an endogenous ligand for the orphan receptor, GPR103 and it is one of the RFamide peptides, which have been shown to exert important neuroendocrine, behavioural, sensory and autonomic functions. All the information we have till know couldn’ be available if we didn’t know the evolution of this important proteins and the relative interactions, which were discovered recently to be important for the regulation of locomotor activity, sleep and these neuropeptides and receptors exert neuroprotective effect in Alzheimer’s Disease. Anyway, there is a long way to be walked on, because there is a need of additional information, while studing the molecular evolution of these proteins and peptides. What is the Molecular Evolution? In the First chapter the readers can find all about this branch of science and the problems this branch had overcomed in order to bring to the scientific community the molecularisation of the evolution concepts. In the following chapter to the readers is presented a descriptive overview of G-proteins and G-protein coupled receptor. In the other chapters it is also presented 26RFA/QRFP and pyroglutamylated RFamide peptide receptor (QRFPR) molecular evolution, where it is described the molecular phylogeny and the functional implication. It is even shown the unique study regarding the presence on natural selection (positive or negative selection) during the evolution of QRFPRs in mammals and fish species. In the end of the book are shown the recent important finding regarding the function and the role of QRFP and its receptor, together with the experimental approach applied to zebrafish and rodents. Anyway, it is imperative to mention that all the available knowledge from experiments to these animals can be useful for the curation of disorders and other deseases like Alzheimer’s Disease, only if we could understand well their molecular evolution.
Evolution is the so called theory in biology postulating that the various types of plants, animals, and other living things on Earth have their origin in other pre-existing types and that the distinguishable differences are due to modifications in successive generations. The theory of evolution is one of the fundamental keystones of modern biological theory (Ayala, 2014). Nowadays, the field of molecular biology provides the most detailed and convincing evidence available for biological evolution. In its unveiling of the nature of DNA and the workings of organisms at the level of enzymes and other protein molecules, it has shown that these molecules hold information about an organism’s ancestry (Ayala, 2014). This has made it possible to reconstruct evolutionary events that were previously unknown and to confirm and adjust the view of events already known. The precision with which these events can be reconstructed is one reason the evidence from molecular biology is so compelling. The investigation of the evolution at the molecular level revealed all the evolutionary processes taking place at this level, which have shown all living organisms, from bacteria to humans, to be related by descent from common ancestors (Ayala, 2014).
Now, it should be mentioned that the Molecular Evolution is a research field originating in the 1960s in the interface of molecular biology, biochemistry and evolutionary biology and, to a lesser degree, of biophysics and studies on the origin of life and exobiology. Many institutional features—including journals, departments and professional societies linked to the field—allow to speak of it as a discipline (or a sub-discipline of biology, depending on the perspective), and not just as a ‘trans-disciplinary field’ of research (Suárez-Díaz, 2009). However, like many disciplinary formations in the second half of the twentieth century (including the broader field of molecular biology), Molecular Evolution has gone through important transformations, including fragmentation and integration into new research fields, in relatively short spans of time.
In this sense the history of Molecular Evolution is not dissociated from what we may say about the history of molecular biology. Both fields constitute products of the institutional and research opportunities of twentieth-century biology; and both have gone through rapid reconfigurations in this changing context (Suárez-Díaz, 2009). However, within the field of studies of science it is commonly acknowledged that disciplines are spaces, where the social (or professional) and epistemic dimensions of science are deeply and complexly interwoven (Suárez-Díaz, 2009).
Scientific Traditions in the Study of Molecular Evolution
By the end of the 1950s and the beginning of the 1960s the ‘molecular vision of life’ started to permeate the study of biological evolution. In many places (laboratories, research groups) and from very different perspectives researchers began to apply the experimental techniques and instruments of molecular biology and biochemistry to many problems of biological evolution. This process can be seen as the molecularization of evolutionary biology (de Chadarevian and Kamminga, 1998). Problems that had been the subject of population genetics, paleontology, and systematics began to be tackled with molecular tools (Suárez-Díaz, 2009).
In some cases, as in the study of genetic variability in populations, the techniques used came from an earlier phase in the history of molecular biology, for example, electrophoresis. In others cases, however, the development of molecular tools came in hand with its application to evolutionary studies, as in the amino acid sequencing of proteins or the use of nucleic acid hybridization (Suárez-Díaz, 2009). Nevertheless this does not mean that efforts to study evolution at the molecular level had not been attempted in the past.
The first uses of molecules in the study of diversity and evolution, then, originated from the study of human blood groups, which in turn had resulted from the practice of blood transfusion connected to the needs of war, and the study of human (Kay, 1993) populations. Later on, and also within the context of hematology and the needs of Second World War, the study of hemoglobin and the identification and collection of ‘abnormal hemoglobins’ became the exemplary locus of the first studies of molecular diversity and ‘molecular diseases’. A collective endeavor on the subject was well institutionalized before the 1960s, in particular in the case of Britain and Germany (Mazumdar, 1995; de Chadarevian, 1998). In terms of historical prevalence and collective importance, then, the serological tests and the techniques developed within hematology and immunology, including paper and later gel-electrophoresis, had a primacy over the comparisons of the few protein sequences attempted in the mid-1960s by the new generation of molecular biologists, biochemists and biophysicists interested in evolution. However, all these scientists interested in evolution and their few protein sequences would be determinant for the future development of this new emerging discipline of Evolution.
The first study along the lines of the new focus on proteins (broadly speaking) and nucleic acids was written by biochemist and Nobel Prize winner Christian B. Anfinsen. In 1959 he published “The molecular basis of evolution”, covering the evolution of genes and proteins, and referring to the primary and tertiary structures of these molecules (Anfinsen, 1959). Anfinsen had contributed to establish the connection between the primary and tertiary structure of proteins (in the case of ribonuclease) and promoted an evolutionary view in which the study of genes (nucleic acids) could be of help in the study of the evolution of proteins (phenotypes).
However, he made an ample recognition of the importance of more traditional fields in the understanding of evolution, like genetics and paleontology (in this later case following George G. Simpson’s views) (Suárez-Díaz, 2009). Equally important, Anfinsen’s book, as he described it in his Introduction, reflected ideas that were being discussed by many other scientists at the time, demanding a more general focus on the evolution of proteins and nucleic acids and the application of molecular experimental techniques (Florkin, 1949; Jukes, 1966).
The use of molecular techniques in problems of evolution, however, did not take place in the same manner or with shared goals everywhere. It was a process that occurred simultaneously in many fields of research, and maybe it is one of the most conspicuous effects of the molecular revolution in biology at large.
The birth of Molecular Evolution is tied to the efforts to ‘molecularize’ the study of evolution in at least three sorts of traditions: experimental (associated with the input from biochemistry, biophysics and molecular biology), theoretical (concerned with the development of mathematical models of population genetics) and comparative traditions (related to systematics and the problems of historical-comparative disciplines such as paleontology) (Suárez-Díaz, 2009).
Some of the crucial events, which contributed to the modernization of the evolution approach are reported below.
One of the fathers of the Molecular Evolution discipline was Emanuel Margoliash, from Abbot Laboratories in Chicago, Illinois. He was a biochemist with evolutionary interests, who decided to sequence cytochrome c molecules of several species. Margoliash was convinced that in order to reconstruct the history of life he needed to focus on a single molecule with evolutionary significance.
Cytochrome c was an excellent candidate. Not only it was a small protein (as compared to hemoglobin), but it was present across the whole biological universe, from bacteria to man, and it seemed that its function as an electron carrier had also remained more or less the same. Very soon Margoliash began to accumulate data on sequences of cytochrome c. In 1967 he joined forces with Walter Fitch, who had previously developed a computer program for assessing the relationship between two molecules, and they published one of the first computer molecular phylogenetic trees (Figure 1), and certainly they provided one of the most influential methods to assess evolutionary distance (Fitch and Margoliash, 1967).
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Figure 1. Phylogeny as reconstructed from observable mutations in the cytochrome c gene. Each number on the figure is the corrected mutation distance along the line of descent as determined from the best computer fit so far found. Each apex is placed at an ordinate value representing the average of the sums of all mutations in the lines of descent from that apex (for more see: Fitch and Margoliash, 1967).
As the development of molecular biology and genetic engineering continued in the 1980s, and was transformed during the 1990s with the advent of the genome projects and the development of databases and bioinformatics, the study of molecular evolution underwent a series of drastic changes. Basically, these related to the magnitude of the quantitative and statistical analysis needed to handle the huge amounts of data available on protein and mostly DNA sequences (Suárez-Díaz, 2009). However, it is less recognized the role of the evolutionary perspective and the analytical tools developed within Molecular Evolution as an important input in the development of functional and comparative genomics, the standardization of analytical methods and the common use of molecular techniques were crucial factors in the formation of the research field of molecular evolution.
The field was characterized by the appropriation of the traditional problems of evolutionary biology with new batteries of tools, belonging to different stages in the history of molecular biology: material tools in the first instance, including instruments and experimental techniques, but later on also bioinformatics, computers and analytical tools (Hagen, 2001). Some of this experimental techniques and analytical tools will be explained in the following chapters.
Moreover, the comparison of amino-acid sequences, the development of nucleic acid hybridization and the application of gel electrophoresis to the problem of variability at the population level, not only affected the nature of the problems of biological evolution in the 1960s, but it also created new evolutionary problems at the molecular level (Suárez-Díaz, 2009).
The techniques, the skills for reproducing them, and the results obtained, traveled frequently between research teams, and helped to build a set of common problems.
Disagreement between Organismal and Molecular Evolutionists
A more general disagreement between them concerned the value of molecules (mostly proteins) as characters to study evolution. In different ways molecular evolutionists during the 1960s stressed not only the importance, but the superiority of molecular traits over morphological characters in the reconstruction of phylogenetic relationships. Either by proposing shorter times in the divergence of hominids and primates (as claimed by Zuckerkandl et al., 1960; Goodman, 1963; Sarich and Wilson, 1967), or by claiming that a single molecule will provide enough evidence to reconstruct the history of life (as argued in Margoliash, 1963).
The insistence of Mayr, Dobshansky and Simpson that morphological characters were ‘polygenic’ and so, that one could not say much on evolution by focusing on a single gene, was taken by Zuckerkandl instead as an advantage of the molecular approach.
He argued, against the organismal biologists, that molecular characters were not polygenic and in this sense they were ‘cleaner’ evidence for evolution; this meant that the complex interweaving of
causes and effects producing morphological characters could be disentangled at the molecular level (Dietrich, 1998; Suárez-Díaz, 2007).
Furthermore, Zuckerkandl gain more territory in scientific community interested in evolution, when he published a paper called ‘Molecules as documents of evolutionary history’, where Zuckerkandl attempted a direct response to the traditional evolutionists; in particular, to Simpson (Suárez-Díaz, 2007). In a highly rhetorical manner he argued that the largest amount of information understood as history (Suárez-Díaz, 2007) was preserved in the semantophoretic molecules or semantides (molecules with meaning, literally) that is, molecules that carry the information of genes or a transcript thereof.
Genes are the primary semantides, messenger-RNA molecules are secondary semantides and polypeptides (at least most of them) were classified as tertiary semantides. Episemantic molecules, in turn, were molecules synthesized under the control of tertiary semantides, while asemantic molecules were characterized as molecules that are not produced by the organism, but are present in it, and therefore do not express, either directly or indirectly, any of the information that this organism contains. In brief, ‘information’, and thus history, was lost as it passed from semantides to asemantides.
Shortly, Zuckerkandl argued that there were two great advantages of using semantide s as evolutionary characters:
1) the opportunity of having quantitative data on the differences and affinities among homologous molecules, with the possibility of applying the concept of information to evolutionary biology, and 2) the fact that semantides constituted a kind of direct evidence, with no need of independent evidence as phylogenetic characters.
Moreover, in Zuckerkandl’s defense, information acted as substitution for the concept of ‘history’ (Suárez-Díaz, 2007). Proteins and nucleic acids were the great reservoirs, the documents of biological history. They were not historical evidences on the same level as morphological characters, however; they were superior, in the sense that a single molecule could serve as evidence for reconstructing a lineage’s past.
The information contained in the phenotype could be deduced
and obtained, in a more direct manner, from proteins and nucleic acids, than from morphological characters.
Such claims were intimately tied to the advancement of a research program in molecular evolution. Drawing upon the ‘informational molecules research program’ as a political resource, the molecular evolutionists advanced an institutional goal: the reconfiguration of the evolutionary field and the consolidation of a disciplinary space.
The concept of informational molecules facilitated the cooperation and communication of comparative, experimental and theoretical approaches to molecular evolution in several ways. It was linked to new types of representations, including material representations (for instance, proportions of hybridization between DNAs from different species; or alignments and determination of similarity of amino acid sequences between two or more proteins; or an electrophoretic gel displaying allelomorphisms), and it was linked also to a special group of material things (like samples of protein or nucleic acid, instead of bones or specimens of plants and animals) (Suárez-Díaz, 2009).
Representations, and less often material things, traveled along the division of labor existing between these traditions. For instance, the new representation of the eukaryotic genome as containing a high proportion of non-functional DNA was incorporated in the new mathematical models of theoretical population biologists; and the comparative molecular evolutionists increasingly benefited from the improvement of sequencing techniques and the accumulation of sequences data coming from experimental traditions (Suárez-Díaz, 2009).
In the following years the research on informational molecules established several genomic phenomena, which challenged the traditional views of adaptive evolution, such as the aforementioned existence of large amounts of DNA with no apparent function and, later, the prevalence of unexpected genetic mechanisms such as lateral (or horizontal) genetic transfer (LGT) among bacteria (O’Malley and Boucher, 2005). The face of evolutionary biology had thus been completely transformed by the end of the 1980s.
Not just the problems but the tools, the concepts and the theories of evolution had been thoroughly affected within the new discursive regime dominated by the idea of informational molecules. Also, the authority and legitimacy associated to the concept of informational molecules played a central role in the construction of the new socio-professional identity (Biagioli, 1994).
Having this concept at the center of their disciplinary domain distinguished the molecular evolutionists from the traditional organismal traditions.
Evolutionary biologists typically distinguish two main types of natural selection: (1) purifying selection, which acts to eliminate deleterious mutations and (2) positive (Darwinian) selection, which favors advantageous mutations. Positive selection can, in turn, be further subdivided into directional selection, which tends toward fixation of an advantageous allele, and balancing selection, which maintains a polymorphism (Hughes, 2007). These are the main concepts, where it is build the neutral theory accepted by most of the scientists nowdays.
The neutral theory of molecular evolution (Kimura, 1983) predicts that purifying selection is ubiquitous, but that both forms of positive selection are rare, whereas not denying the importance of positive selection in the origin of adaptations.
However, it would be difficult to understand all these concepts and the neutral theory, without a short description of the Neo-Darwinism theory. The modern concept of natural selection dates from the Neo-Darwinian synthesis of the 1920–1930’s, when the original insight of Darwin and Wallace was combined with Mendelian genetics to model evolution as the change in gene frequencies in populations. The importance of these pioneering studies to evolutionary biology should not be minimized, but at the same time, it is necessary to realize that there were limitations to the original Neo-Darwinists’ understanding of the evolutionary process (Hughes, 2007).
The original Neo-Darwinists knew nothing about the physical nature of the gene and little of how genes actually affect phenotypic traits. Also, at that time, there was little knowledge regarding such simple matters as the census numbers of natural populations and the frequency of population bottlenecks.
As a result of these gaps in knowledge, certain unrealistic views became part and parcel of the Neo-Darwinist account of natural selection, and unfortunately, many of these are still with us, at least implicitly.
The dawn of the molecular era in biology also saw the first serious challenges to the Neo-Darwinist worldview, in Motoo Kimura’s neutral theory of molecular evolution (Kimura, 1968, 1983). Before proposing the neutral theory, Kimura had devoted over a decade to the study of evolutionary dynamics in finite populations, a study for which he adapted mathematical tools (such as the diffusion approximation) that were new to population genetics (Kimura, 1955, 1957, 1964). In developing a sophisticated understanding of the role of population size in the evolutionary change of gene frequencies, Kimura made a contribution to evolutionary biology that is arguably second only to Darwin’s (Hughes, 2007).
The neutral theory predicts both (1) that most polymorphisms are selectively neutral and are maintained by genetic drift; and (2) that most changes at the molecular level that are fixed over evolutionary time are selectively neutral and are fixed by drift. Thus, the neutral theory provides a conceptual framework uniting ecological and evolutionary time frames. It is often stated even today that the neutral theory predicts that most mutations are selectively neutral. But this is not a prediction of the neutral theory. The neutral theory predicts that the majority of mutations that are fixed over evolutionary time are selectively neutral.
When the neutral theory was first proposed, the extent of noncoding DNA in the genomes of eukaryotes was not known. Now, given the evidence that a substantial majority of the nucleotides in a typical mammalian genome may be nonfunctional, we may hypothesize that most mutations occurring in nonfunctional regions are selectively neutral. And, given the abundance of nonfunctional regions, it follows that a majority of mutations in such genomes are probably selectively neutral. But this is not a consequence of the neutral theory per se (Hughes, 2007).
In fact, as regard to the coding regions, the neutral theory predicts that most mutations are not selectively neutral. Rather, because most mutations in coding regions are nonsynonymous (amino-acid-altering) and thus disrupt protein structure, most mutations in coding regions are selectively deleterious. One of the most important predictions of the neutral theory is thus that purifying selection will predominate in coding regions (and in other functionally important regions as well).
However, the tendency of the molecular biologist and the evolutionary biologist is to look for the presence of the positive selection at the level of gene, protein and genome.
For example, the “red” and “green” color vision genes in humans are contiguously located on the X chromosome and are believed to have been generated by gene duplication that occurred just before humans and Old World monkeys diverged. The proteins (opsins) encoded by these two genes are known to have 15 amino acid differences (Nathans et al., 1986). However, only three amino acid differences are responsible for the functional difference of the two proteins, and other amino acid differences are virtually irrelevant (R. Yokoyama and S. Yokoyama, 1990). Yokoyama and Radlwimmer (2001) have shown that most evolutionary changes of red-green color vision in vertebrates can be explained by amino acid changes at five critical sites of the protein. Some other examples of adaptive evolution by a few amino acid substitutions are given in Table 1.
Table 1. Examples of functional changes caused by one or a few amino acid changes.
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It has sometimes been argued that searching for positive selection on the human genome can be justified, because such studies yield insights into the basis of human complex diseases (Kelley et al., 2007). The rationale behind such an expectation is rarely clarified (Hughes, 2007). On the other hand, there is strong evidence for the presence of abundant slightly deleterious variants in the human population. Given that many of these variants are known to be subject to ongoing purifying selection, it seems much more plausible to examine these variants as candidates for a role in complex disease than to search for positively selected variants (Hughes et al., 2003; Yampolsky et al., 2005).
Anyway, it is by now such a truism of molecular biology that functionally important sequences are ‘conserved’ or ‘functionally constrained’ (that is, evolve slowly), that many biologists probably do not realize that this generalization is a prediction of the neutral theory. Moreover, few remember that the selectionists made the opposite prediction; that the most functionally important regions of proteins should evolve rapidly (Hughes, 2007).
Some of the most basic techniques of bioinformatics depend on the fact that the neutralists were right in this case. Homology searches and sequence alignments depend on the fact that functionally important sequences are conserved over evolutionary time. If the selectionists had been right, these everyday tools of modern biology would be impossible (Hughes, 2007)
G Protein Coupled Receptors
G-protein-coupled receptors (GPCRs) constitute a large and diverse family of proteins whose primary function is to transduce extracellular stimuli into intracellular signals. They are among the largest and most diverse protein families in mammalian genomes. On the basis of homology with rhodopsin, they are predicted to contain seven membrane-spanning helices, an extracellular N-terminus and an intracellular C-terminus. This gives rise to their other names, the 7-TM receptors or the heptahelical receptors (Figure 2). GPCRs transduce extracellular stimuli to give intracellular signals through interaction of their intracellular domains with heterotrimeric G proteins, and the crystal structure of one member of this group, bovine rhodopsin, has recently been solved (Palczewski et al., 2000; Cherezov et al., 2007).
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Figure 2. The crystal structure of a human beta2-adrenergic receptor-T4 lysozyme fusion protein bound to the partial inverse agonist carazolol at 2.4 angstrom resolution. (Cherezov et al., 2007)
The presence of GPCRs in the genomes of bacteria, yeast, plants, nematodes and other invertebrate groups argues in favor of a relatively early evolutionary origin of this group of molecules. The diversity of GPCRs is dictated both by the multiplicity of stimuli to which they respond, as well as by the variety of intracellular signalling pathways they activate. These include light, neurotransmitters, odorants, biogenic amines, lipids, proteins, amino acids, hormones, nucleotides, chemokines and, undoubtedly, many others.
In addition, there are at least 18 different human Gα proteins to which GPCRs can be coupled (Hermans, 2003; Wong, 2003). These Gα proteins form heterotrimeric complexes with Gβ subunits, of which there are at least 5 types, and Gγ subunits, of which there are at least 11 types (Hermans, 2003).
Estimates of the number of GPCRs in the human genome vary widely. Based on their sequences, as well as on their known or suspected functions, there are estimated to be five or six major classes of GPCR. In a recent analysis of the GPCRs in the human genome, more than 800 GPCRs were listed (Fredriksson et al., 2003). Of this total, 701 were in the rhodopsin family (type A) and, of these, 241 were non-olfactory (Fredriksson et al., 2003). According to this analysis, there are approximately 460 type A olfactory receptors, although estimates range from 322 (Glusman et al., 2001; Takeda et al., 2002) to 900 (Venter et al., 2001), of which 347 have already been cloned (Zozulya et al., 2001).
This large number of olfactory receptors accounts for the ability of humans to detect a wide variety of exogenous (olfactory) ligands. A study similar to that of Fredriksson et al. (Fredriksson et al., 2003) identified 367 human endoGPCRs and 392 mouse endoGPCRs (Vassilatis et al., 2003); the term endoGPCR refers to GPCRs for endogenous (non-olfactory) ligands. In view of the known existence of alternatively spliced variants and editing isoforms of GPCRs, it is likely that the true number of GPCRs will never be known and is much higher than estimated.
Below (Figure 3) is shown a poster prepared by Kroeze and colleagues (2003), where the presented tree illustrates the relationships among the primary protein sequences of 274 type A rhodopsin-like GPCRs; for clarity, the secretin family receptors (of which there are 15), the adhesion receptor family (24), the glutamate receptor family (15) and the frizzled/taste2 receptor family (24) were not included. Kroeze and colleagues (2003), while constructing this tree, used the list of receptors, which was previously used by Fredriksson et al. (2003) and this served as a starting point. Then, the newly discovered `orphan' receptors were added to the list (http://kidb.bioc.cwru.edu/rothlab/jalview/viewJalView.html).
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Figure 3. A poster prepared by Kroeze and colleagues (2003), where the presented tree illustrates the relationships among the primary protein sequences of 274 type A rhodopsin-like GPCRs. (I changed the resolution into 300dpi, anyway if this figure resolution is a problem you can eliminate it..)
First, these scientists with interest in molecular evolution of GPCRs obtained the protein sequence of each receptor and then the sequences of the N- and C-termini, which are of variable length and show little similarity among the receptors, were trimmed manually. After that, the protein sequences were then aligned and the tree was drawn using the ClustalW server (http://clustalw.genome.ad.jp). However, the G-protein-coupling information in the poster is derived from the review by Wong (Wong, 2003).
The groupings of the receptors in the poster are thus similar, but not identical, to those of Fredriksson et al. (2003). For example, Fredriksson's α, β, γ and δ groups, which appear to be `monophyletic' in their tree, were not monophyletic in ours; this is likely to be due to slight differences in the options used in the two alignments, and the relative imprecision of the location of the roots of the branches in both trees.
Furthermore, it is interesting to know that the orphan receptors GPR57 and GPR58 were grouped with the trace amine receptors, and comparison of their sequences indicates that these orphans probably constitute the human equivalent of the type 2 trace amine receptors of rodents. Thus, trees of this type may serve to help in the process of `de-orphanizing' receptors.
How Do GPCRs Work?
The first step in signal transduction is ligand binding. The nature of GPCR ligand-binding sites is best studied by a combination of site-directed mutagenesis, molecular modelling of the receptors and screening of large numbers of potential ligands. For those who would be very much interested about the effects of mutations, it would be extremely recommended the most comprehensive database of the effects of mutations in GPCRs upon ligand binding, which can be found at http://wwwgrap.fagmed.uit.no. Agonist binding is followed by a change in the conformation of the receptor that may involve disruption of a strong ionic interaction between the third and sixth transmembrane helices (Ballesteros et al., 2001; Shapiro et al., 2002), which facilitates activation of the G-protein heterotrimer. Depending on the type of G protein to which the receptor is coupled, a variety of downstream signalling pathways can be activated (Marinissen and Gutkind, 2001; Neves et al., 2002). Signalling is then attenuated (desensitized) by GPCR internalization, which is facilitated by arrestin binding (Ferguson, 2001). Signalling, desensitization and eventual resensitization (Figure 4) are regulated by complex interactions of various intracellular domains of the GPCRs with numerous intracellular proteins (Hall and Lefkowitz, 2003; Bockaert et al., 2003; Ritter and Hall, 2009).
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Figure 4. Following agonist-induced receptor endocytosis, some G protein-coupled receptors (GPCRs) are targeted for proteolytic and/or lysosomal degradation, whereas other GPCRs rapidly recycle back to the plasma membrane. a | The interaction between GPCR-associated sorting protein 1 (GASP1) and δ-type opioid receptor (δOPR) promotes the endocytic targeting of agonist-internalized δOPRs to lysosomes, where the receptors are degraded. However, in a distinct cellular compartment (or distinct cell type) that lacks GASP1, as shown on the left, δOPRs are rapidly recycled back to the plasma membrane. b | By contrast, the interaction between the GPCR-interacting protein Na+–H+ exchange regulatory factor 1 (NHERF1; also known as EBP50 and SLC9A3R1) and the β2-adrenergic receptor (β2AR) promotes the rapid recycling of receptors following agonist-promoted internalization. However, in a distinct cellular compartment (or distinct cell type) that lacks NHERF1, as shown on the left, β2ARs are preferentially targeted to lysosomes for degradation (Ritter and Hall, 2009).
Although many studies have used β-adrenergic receptors as prototypical GPCRs, it has become increasingly clear that much more can be learned by systematic study of other receptors. The studies of the serotonin 5-HT2A receptor, for instance, showed that GPCR internalization and desensitization can occur by arrestin-independent pathways (Bhatnagar et al., 2001; Gray et al., 2003) and similar findings have been reported for other GPCRs (Lee et al., 1998). Interactions of GPCRs with other proteins, including cytoskeletal components such as PSD-95 (Hall and Lefkowitz, 2002; Xia et al., 2003), are increasingly being found to be important for regulating the activity, targeting and trafficking of GPCRs.
Attractive Targets for Magic Bullets
Although the biology of GPCRs is certainly intriguing, their ultimate importance is underscored by the fact that at least one third (Robas et al., 2003) and perhaps as many as half (Flower, 1999) of currently marketed drugs target GPCRs, although only 10% of GPCRs are known drug targets (Vassilatis et al., 2003). As new functions for GPCRs are discovered, especially for the orphan GPCRs for which no function is currently known, the number of drugs that target GPCRs can only be expected to increase. This is a focus of intense research effort, both in academia and in industry.
In addition to biological studies of the types summarized above, much excitement remains in the field because of the continuing de-orphanization of GPCRs and the subsequent elucidation of their pharmacology and physiology.
Once a large enough panel of GPCRs has been obtained and comprehensively characterized, a systematic analysis of the `receptorome' (the portion of the proteome encoding receptors) can yield important discoveries. Kroeze et al. (2003) used such an approach to discover the molecular mechanisms responsible for serious drug side-effects for example, phen/fen-induced heart disease (Rothman et al., 2000) and weight gain associated with the use of atypical antipsychotics (Kroeze et al., 2003). Additionally, screening the receptorome has been used to elucidate the actions of natural compounds and to obtain validated molecular targets for drug discovery (e.g. Roth et al., 2002).
A Short History
As with the G-protein-coupled receptors (GPCRs), heterotrimeric guanine nucleotide-binding proteins (G-proteins) represent an ancient protein family that has been highly conserved over evolution. The capacity of a number of bacterial exotoxins to covalently modify the α-subunit of many of the heterotrimeric G-proteins, and hence alter their function, attests to this. Such toxins were key tools in the discovery and classification of the G-proteins and remain important reagents in many studies of G-protein function (Milligan and Kostenis, 2006).
It was clear from the work of Sutherland and others in the late 1950s and early 1960s, which resulted in the award of the Nobel Prize for Physiology or Medicine in 1971, that a range of hormones was able to stimulate production of cyclic AMP (cAMP). It was not evident at that time, however, that this was a GTP-dependent process that required the intermediacy of a G-protein. This reflected the fact that ATP isolated from rabbit muscle and used as substrate for the generation of cAMP in such assays was contaminated with sufficient GTP to mask this requirement. It was not until enzymic synthesis of ATP became commonplace that the absolute requirement for GTP became apparent (Milligan and Kostenis, 2006).
Although the requirement for a GTP-dependent step was now evident, identification and characterisation of the putative GTP-dependent signal-transducing protein remained a tremendous challenge. However, as with many key steps in science, the confluence of information from apparently disparate strands of work underpinned this advance.
Studies on the action of an exotoxin produced by Vibrio cholerae, the bacterium responsible for the symptoms of the disease cholera, showed both that addition of the toxin to cells produced sustained generation and elevation of cAMP levels, and that it did so via its action as a mono-ADP-ribosyltransferase, that is, an enzyme able to catalyse the transfer of the ADP-ribose element of nicotinamide adenine dinucleotide (NAD+) to a protein substrate (Gill and Meren, 1978).
In parallel, mutagenesis of the mouse lymphoma cell line S49 (Coffino et al., 1975) began to dissect and identify molecular components of the trans-plasma membrane signal transduction cascade by which β -adrenoceptor, agonists cause elevation of cAMP. Sustained elevation of cAMP in S49 cells results in their death. Thus, mutant lines that continued to prosper in the presence of β-adrenoceptor agonists were isolated. Although not the first mutant identified, the key initial mutant cell line was (with hindsight erroneously) named cyc− because, as it failed to generate cAMP in response to isoprenaline and other agonists, but could be shown to still express a ligand-binding site with the characteristics of the β 2-adrenoceptor, the most obvious conclusion was that it must lack expression of the cAMP-generating enzyme, adenylyl cyclase. However, a range of studies indicated that direct regulation of adenylyl cyclase/cAMP production could still occur in these cells (Milligan and Kostenis, 2006).
Furthermore, while treatment of membranes of wild-type S49 cells with activated cholera toxin and [32P]NAD+ resulted in incorporation of radioactivity into a polypeptide of some 45 kDa, this did not occur when membranes of S49 cyc− cells were used. Membranes of S49 cyc− cells thus lacked a key component of the cAMP generation cascade and hence provided an ideal background for reconstitution studies designed to purify the cholera toxin substrate (Milligan and Kostenis, 2006).
Using rabbit liver as a source, this purification was a tour de force and identified a 45 kDa polypeptide corresponding to the cholera toxin substrate and transducing protein (Northup et al., 1980). However, despite efforts employing a range of chromatographic steps, the 45 kDa polypeptide copurified with a 35 kDa and (although originally overlooked, because of its rapid mobility through SDS–PAGE) an 8–10 kDa polypeptide. The 45 kDa protein was thus defined as the α -subunit of the adenylyl cyclase stimulatory G-protein Gs and the corresponding 35 and 8–10 kDa polypeptides the β - and γ -subunits that make up the functional G-protein heterotrimer. For this, and a host of other key studies on the function and structure of hetero-trimeric G-proteins, Alfred G. Gilman (Gilman, 1995) was awarded the Nobel Prize for Physiology or Medicine in 1994 along with Martin Rodbell (Rodbell, 1995).
A number of developments in what are now viewed as key underpinning aspects of the basic pharmacology of receptor ligand-binding studies could now begin to be understood in molecular terms.
For example, in competition binding studies using [3H]antagonist/agonists at the β 2-adrenoceptor, the ‘low' Hill slope for full agonist ligands observed in membranes of wild-type S49 cells was converted into a single site that displayed only low affinity for the agonist in membranes of S49 cyc− cells. As this was equivalent to the effects of adding guanine nucleotides to assays performed on wild-type S49 cell membranes, such studies defined the receptor/G-protein complex as a high-affinity site for agonists, the isolated receptor as a low-affinity site and indicates that classical antagonists did not discriminate between the two. Such studies were therefore integral to the development of the ‘two-state' receptor model and subsequent adaptations of this concept (Rang, 2006).
Although it had been recognized from the early studies on cAMP production that certain receptor ligands were able to reduce, rather than increase, cAMP levels, and that ligand-binding studies on such receptors often produced data similar to the ‘guanine nucleotide shifts' in agonist affinity discussed above, serious efforts to identify an equivalent adenylyl cyclase inhibitory ‘Gi' G-protein again required both information from an apparently unrelated research area and the concretion of relevant information in a timely review from Martin Rodbell (Rodbell, 1980).
Studies on the action of ‘islet-activating protein', an exotoxin produced by Bordetella pertussis, the causative agent of whooping cough, showed it to be a mono-ADP-ribosyltransferase able to modify covalently a 41 kDa polypeptide present in the membranes of essentially all cells (Katada and Ui, 1982). Furthermore, in a physiological context, ‘islet-activating protein' (now known generically as ‘pertussis toxin') attenuated α 2-adrenoceptor regulation of insulin secretion from islet cells, suggesting that the molecular target for pertussis toxin might be ‘Gi'. Although a functional reconstitution assay akin to that used for the purification of Gs was not readily available, purification of the pertussis toxin substrate in fractions enriched in high-affinity GTPase activity resulted in the identification of the 41 kDa polypeptide as the α -subunit of ‘Gi', along with 35 and 8–10 kDa polypeptides that appeared identical to the β - and γ -subunits of Gs (Bokoch et al., 1984).
G α -protein family
Although inhibition of a signal or a biological process by pretreatment with pertussis toxin rapidly became diagnostic of the involvement of ‘Gi', it was soon apparent that ‘Gi'α was not a single molecular species. A number of studies, particularly using brain as a rich source of polypeptides that were substrates for ADP-ribosylation by pertussis toxin, purified more than one polypeptide with apparent molecular mass close to 40 kDa (Huff et al., 1985). Although these could have represented nothing more than proteolytic cleavage products from a single ‘Gi'α-protein, they were recognised differentially by various antisera including those raised against the C-terminal region of rod transducin (Gt1) α (Pines et al., 1985). As with the G-protein-coupled photon receptor rhodopsin, high-level expression in the specialised architecture of mammalian rod outer segments had allowed the purification, detailed characterisation and cloning of Gt1α in the advance of other G-proteins (Figure 5). The major, ‘non-Gi', pertussis toxin substrate from the brain was thus designated Goα for G‘other' because its function was unclear.
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Figure 5. Homology of mammalian G-protein α -subunits. The relatedness of individual mammalian G-protein α -subunits is shown as an unrooted homology tree. The date of cloning of cDNAs corresponding to each family member is shown in parentheses (Milligan and Kostenis, 2006).
Cholera toxin-catalysed [32P]ADP-ribosylation studies had indicated at least two forms of Gs α with amounts of the two forms varying between tissues. Cloning of cDNAs encoding Gs α now uncovered that these variants were derived from a single gene via alternative splicing of exon 3 (Bray et al., 1986). By contrast, cloning of cDNAs encoding the various pertussis toxin-sensitive ‘Gi' G-protein α -subunits indicated each of what became known as Gi1α, Gi2α and Gi3α to be the products of different genes. ‘Goα‘ was also the product of a separate gene that can be differentially spliced to generate at least two polypeptides, Go1α and Go2α (Figure 5). Although the Gi2α and Gi3α gene products are widely expressed, both Gi1α and the forms of ‘Goα' have more restricted distribution patterns that can generally be described as ‘neuroendocrine' (Milligan and Kostenis, 2006).
Studies on specialised sensory systems, including olfactory, visual and lingual tissues, uncovered other G-protein α subunits, Golfactory, cone transducin (also called Gt2) and Ggustducin, respectively, with highly restricted distribution patterns that were highly related to, but distinct from, Gs or the previously identified Gi family members. Although receptor-mediated production of inositol 1,4,5 trisphosphate, and hence elevation of intracellular [Ca2+], seemed conceptually similar to receptor regulation of cAMP production, an absolute requirement for a GTP-dependent step and therefore a G-protein was significantly more recalcitrant to demonstration.
In part, this reflected that a membrane-based assay for ligand function was substantially more difficult to establish, that in most cell types neither cholera toxin nor pertussis toxin pretreatment modified this cascade and that ‘guanine nucleotide shifts' of agonist affinity in [3H] antagonist/agonist competition binding studies were generally small (and often negligible) for receptors that link predominantly to this pathway (Milligan and Kostenis, 2006). However, determined purification efforts resulted in the identification and characterisation of Gqα and G11α as 42 kDa polypeptides that fulfilled the criteria for phosphoinositidase Cβ-linked G-proteins (Taylor et al., 1990).
Essentially in parallel, and taking advantage of the high homology of other cloned Gα sequences, Mel Simon and colleagues cloned both Gqα and G11α (Strathmann and Simon, 1990) and showed these to be widely expressed. They also cloned the related G14α and G16α that have much more limited expression patterns (Figure 5), although both can also link receptors to the elevation of intracellular [Ca2+]i.
Finally, further efforts based on homology cloning identified two additional Gα-subunits, G12α and G13α, that form a separate subfamily (Figure 5) and are involved in communications between heterotrimeric G-protein-linked signalling pathways and cell responses regulated by monomeric GTP-binding proteins, including cellular shape and morphology and cell proliferation (Riobo and Manning, 2005).
Families of β - and γ -subunits
At least five different β- and 12 γ-subunits have been described till now (Milligan and Kostenis, 2006). Although a number of possible pairings have been indicated not to form, and tissue expression patterns may further limit the actual number of pairings in particular cells and tissues, there is still the potential for coexpression of a substantial number of pairs.
Despite this, early studies suggested that β / γ complexes isolated along with different α -subunits or from different tissues were functionally interchangeable, except that β 1/ γ 1 (the combination associated with Gt1 α in rod outer segments) was generally less potent functionally than β / γ complexes isolated from the brain, for example. The β -subunits have a β -propeller structure, containing seven so-called WD (tryptophan-aspartate)-40 repeats, and the crystal structure of the β 1/ γ 1 complex (Sondek et al., 1996) showed that the γ -subunit interacted with the β -subunit via an N-terminal coiled coil and a series of other extensive contacts along much of the length of the γ subunit sequence (Figure 6). Although β 1− β 4 are highly homologous, β 5 is substantially less so, suggesting that it may play a different role(s). Although it can certainly interact with a number of the γ -subunits, unlike the other β -subunits, it dissociates from the γ and is also able to interact with a number of regulator of G-protein signalling (RGS) protein family members that contain a G-protein γ -subunit-like domain.
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Figure 6. β/γ and receptor contact sites on Gα. Top: Sequence alignment of the N- and C- terminal regions of selected Gα-subunits. Residues that are subject to N-linked myristoylation, thio-palmitoylation or N-linked palmitoylation are highlighted in orange, green and yellow, respectively. In each case, M (black) is the protein synthesis initiator, methionine, that is eliminated during protein synthesis. Residues comprising the N-terminal αN helix are highlighted in red and residues at the extreme C-terminus of Gα are shown in blue. The αN helix is required for binding β/γ-subunits, and particular β/γ contacts are boxed in black, the extreme C-terminus plays a key role in specific receptor recognition. In the secondary structure diagram below the aligned sequences, β/γ and receptor interaction sites are highlighted in red and blue, respectively. Only selected domains of Gα are shown, and for simplicity the domains between αA and the α2 helix have been omitted as indicated by the dotted line. Bottom: Illustration of the N-terminal αN helix (red) and the C-terminal receptor contact region (blue) in the context of the tertiary and quaternary structure of the resting state, inactive Gi1αβ1γ2 heterotrimer. The GDP molecule is buried between the GTPase and helical domain of Gα (green), the β-subunit is coloured yellow and the γ-subunit is shown in orange. The diagram was generated using the coordinates from the PDB file 1GP2 and visualised with WebLab ViewerPro (Milligan and Kostenis, 2006).
G-proteins and Disease
By far the most prevalent disease associated with alteration in G-protein activity and amount is cholera (Milligan and Kostenis, 2006). As described above, ADP-ribosylation of the α-subunit of Gs catalysed by the activated A-subunit of the exotoxin of V. cholerae, that is ingested via contaminated water results in the persistent stimulation of adenylyl cyclase activity. This results in extrusion of water from cells of the intestinal epithelium and the watery diarrhoea and dehydration associated with the condition.
A number of other bacterial exotoxins can produce similar effects via the same mechanism. As well as regulation of the activity of G-proteins by extraneous factors, alteration in levels of G-protein subunits has been reported to be associated, or at least correlated, with disease processes, including an upregulation of Giα-subunits in heart failure.
Furthermore, a series of relatively rare endocrine conditions are linked to poor expression or mutation of a variety of Gα-subunits (Spiegel and Weinstein, 2004). Although most reports have centred on the function of the α -subunits, a relatively common polymorphic variant of the β3 -subunit has been associated with various cardiovascular phenotypes and aspects of the metabolic syndrome, but as with many such studies, the contribution of this is likely to be modified by a series of other variations that are rarely examined in parallel.
RFamide Neuropeptide Family and 26-amino acid residue RFamide peptide (26RFa/QRFP)
Peptides, defined by their carboxy-terminal arginine (R) and amidated phenylalanine (F) residues (RFamide), have been identified in the nervous systems of animals within all major phyla. The first recognized member of the RFamide neuropeptide family was the cardio-excitatory peptide, Phe-Met- Arg-Phe-amide (FMRFamide), isolated from ganglia of the clam Macrocallista nimbosa (Price and Greenberg, 1977).
Vertebrates and more especially invertebrates can each express an array of RFamide peptides, owing to the fact that multiple genes encoding RFamides are often present in a single species, and multiple mature RFamide peptides can be generated by a single polypeptide precursor.
These peptides seem to act as neurotransmitters and neuromodulators (Walker et al., 2009). Immunohistochemical studies that used antisera against FMRFamide suggested that the nervous system of vertebrates also contain neuropeptides immunologically related to FMRFamide (Raffa, 1988; Rastogi et al., 2001). In fact, several neuropeptides harbouring the RFamide sequence at their C-terminus have been characterised in the brain of various vertebrates.
In the past, the existence of five groups within the RFamide peptide family has been recognised in vertebrates (Figure 7), namely the neuropeptide FF (NPFF) group, the prolactin-releasing peptide (PrRP) group, the gonadotropin-inhibitory hormone (GnIH) group, the kisspeptin group, and the 26RFa/QRFP group (Chartrel et al., 2011; Leprince et al., 2013; Ukena et al., 2014).
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Figure 7. Phylogenetic tree of the RFamide peptide family in vertebrates. Studies over the past decade have demonstrated that the brain of vertebrates produces a variety of RFamide peptides. To date, five groups have been identified within this family: the neuropeptide FF (NPFF) group, the prolactin-releasing peptide (PrRP) group, the gonadotropin-inhibitory hormone (GnIH) group, the kisspeptin group, and the 26RFa/QRFP group (Ukena et al., 2014).
This is the original image of Ukena et al., 2014
Human kiss1 (this is 8 point)
Generally, RFamide peptides have been shown to exert important neuroendocrine, behavioural, sensory and autonomic functions (Ukena and Tsutsui, 2005; Tsutsui and Ukena, 2006).
In mammals, 26RFa/QRFP has been found to be a high-affinity endogenous ligand for the previously identified QRFPR. In humans, 26RFa/QRFP has been found to be an endogenous ligand for the orphan receptor, GPR103-QRFPR, which is a class-A G protein-coupled receptor (GPCR; Jiang et al. 2003). In rodents and monkeys, 26RFa/ QRFP plays diverse biological roles, including regulation of food intake and energy homeostasis, hormone secretion, nociception and bone formation.
Recently, the mature sequences of 26RFa/QRFP have been identified by structural analysis in quail and zebra finch. In birds, as well as in mammals, 26RFa/ QRFP-producing neurons are only located in the hypothalamus, while QRFPR is widely distributed throughout the brain. In birds, 26RFa/QRFP also exerts an orexigenic action, as it does in rodents, and a similar effect of 26RFa/QRFP has been suggested in fish, because of upregulation of 26RFa/qrfp mRNA by a negative energy state (Ukena et al. 2014).
Unity and diversity of the structure of 26RFa/QRFP in vertebrates
The 26-amino acid residue RFamide peptide, 26RFa/QRFP, was identified for the first time in the brain of an amphibian species (Chartrel et al. 2003). An antibody against the RFamide motif was used to screen peptide fractions purified from a brain extract of the European green frog (Rana esculenta).
After HPLC purification, the sequence of the isolated substance was analyzed by mass spectrometry MS/MS fragmentation; it turned out to be a 26-amino acid peptide possessing the RFamide motif at its C-terminus, namely VGTALGSLAEELNGYNRKKGGFSFRFamide. This neuropeptide had not been reported in any animals previously and was designated as 26RFa (Figure 8A; Chartrel et al. 2003).
The amino acid sequence of frog 26RFa was employed to identify the cDNA encoding the counterpart of 26RFa in rat and humans (Chartrel et al. 2003). Concurrently, two other research groups independently identified 26RFa/QRFP precursors using a bioinformatic approach in the rat, mouse, bovine, and human genomes and paired 26RFa/QRFP with a previously identified orphan G protein-coupled receptor (GPCR), GPR103, also known as AQ27 or SP9155 (Fukusumi et al. 2003, Jiang et al. 2003; Figure 8B).
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Figure 8. Alignments of the amino acid sequences of identified 26RFa/QRFP peptides (A) and their precursor proteins (B) deduced from mammalian (human, bovine, rat, and mouse), avian (chicken, quail, and zebra finch), amphibian (Xenopus), and fish (goldfish) cDNAs. The predicted signal peptide sequences are underlined with a dashed line. <E represents pyroglutamic acid. The positions of identified mature peptides in the precursor proteins are underlined with solid lines. The human and Xenopus 26RFa/QRFP precursors may also generate a nine-amino acid peptide, termed 9RFa (boxed). Fully conserved amino acids are highlighted with red boxes and highly conserved amino acids with gray boxes respectively. The Lys (K)-Arg (R) dibasic processing sites in birds and Xenopus, the single Arg (R) putative processing sites in mammals and fish, and the Gly (G) C-terminal amidation signals are shown in bold. Gaps marked by hyphens were inserted to optimize homology. The GenBank accession numbers of these sequences are as follows: human 26RFa/QRFP, NP_937823; bovine 26RFa/QRFP, NP_937865; rat 26RFa/QRFP, NP_937843; mouse 26RFa/QRFP, NP_906269; chicken 26RFa/QRFP, XP_001235089; quail 26RFa/QRFP, BAI81890; zebra finch 26RFa/QRFP, BAK32798; Xenopus tropicalis 26RFa/QRFP, XP_002936227; and goldfish 26RFa/Qrfp, ACI46681.
The reason why sometimes in the scientific books and papers sometimes it is shown as GPR103 and sometimes as QRFPR is just linked to the fact that GPR103 has been renamed QRFPR by the HUGO Gene Nomenclature Committee (http://www.genenames.org/).
The mature 43-amino acid residue RFamide peptide was identified from the culture medium of CHO cells that expressed the human peptide precursor (Fukusumi et al. 2003). As the N-terminal amino acid was pyroglutamic acid, this RFamide peptide was also named pyroglutamylated RFamide peptide (QRFP; Fukusumi et al., 2003). Subsequently, the cDNAs encoding the 26RFa/QRFP precursors have been characterized in goldfish (Liu et al., 2009), quail (Ukena et al. 2010), chicken (Ukena et al., 2010), and zebra finch (Tobari et al. 2011) (Figure 8B). Although the 26RFa/qrfp cDNA has not been characterized in the European green frog, the corresponding sequence in the African clawed frog (Xenopus tropicalis) is present in the database (Figure 8B). Furthermore, homologous sequences have been listed in the genome database of reptilian (lizard) and fish (stickleback, medaka, fugu, and zebrafish) species (Liu et al., 2009). These data have revealed the existence of the 26RFa/QRFP-encoding gene in representative species of the whole vertebrate phyla, including fish, amphibians, reptilians, birds, and mammals (Chartrel et al., 2011, Ukena et al., 2011).
- Quote paper
- Associate Professor Rigers Bakiu (Author), 2016, Molecular Evolution of Pyroglutamylated RFamide Peptide and Orphan G Protein Coupled Receptor, Munich, GRIN Verlag, https://www.grin.com/document/347085