**The Power of an Evolutionary Perspective in Studies of Endocrinology**

Jing He1, David M. Irwin1,2 and Ya-Ping Zhang1,3

*1State Key Laboratory of Genetic Resource and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences 2Department of Laboratory Medicine and Pathobiology and Banting and Best Diabetes Centre, University of Toronto 3Laboratory for Conservation and Utilization of Bioresource, Yunnan University 1,3China 2Canada* 

## **1. Introduction**

Much of our understanding of the molecular basis of endocrinology has been the product of highly productive studies that have focused on specific molecules (e.g., hormones) and their specific immediate interacting partners. However, biomolecules are not isolated particles, but instead they are elements of highly integrated interaction networks, and specific interactions among them drive virtually all cellular functions and underlie phenotypic complexity and diversity. Many hormones, and their specific receptors and other interacting proteins, are known to be evolutionarily related, which raises intriguing questions concerning how specificity originated within these systems.

Our previous studies have illustrated that biochemical entities are developmentally and evolutionarily fluid, with capabilities to be altered both in composition and behavior. Gene birth and death are widespread phenomena in genome evolution and accounts for the great diversity of gene families involved in endocrinology. While concordance of evolutionary histories both in pattern and process of hormones, receptors and interacting proteins might be expected for integrated systems, studies have shown that the evolutionary history of receptors need not mirror that of their ligands. Simultaneous emergence, or loss, of multiple interacting partners by multiple gene duplication or gene loss is unlikely in evolution. Gene duplication is essential in the development of complex endocrinology. It is creative in producing elements that allow evolutionary tinkering and thus plays a major role in gene co-option (i.e., recruitment for novel functions) facilitating the evolution of greater biological complexity. Alternatively, if an interacting partner is lost, the retained partner may either be subsequently lost or, more interestingly, serve as raw material in evolution and become recruited into a new interaction yielding a new function. Thus, a stepwise process of elaboration through mutation and optimization ensues, adapting genes (and their encoded proteins) into the physiology of an organism.

Here we review several recent advances in our understanding of the evolution of hormone signalling pathways that illustrate the power of an evolutionary perspective. Among our

The Power of an Evolutionary Perspective in Studies of Endocrinology 5

the assemblage of the complex endocrine systems and the main events that have prompted their diversification, and yields better insight into their biological functions and potential

Genomes are the entirety of organisms' hereditary information, and encode all the information necessary to give rise to biomolecular products. The structure and content of genomes do not remain static, and continually change through evolutionary time. Major evolutionary mechanisms that have been instrumental in shaping the genome include gene

As genome size increases, gene content tends to increase, although at a disproportionately lower rate in eukaryotes compared to non-eukaryotes (Gregory 2005; Hou and Lin 2009; Konstantinidis and Tiedje 2004; Lynch and Conery 2003). The gene number increases with genome size, and morphological complexity is mostly generated by expanding the sizes of gene families rather than due to a growth of the number of unique gene types, hence multicellular organisms employ large sets of similar gene products while exhibiting extraordinary biodiversity. The elements of endocrine systems often group into families (e.g., hormones and their specific receptors), whose members have diverged to various extents in regulation and function (Danks et al. 2011; Hoffmann and Opazo 2011; Irwin 2010;

Gene duplication is the most important mechanism for generating new genes and new biochemical processes and has facilitated the evolution of biodiversity and complexity (Li 2006; Ohno 1970). The most obvious contribution is supplying the raw genetic material for the various evolutionary forces (e.g., mutation, genetic drift and natural selection) to act upon, which lead to specialized or new gene functions (Zhang 2003). Without gene duplication, the capability and plasticity of organisms in adapting to changing environments would be severely limited. Gene duplication can also contribute to species divergence (Ting et al. 2004) along with the origin of species-specific features (Zhang et al. 2002). Duplication may involve part of gene, a single gene, part of a chromosome, an entire chromosome, or the whole genome. The first four scenarios are also known as regional duplications, because they do not result in a doubling of the entire genome. Hereinafter, the role of whole genome duplication and regional duplication will be discussed respectively.

Genome duplication is often considered to be more important than regional duplication in evolution, as it allows for the duplication of the entire regulatory systems. Regional duplications, on the other hand, generally allow for part of a regulatory system to be duplicated, which may lead to imbalances that may disrupt normal function. The significance of whole genome duplication has been highlighted by the studies of invertebrate chordates and the base of vertebrate evolution (Garcia-Fernández and Holland 1994), as well as fish diversification (Dehal and Boore 2005; Jaillon et al. 2004). Genome duplication facilitated the appearance and diversification of complex features, such as the endocrine system (Holland et al. 2008). The amphioxus genome exhibits considerable

**3. Evolutionary mechanisms for the diversification of endocrine systems** 

cross-talk between them through evolutionary related interacting proteins.

duplication, gene loss and evolutionary shift.

Kim et al. 2011; Sundström, Dreborg and Larhammar 2010).

**3.1 Gene duplication** 

**3.1.1 Whole genome duplication** 

examples are the motilin and ghrelin signalling pathways where we have demonstrated that both the hormones (motilin and ghrelin) and their specific receptors descended from common ancestors through independent gene duplications. The motilin receptor originated well before the evolution of the hormone, and the motilin-motilin receptor specificity has arisen, as the result of ligand-receptor coevolution, after the hormone gene duplicated. Similarly, motilin and its specific receptor gene specifically lost on the rodent lineage followed a stepwise process. Once one of the interacting partners is lost, the retained partner may subsequently be lost, or serve as raw material in evolution and become recruited into a new function.

Given the evolutionary dynamics of the genome and the plasticity of biomolecule networks, an evolutionary perspective is necessary to understand many aspects of the molecular basis of endocrinology. An integrated evolutionary comparative strategy helps enhance our understanding of the assemblage of the complex endocrine systems, provide important clues in interaction capacity exploration, and identify the main diversification events of the endocrine systems and potential cross-talk between them through evolutionary related interacting proteins. In addition, knowledge of how elements of the endocrine systems that underlie cellular functions are evolutionarily and developmentally interact, help not only in choosing appropriate species to examine function, but also provide genetic makers to probe the emergence of specific traits and characteristics, uncovering the genetic basis that underlie the morphological and behavior changes, and thus enhance our understanding of how organisms adapt to changing environments.

## **2. The molecular basis of endocrine systems**

Endocrinology is the branch of biology that focuses on regulatory systems involving a group of specialized chemical substances called hormones that travel though blood. New developments in endocrinology have been dominated by progress in molecular genetics. Although these investigations often relate to rare single gene disorders, they have resulted in major advances in our understanding of the cellular mechanisms of hormone action (Cegla, Tan and Bloom 2010; Hodson et al. 2010; Peter and Vallo 1998). Most endocrinology studies are medically orientated, and thus anthropocentric, with little or no comparative or evolutionary perspective, and therefore provide few insights into our comprehension of the enigmatic origin and diversification of these systems (Markov et al. 2008).

Much of our understanding of the molecular basis of endocrinology has come from highly productive studies that have focused on specific molecules and their immediate interacting partners, in fact, hormonal systems are a central part. Indeed the specificity of each hormone ligand and receptor pair is maintained in divergent species (Moyle et al. 1994), while biochemical entities are developmentally and evolutionarily fluid, with a much wider range of capabilities for alteration in both composition and behavior (Avise and Ayala 2007; Wilkins 2007). Thus intriguing questions concerning how the diversity and specificity occur within these systems remain to be answered.

Genomes are documents of life history, and their structures continually change throughout evolution. Humans represent only a leaf on the tree of life. The anthropocentrism view of evolution, where humans are the pinnacle of gradually developed complexity, is a onesided and incorrect view (Markov et al. 2008). In the light of evolution, an approach that considers each taxa equally, adopts a comparative strategy, integrates information from diverse organisms and various scientific approaches, helps enhance our understanding of the assemblage of the complex endocrine systems and the main events that have prompted their diversification, and yields better insight into their biological functions and potential cross-talk between them through evolutionary related interacting proteins.

## **3. Evolutionary mechanisms for the diversification of endocrine systems**

Genomes are the entirety of organisms' hereditary information, and encode all the information necessary to give rise to biomolecular products. The structure and content of genomes do not remain static, and continually change through evolutionary time. Major evolutionary mechanisms that have been instrumental in shaping the genome include gene duplication, gene loss and evolutionary shift.

## **3.1 Gene duplication**

4 Contemporary Aspects of Endocrinology

examples are the motilin and ghrelin signalling pathways where we have demonstrated that both the hormones (motilin and ghrelin) and their specific receptors descended from common ancestors through independent gene duplications. The motilin receptor originated well before the evolution of the hormone, and the motilin-motilin receptor specificity has arisen, as the result of ligand-receptor coevolution, after the hormone gene duplicated. Similarly, motilin and its specific receptor gene specifically lost on the rodent lineage followed a stepwise process. Once one of the interacting partners is lost, the retained partner may subsequently be lost, or serve as raw material in evolution and become recruited into a

Given the evolutionary dynamics of the genome and the plasticity of biomolecule networks, an evolutionary perspective is necessary to understand many aspects of the molecular basis of endocrinology. An integrated evolutionary comparative strategy helps enhance our understanding of the assemblage of the complex endocrine systems, provide important clues in interaction capacity exploration, and identify the main diversification events of the endocrine systems and potential cross-talk between them through evolutionary related interacting proteins. In addition, knowledge of how elements of the endocrine systems that underlie cellular functions are evolutionarily and developmentally interact, help not only in choosing appropriate species to examine function, but also provide genetic makers to probe the emergence of specific traits and characteristics, uncovering the genetic basis that underlie the morphological and behavior changes, and thus enhance our understanding of

Endocrinology is the branch of biology that focuses on regulatory systems involving a group of specialized chemical substances called hormones that travel though blood. New developments in endocrinology have been dominated by progress in molecular genetics. Although these investigations often relate to rare single gene disorders, they have resulted in major advances in our understanding of the cellular mechanisms of hormone action (Cegla, Tan and Bloom 2010; Hodson et al. 2010; Peter and Vallo 1998). Most endocrinology studies are medically orientated, and thus anthropocentric, with little or no comparative or evolutionary perspective, and therefore provide few insights into our comprehension of the

Much of our understanding of the molecular basis of endocrinology has come from highly productive studies that have focused on specific molecules and their immediate interacting partners, in fact, hormonal systems are a central part. Indeed the specificity of each hormone ligand and receptor pair is maintained in divergent species (Moyle et al. 1994), while biochemical entities are developmentally and evolutionarily fluid, with a much wider range of capabilities for alteration in both composition and behavior (Avise and Ayala 2007; Wilkins 2007). Thus intriguing questions concerning how the diversity and specificity occur

Genomes are documents of life history, and their structures continually change throughout evolution. Humans represent only a leaf on the tree of life. The anthropocentrism view of evolution, where humans are the pinnacle of gradually developed complexity, is a onesided and incorrect view (Markov et al. 2008). In the light of evolution, an approach that considers each taxa equally, adopts a comparative strategy, integrates information from diverse organisms and various scientific approaches, helps enhance our understanding of

enigmatic origin and diversification of these systems (Markov et al. 2008).

new function.

how organisms adapt to changing environments.

within these systems remain to be answered.

**2. The molecular basis of endocrine systems** 

As genome size increases, gene content tends to increase, although at a disproportionately lower rate in eukaryotes compared to non-eukaryotes (Gregory 2005; Hou and Lin 2009; Konstantinidis and Tiedje 2004; Lynch and Conery 2003). The gene number increases with genome size, and morphological complexity is mostly generated by expanding the sizes of gene families rather than due to a growth of the number of unique gene types, hence multicellular organisms employ large sets of similar gene products while exhibiting extraordinary biodiversity. The elements of endocrine systems often group into families (e.g., hormones and their specific receptors), whose members have diverged to various extents in regulation and function (Danks et al. 2011; Hoffmann and Opazo 2011; Irwin 2010; Kim et al. 2011; Sundström, Dreborg and Larhammar 2010).

Gene duplication is the most important mechanism for generating new genes and new biochemical processes and has facilitated the evolution of biodiversity and complexity (Li 2006; Ohno 1970). The most obvious contribution is supplying the raw genetic material for the various evolutionary forces (e.g., mutation, genetic drift and natural selection) to act upon, which lead to specialized or new gene functions (Zhang 2003). Without gene duplication, the capability and plasticity of organisms in adapting to changing environments would be severely limited. Gene duplication can also contribute to species divergence (Ting et al. 2004) along with the origin of species-specific features (Zhang et al. 2002). Duplication may involve part of gene, a single gene, part of a chromosome, an entire chromosome, or the whole genome. The first four scenarios are also known as regional duplications, because they do not result in a doubling of the entire genome. Hereinafter, the role of whole genome duplication and regional duplication will be discussed respectively.

## **3.1.1 Whole genome duplication**

Genome duplication is often considered to be more important than regional duplication in evolution, as it allows for the duplication of the entire regulatory systems. Regional duplications, on the other hand, generally allow for part of a regulatory system to be duplicated, which may lead to imbalances that may disrupt normal function. The significance of whole genome duplication has been highlighted by the studies of invertebrate chordates and the base of vertebrate evolution (Garcia-Fernández and Holland 1994), as well as fish diversification (Dehal and Boore 2005; Jaillon et al. 2004). Genome duplication facilitated the appearance and diversification of complex features, such as the endocrine system (Holland et al. 2008). The amphioxus genome exhibits considerable

The Power of an Evolutionary Perspective in Studies of Endocrinology 7

loss of multiple interacting partners is unlikely, despite the intimate association between them. Gene loss, a dramatic genetic event, leads to the immediate loss of specific interactions, and probably affects interaction turnover as greatly as gene duplication. If a signaling protein is lost, then what are the evolutionary consequences for the retained

Genes are not only duplicated and lost, there are many genes which have been conserved and are unambiguous orthologues in a wide variety of taxonomic species, but evolutionary shifts occur frequently and orthologues genes can have distinct functions in different taxa (Macqueen et al. 2010; Zhou et al. 2008). Species adapt to diverse ecosystems and environments, and have differing genetic backgrounds. As selective constraints vary, it impacts the pattern of gene evolution, and changes in selection can yield changes in function (Irwin 2001). In some cases, positive selection appears to underlie the evolutionary shift (Wallis 2001; Liu et al. 2001); while in others, inefficient purifying selection and increased genetic drift, associated with a reduction in effective population size, are the cause (Macqueen et al. 2010). The genetic network of the endocrine systems are developmentally and evolutionarily fictile, the elemental composition is prone to be altered via gene gain and loss, and its physiological properties frequently change through mutations in endocrine gene coding sequences and/or regulatory systems, and turnover of interacting biomolecules. Using a comparative strategy, integrating information on species phylogenetic relationships, gene evolutionary history, gene sequences and functional properties such as expression, interaction and physiological data, should enhance our understanding of how they evolved and yield better insight into their biological functions. The large amounts of accumulating

genetic information is a powerful resource for addressing these questions.

structure of their precursor genes show considerable similarity (Fig. 1).

**motilin/ghrelin hormone family and their receptors 4.1 Gene duplication plays a major role in gene co-option** 

**4. Case studies for evolutionary endocrinology: Lessons from the** 

Ghrelin and motilin represent a novel gastrointestinal hormone family in mammals (Inui 2001). Not only are ghrelin and motilin structurally related, but, the sequence and overall

Ghrelin is derived by posttranslational cleavage from its precursor preproghrelin (GHRL), and is a circulating peptide hormone that is secreted mainly by the stomach and acts upon the hypothalamus and hindbrain (Nakazato et al. 2001; Kojima and Kangawa 2005). Growth hormone secretagogue receptor (GHSR) is the specific receptor for ghrelin and a G proteincoupled receptor, and upon stimulation releases growth hormone (GH) from the pituitary (Howard et al. 1996; Kojima et al. 1999; Sun, Ahmed and Smith 2003). Evidence from mammals suggests that ghrelin also acts to stimulate gastric motility, increase appetite and food intake, and induce a positive energy balance leading to body weight gain (Murray et al. 2003; Peeters 2005). Prepromotilin (MLN) is posttranslationaly processed to yield a secreted peptide that is then cleaved at a paired basic amino acid site and gives rise to motilin (Poitras 1993). Motilin primarily acts to increase gastrointestinal motility by activating neural pathways or via the direct stimulation of smooth muscles. In human and dog it has been suggested that motilin has a physiological role in the regulation of a motor pattern

partners?

**3.3 Evolutionary shift** 

**4.1.1 Ghrelin and motilin** 

synteny with the human genome, but lacks the whole-genome duplications characteristic of vertebrates (Dehal and Boore 2005). Holland et al. (2008) examined the existence of endocrine components based on the amphioxus genome, and reasoned that ancestral chordate only possess a basic set of endocrine functions. Hereby a fully functional endocrine system must have arisen after the divergence of cephalochordate, which was driven in all probability by subsequent genome duplications.

## **3.1.2 Regional duplication**

While whole genome duplication events are not uncommon, nevertheless they only infrequently contribute to the evolution of well-developed bisexual organisms, as they likely disrupt the mechanism of sex determination and would be quickly eliminated (Li 1997). Regional duplications make up the gap, providing new genetic material for local elaboration and optimization, and fuel the evolution of lineage-specific variability (Li 1997; Ohno 1970). Whole genome duplications and small-scale duplications have very different consequences. Selective retention of different duplicates, and enrichment of signaling proteins and transcription factors, have been observed in yeast, plants, early vertebrate and fish following whole genome duplications (Conant 2010; Gout, Duret and Kahn 2009; Huminiecki and Heldin 2010; Kassahn et al. 2009; Manning and Scheeff 2010). This indicates that the individual duplication of signaling proteins and transcriptional regulators may be deleterious, since interactions between them are relatively transient and subtle, requiring a dosage balance from the whole genome duplication to survive. It is reasonably to expect that, simultaneous duplications of ligands and downstream signaling genes are required to allow the expansion of the complex endocrine systems. As it is, only part of the regulatory system has been duplicated in regional duplication, then, what are the evolutionary consequences of such an imbalanced outcome?

## **3.2 Gene loss**

Studies on genome evolution have focused on the creation of new genes, including changes in regulatory mechanisms, and often neglect the role of selective gene loss in shaping these genomes. Gene loss or pseudogenization is a widespread phenomenon in genome evolution (Wang, Grus and Zhang 2006). The differential fixation of mutational gene loss after genome duplication illustrates the power of these types of events (Semyonov et al. 2008; van Hoek and Hogeweg 2009). Within gene families gene turnover, caused by differential gene gain and loss, leads to diverse patterns of gene distributions on different lineages, and contributes significantly to the evolution of biodiversity and may be the basis for reproductive isolation and speciation in geographically isolated populations (Gagneux and Varki 2001; Gout, Duret and Kahn 2009; Hahn, Demuth and Han 2007; Kettler et al. 2007; Powell et al. 2008). Sometimes, the ubiquitous, and near-stochastic, gene loss process can lead to the loss of single copy genes. By taking advantages of the availability of large amounts of vertebrate genomic information, it has been shown that many important human endocrine genes have been found to be missing or inactivated in other vertebrates, and vice versa (He et al. 2010; Irwin 2010; Pitel et al. 2010). In the same way, evolutionary comparisons among different taxa can help identify novel elements of endocrine systems that are not possessed by model animals. Endocrine entities are not isolated particles, but are elements of highly integrated interaction networks, and play their role through specific interactions (Carroll, Bridgham and Thornton 2008). As a random process, the simultaneous loss of multiple interacting partners is unlikely, despite the intimate association between them. Gene loss, a dramatic genetic event, leads to the immediate loss of specific interactions, and probably affects interaction turnover as greatly as gene duplication. If a signaling protein is lost, then what are the evolutionary consequences for the retained partners?

## **3.3 Evolutionary shift**

6 Contemporary Aspects of Endocrinology

synteny with the human genome, but lacks the whole-genome duplications characteristic of vertebrates (Dehal and Boore 2005). Holland et al. (2008) examined the existence of endocrine components based on the amphioxus genome, and reasoned that ancestral chordate only possess a basic set of endocrine functions. Hereby a fully functional endocrine system must have arisen after the divergence of cephalochordate, which was driven in all

While whole genome duplication events are not uncommon, nevertheless they only infrequently contribute to the evolution of well-developed bisexual organisms, as they likely disrupt the mechanism of sex determination and would be quickly eliminated (Li 1997). Regional duplications make up the gap, providing new genetic material for local elaboration and optimization, and fuel the evolution of lineage-specific variability (Li 1997; Ohno 1970). Whole genome duplications and small-scale duplications have very different consequences. Selective retention of different duplicates, and enrichment of signaling proteins and transcription factors, have been observed in yeast, plants, early vertebrate and fish following whole genome duplications (Conant 2010; Gout, Duret and Kahn 2009; Huminiecki and Heldin 2010; Kassahn et al. 2009; Manning and Scheeff 2010). This indicates that the individual duplication of signaling proteins and transcriptional regulators may be deleterious, since interactions between them are relatively transient and subtle, requiring a dosage balance from the whole genome duplication to survive. It is reasonably to expect that, simultaneous duplications of ligands and downstream signaling genes are required to allow the expansion of the complex endocrine systems. As it is, only part of the regulatory system has been duplicated in regional duplication, then, what are the evolutionary

Studies on genome evolution have focused on the creation of new genes, including changes in regulatory mechanisms, and often neglect the role of selective gene loss in shaping these genomes. Gene loss or pseudogenization is a widespread phenomenon in genome evolution (Wang, Grus and Zhang 2006). The differential fixation of mutational gene loss after genome duplication illustrates the power of these types of events (Semyonov et al. 2008; van Hoek and Hogeweg 2009). Within gene families gene turnover, caused by differential gene gain and loss, leads to diverse patterns of gene distributions on different lineages, and contributes significantly to the evolution of biodiversity and may be the basis for reproductive isolation and speciation in geographically isolated populations (Gagneux and Varki 2001; Gout, Duret and Kahn 2009; Hahn, Demuth and Han 2007; Kettler et al. 2007; Powell et al. 2008). Sometimes, the ubiquitous, and near-stochastic, gene loss process can lead to the loss of single copy genes. By taking advantages of the availability of large amounts of vertebrate genomic information, it has been shown that many important human endocrine genes have been found to be missing or inactivated in other vertebrates, and vice versa (He et al. 2010; Irwin 2010; Pitel et al. 2010). In the same way, evolutionary comparisons among different taxa can help identify novel elements of endocrine systems that are not possessed by model animals. Endocrine entities are not isolated particles, but are elements of highly integrated interaction networks, and play their role through specific interactions (Carroll, Bridgham and Thornton 2008). As a random process, the simultaneous

probability by subsequent genome duplications.

consequences of such an imbalanced outcome?

**3.2 Gene loss** 

**3.1.2 Regional duplication** 

Genes are not only duplicated and lost, there are many genes which have been conserved and are unambiguous orthologues in a wide variety of taxonomic species, but evolutionary shifts occur frequently and orthologues genes can have distinct functions in different taxa (Macqueen et al. 2010; Zhou et al. 2008). Species adapt to diverse ecosystems and environments, and have differing genetic backgrounds. As selective constraints vary, it impacts the pattern of gene evolution, and changes in selection can yield changes in function (Irwin 2001). In some cases, positive selection appears to underlie the evolutionary shift (Wallis 2001; Liu et al. 2001); while in others, inefficient purifying selection and increased genetic drift, associated with a reduction in effective population size, are the cause (Macqueen et al. 2010).

The genetic network of the endocrine systems are developmentally and evolutionarily fictile, the elemental composition is prone to be altered via gene gain and loss, and its physiological properties frequently change through mutations in endocrine gene coding sequences and/or regulatory systems, and turnover of interacting biomolecules. Using a comparative strategy, integrating information on species phylogenetic relationships, gene evolutionary history, gene sequences and functional properties such as expression, interaction and physiological data, should enhance our understanding of how they evolved and yield better insight into their biological functions. The large amounts of accumulating genetic information is a powerful resource for addressing these questions.

## **4. Case studies for evolutionary endocrinology: Lessons from the motilin/ghrelin hormone family and their receptors**

## **4.1 Gene duplication plays a major role in gene co-option 4.1.1 Ghrelin and motilin**

Ghrelin and motilin represent a novel gastrointestinal hormone family in mammals (Inui 2001). Not only are ghrelin and motilin structurally related, but, the sequence and overall structure of their precursor genes show considerable similarity (Fig. 1).

Ghrelin is derived by posttranslational cleavage from its precursor preproghrelin (GHRL), and is a circulating peptide hormone that is secreted mainly by the stomach and acts upon the hypothalamus and hindbrain (Nakazato et al. 2001; Kojima and Kangawa 2005). Growth hormone secretagogue receptor (GHSR) is the specific receptor for ghrelin and a G proteincoupled receptor, and upon stimulation releases growth hormone (GH) from the pituitary (Howard et al. 1996; Kojima et al. 1999; Sun, Ahmed and Smith 2003). Evidence from mammals suggests that ghrelin also acts to stimulate gastric motility, increase appetite and food intake, and induce a positive energy balance leading to body weight gain (Murray et al. 2003; Peeters 2005). Prepromotilin (MLN) is posttranslationaly processed to yield a secreted peptide that is then cleaved at a paired basic amino acid site and gives rise to motilin (Poitras 1993). Motilin primarily acts to increase gastrointestinal motility by activating neural pathways or via the direct stimulation of smooth muscles. In human and dog it has been suggested that motilin has a physiological role in the regulation of a motor pattern

The Power of an Evolutionary Perspective in Studies of Endocrinology 9

FNAPFDVGIKLSGVQYQQHS

FNAPFDVGIKLSGVQYQQHS

FNAPFDVGIKLSGVQYQQHS

FNAPFDVGIKLSGVQYQQHS

FNAPFDVGIKLSGVQYQQHS

FNAPFDVGIKLSGVQYQQHS

FNAPFDVGIKLSGVQYQQHS

FNAPFDVGIKLSGVQYQQHS

FNSPLDVGIKLSGAQYQQHS

FNVPFDVGIKLSGVQYQQHS

FNAPFDVGIKLSGIQYQQHSQ

FNAPFDVGIKLSGAQYQQHG

FNAPFDVGIKLSGAQYQQHG

FNAPFDVGIKLSGAQYQQHG

FNAPFDVGIKLSGAQYQQHG

FNAPFDVGIKLSGAQYHQHG

FNAPFDVGIKLSGPQYHQHG

FNAPFDVGIKLSGAQYHQHS

FNAPFDIGIKLSGAQSLQHGQ

FNAPFNIGIKLSGAQSLQHGQ

FNAPFNIGIKLSGAQSLQHGQ

QALG

QALG

QALG

QALG

QALG

QALG

QALG

QALG

QALG

QALG

ALG

RALG

RALG

RALG

RALG

QALG

QALG

QALG

TLG

TLG

TLG

Species Ghrelin sequence Obestatin homolog sequence

*Homo sapiens* GSSFLSPEHQRVQQRKESKKPP AKLQP

*Pan troglodytes* GSSFLSPEHQRVQQRKESKKPP AKLQP

*Pongo pygmaeus* GSSFLSPEHQRVQQRKESKKPP AKLQP

*Hylobates lar* GSSFLSPEHQRVQQRKESKKPP AKLQP

*Macaca fuscata* GSSFLSPEHQRAQQRKESKKPP AKLQP

*Papio hamadryas* GSSFLSPEHQRAQQRKESKKPP AKLQP

*Saimiri sciureus* GSSFLSPEHQRIQQRKESKKPPA KLQP

*Macaca mulatta* GSSFLSPEHQRAQQRKESKKPP AKLQP

*Otolemur garnettii* GSSFLSPDHQKIQQRKESKKPPA KLQP

*Cebus paella* GSSFLSPEHQRMQQRKESKKPP AKLQS

*Aotus trivirgatus* GSSFLSPEHQRIQQRKESKKPPA KLQP

AKLQP

*Mus musculus* GSSFLSPEHQKAQQRKESKKPP AKLQP

*Rattus norvegicus* GSSFLSPEHQKAQQRKESKKPP AKLQP

AKLQP

ARLQP *Felis catus* GSSFLSPEHQKVQQRKESKKPP AKLQP

*Canis familiaris* GSSFLSPEHQKLQQRKESKKPP AKLQP

*Equus caballus* GSSFLSPEHHKVQHRKESKKPP AKLKP

*Rangifer tarandus* GSSFLSPEHQKLQRKEPKKPSGR LKP

*Capra hircus* GSSFLSPEHQKLQRKEPKKPSGR LKP

*Ovis aries* GSSFLSPEHQKLQRKEPKKPSGR LKP

GSSFLSPEHQKAQQRKESKKPQ

GSSFLSPEHQKTQQRKESKKPP

GSSFLSPEHQKAQQRKDAKKPP

*Bos taurus* GSSFLSPEHQKLQRKEAKKPSG FNAPFNIGIKLAGAQSLQHG

*Mesocricetus auratus* 

*Meriones unguiculatus* 

*Oryctolagus cuniculus* 

typical for the fasted state (Poitras 1993). It is of interest to note that motilin also has a weak GH-releasing effect (Samson et al. 1984). GPR38, an orphan G protein coupled receptor, was identified as the motilin receptor, MLNR, through a remarkable process of reverse pharmacology (Feighner et al 1999). GHSR and MLNR, whose sequences are very similar, are members of the β-group of rhodopsin-like receptor family (Holst et al. 2004).

Despite the very close resemblance of the hormones and receptors, to date there is no evidence for any cross-reactivity between the ligands, which corresponds to the fact that the pharmacophore of the peptides are quite different (Peeters 2005). Octanoylation of serine3 is a unique and crucially important feature of ghrelin and studies have demonstrated that without the octanoyl group the potency of GHRL is dramatically decreased (Peeters 2005; Kaiya et al. 2001).

## **4.1.2 Evolution of the motilin/ghrelin hormone gene family**

Genes for ghrelin have been cloned from a number of vertebrate species. Using bioinformatic methods, we have identified additional ghrelin gene sequences from diverse mammalian species and a frog *Xenopus tropicalis* (Table 1). Motilin genes have only been identified and characterized in mammals and birds, even after the use of bioinformatic approaches (Table 2). Ghrelin and motilin genes are both single copy genes in all of the species studied, and reside in conserved gene neighborhoods respectively, strongly supporting their orthology. The amino acid sequences of ghrelin are well conserved among species, especially in the N terminal region, and the same principle holds for motilin (Table 1-2). Interestingly, when the comparative genomic analysis was conducted between human and other vertebrates (chicken, *X*. *tropicalis*, medaka, tetraodon, and zebrafish) aimed at the GHRL and MLN neighborhood regions, it was revealed that homologs of the human GHRL and MLN flanking genes, which are located on different chromosomes in amniotes, were found to reside on the same chromosome near the GHRL locus in medaka, tetraodon, zebrafish, and *X*. *tropicalis*. This observation suggests that there was a duplication of the GHRL gene yielding MLN on the amniote lineage however there was no overlap in the genomic neighborhoods for GHRL and MLN. We could not identify any sequences similar to ghrelin and/or motilin genes in the recently released lamprey and deuterostome draft genomes. GHRL sequences from fish and amphibians posses only a single putative endoproteinase recognition site C-terminal to the signal peptidase cleavage site, thus can produce only a single posttranslational-processed peptide, ghrelin. In contrast, GHRL of amniotes (reptiles, birds, and mammals) possess three putative endoproteinase recognition sites, potentially giving rise to a second posttranslationalprocessed peptide, a 24-residue ghrelin-associated peptide (Fig. 1). The second peptide has recently been identified to be obestatin in mammals (Zhang et al. 2005). All MLNs, which are only found in reptiles, birds and mammals, possess three putative endoproteinase recognition sites, thus potentially give rise to two posttranslational-processed peptides, motilin and a 17 residue peptide in a position homologous to obestatin (Fig 1). Phylogenetic analysis revealed that bullfrog GHRL groups with amniote MLN rather than amnitote GHRL, although the bootstrap support for this conclusion is low (Fig. 2).

Based on the distribution of GHRL and MLN genes in the species studied, comparative genomics analysis between human and other vertebrates, and endoproteinase cleavage sites distribution in GHRL/MLN genes, we surmise that MLN was generated by a gene duplication on the early amniote lineage as illustrated in figure 2 (He et al. 2011). Other potential evolutionary scenarios (e.g., duplication prior to the fish-tetrapod divergence) require a larger number of gene deletion events along with parallel gain or loss of endopeptidease cleavage sites, and thus are less parsimonious.

typical for the fasted state (Poitras 1993). It is of interest to note that motilin also has a weak GH-releasing effect (Samson et al. 1984). GPR38, an orphan G protein coupled receptor, was identified as the motilin receptor, MLNR, through a remarkable process of reverse pharmacology (Feighner et al 1999). GHSR and MLNR, whose sequences are very similar,

Despite the very close resemblance of the hormones and receptors, to date there is no evidence for any cross-reactivity between the ligands, which corresponds to the fact that the pharmacophore of the peptides are quite different (Peeters 2005). Octanoylation of serine3 is a unique and crucially important feature of ghrelin and studies have demonstrated that without the octanoyl group the potency of GHRL is dramatically decreased (Peeters 2005;

Genes for ghrelin have been cloned from a number of vertebrate species. Using bioinformatic methods, we have identified additional ghrelin gene sequences from diverse mammalian species and a frog *Xenopus tropicalis* (Table 1). Motilin genes have only been identified and characterized in mammals and birds, even after the use of bioinformatic approaches (Table 2). Ghrelin and motilin genes are both single copy genes in all of the species studied, and reside in conserved gene neighborhoods respectively, strongly supporting their orthology. The amino acid sequences of ghrelin are well conserved among species, especially in the N terminal region, and the same principle holds for motilin (Table 1-2). Interestingly, when the comparative genomic analysis was conducted between human and other vertebrates (chicken, *X*. *tropicalis*, medaka, tetraodon, and zebrafish) aimed at the GHRL and MLN neighborhood regions, it was revealed that homologs of the human GHRL and MLN flanking genes, which are located on different chromosomes in amniotes, were found to reside on the same chromosome near the GHRL locus in medaka, tetraodon, zebrafish, and *X*. *tropicalis*. This observation suggests that there was a duplication of the GHRL gene yielding MLN on the amniote lineage however there was no overlap in the genomic neighborhoods for GHRL and MLN. We could not identify any sequences similar to ghrelin and/or motilin genes in the recently released lamprey and deuterostome draft genomes. GHRL sequences from fish and amphibians posses only a single putative endoproteinase recognition site C-terminal to the signal peptidase cleavage site, thus can produce only a single posttranslational-processed peptide, ghrelin. In contrast, GHRL of amniotes (reptiles, birds, and mammals) possess three putative endoproteinase recognition sites, potentially giving rise to a second posttranslationalprocessed peptide, a 24-residue ghrelin-associated peptide (Fig. 1). The second peptide has recently been identified to be obestatin in mammals (Zhang et al. 2005). All MLNs, which are only found in reptiles, birds and mammals, possess three putative endoproteinase recognition sites, thus potentially give rise to two posttranslational-processed peptides, motilin and a 17 residue peptide in a position homologous to obestatin (Fig 1). Phylogenetic analysis revealed that bullfrog GHRL groups with amniote MLN rather than amnitote GHRL, although the

Based on the distribution of GHRL and MLN genes in the species studied, comparative genomics analysis between human and other vertebrates, and endoproteinase cleavage sites distribution in GHRL/MLN genes, we surmise that MLN was generated by a gene duplication on the early amniote lineage as illustrated in figure 2 (He et al. 2011). Other potential evolutionary scenarios (e.g., duplication prior to the fish-tetrapod divergence) require a larger number of gene deletion events along with parallel gain or loss of

are members of the β-group of rhodopsin-like receptor family (Holst et al. 2004).

**4.1.2 Evolution of the motilin/ghrelin hormone gene family** 

bootstrap support for this conclusion is low (Fig. 2).

endopeptidease cleavage sites, and thus are less parsimonious.

Kaiya et al. 2001).


The Power of an Evolutionary Perspective in Studies of Endocrinology 11

Species Ghrelin sequence Obestatin homolog sequence

*niloticus* GSSFLSPSQKPQNKVKSSRIG

*mykiss* GSSFLSPSQKPQGKGKPPRVG

*schlegelii* GSSFLSPSQKPQNRGKSSRVG *Anguilla japonica* GSSFLSPSQRPQGKDKKPPRVG

Species Motilin sequence

Table 1. Bioactive peptide sequences from diverse vertebrata ghrelin gene.

*Homo sapiens* FVPIFTYGELQRMQEKERNKGQ

*Pan troglodytes* FVPIFTYGELQRMQEKERNKGQ

*Macaca mulatta* FVPIFTYGELQRMQEKERSKGQ

*Cavia porcellus* FVPIFTYSELRRTQEREQNKRL

*Oryctolagus cuniculus* FVPIFTYSELQRMQERERNRGH

*Felis catus* FVPIFTHSELQRIREKERNKGQ

*Canis familiaris* FVPIFTHSELQKIREKERNKGQ

*Ovis aries* FVPIFTYGEVQRMQEKERYKGQ

*Bos taurus* FVPIFTYGEVRRMQEKERYKGQ

*Sus scrofa* FVPSFTYGELQRMQEKERNKGQ

*Equus caballus* FVPIFTYSELQRMQEKERNRGQ

*Myotis lucifugus* FVPIFTHSELQRMQEKERNKEQ

*Dasypus novemcinctus* FVPIFTYSELQRMQEKEWNKGQ

*Loxodonta africana* FVPIFTYSEIRRMQERERNNGQ

*Monodelphis domestica* FVPIFTYSDVQRMQEKERNKGQ

*Ornithorhynchus anatinus* FIPIFTHSDVQRMQERERNKGQ

*Gallus gallus* FVPFFTQSDIQKMQEKERNKGQ

Table 2. Bioactive peptide sequences from diverse vertebrata motlin gene.

*Oreochromis* 

*Oncorhynchus* 

*Acanthopagrus* 


FNAPFNIGIKLSGAQSLQHGQ

FNAPFDVGIKLSGAQYQEHG

FNAPCDVGIKLSGAQSDQHG

FNAPFDVGIKLSGAQSHWHG

FSAPFDVGLRLSGAQYEQHG

FNAPFDVGIKLSGAQYQQHG

FNVPFDIGIKVSVAQYGEHGR

FNAPFDIGIKVAEAQYQQYG

FNVPFEIGVKITEREYQEYGQ

FNVPFEIGVKITEREYQEYGQ

FHVPFEIGVKITEEEYQEYGQ

FNVPFEIGVKITEEEYQEYGQ

FNVPFEIGVKITEEQYQEYGQ

LNVPFEIGVKITEDQYQEYGQ

TLG

QALG

QPLG

QALG

EALR

RSLG

ALD

HALE

ALE

ALE

TLE

TLE

MLE

VLE

Species Ghrelin sequence Obestatin homolog sequence RLKP QTLG

GSSFLSPEHQKVQRKESKKPPA

GSSFLSPEHQKGQQRKEPKKPP

GSSFLSPEHQKTQLRKEFKKPAT

GSSFLSPEHPKTQRKETKKPSVK

GSSFLSPAYKNIQQQKDTRKPT

GSSFLSPEFKKIQQQNDPTKTTA

GSSFLSPDYKKIQQRKDPRKPTT

GSSFLSPEYQNTQQRKDPKKHT

*Bubalus bubalis* GSSFLSPEHQKLQRKEPKKPSGR LKP

*Sus scrofa* GSSFLSPEHQKVQQRKESKKPA AKLKP

*Kogia breviceps* GSSFLSPEHQKLQRKEAKKPSG RLKP *Myotis lucifugus* GSSFLSPEHQKAQQRKESKKPP AKLQP

GKVQP

KLQP

LQP

*Echinops telfairi* GSSFLSPGHPKVQPQRKESKTPA GKLQA

*Loxodonta africana* GSSFLSPKNQKLQQRKESKKPP AKLQP

*Gallus gallus* GSSFLSPTYKNIQQQKDTRKPTA RLH

ARLHP

*Anser sp.* GSSFLSPEFKKIQQQNDPAKAT AKIH

*Rana catesbeiana* GLTFLSPADMQKIAERQSQNKL RHGNMN *Rana esculenta* GLTFLSPADMRKIAERQSQNKL RHGNMN *Danio rerio* GTSFLSPTQKPQGRRPPRVG *Carassius auratus* GTSFLSPAQKPQGRRPPRMG *Ictalurus punctatus* GSSFLSPTQKPQNRGDRKPPRV

KIH

KLH

KLN

G

*mossambicus* GSSFLSPSQKPQNKVKSSRIG

KLQP

*Ailuropoda melanoleuca* 

*Erinaceus europaeus* 

*Dasypus novemcinctus* 

*Monodelphis domestica* 

*Meleagris gallopavo* 

*Dromaius novaehollandiae* 

*elegans* 

*Oreochromis* 

*platyrhynchos* 

*Trachemys scripta* 

*Anas* 


Table 1. Bioactive peptide sequences from diverse vertebrata ghrelin gene.


Table 2. Bioactive peptide sequences from diverse vertebrata motlin gene.

The Power of an Evolutionary Perspective in Studies of Endocrinology 13

Fig. 2. Schematic representation of the motilin/ghrelin gene family phylogenetic

MLN, prepromotilin.

relationships. Bootstrap percentages are shown on interior branches. GHRL, preproghrelin.

Fig. 1. Schematic representation of ghrelin and motilin preproproteins. The preproproteins of ghrelin and motilin are represented by boxes divided into protein domains, proportional to their length. The open and filled triangles indicate the locations of cleavage sites used by signal peptidase and proprotein convertase, respectively. The sequences of the putative endoproteinase cleavage sites of various vertebrate classes are shown bellow. Alternative processing sites in birds and bony fish are indicated, "↑" denotes intron position with intron phase shown beside the arrow. SP, signal peptide. "/", lack of putative endoproteinase recognition sites.

Fig. 1. Schematic representation of ghrelin and motilin preproproteins. The preproproteins of ghrelin and motilin are represented by boxes divided into protein domains, proportional to their length. The open and filled triangles indicate the locations of cleavage sites used by signal peptidase and proprotein convertase, respectively. The sequences of the putative endoproteinase cleavage sites of various vertebrate classes are shown bellow. Alternative processing sites in birds and bony fish are indicated, "↑" denotes intron position with intron phase shown beside the arrow. SP, signal peptide. "/", lack of putative endoproteinase

recognition sites.

Fig. 2. Schematic representation of the motilin/ghrelin gene family phylogenetic relationships. Bootstrap percentages are shown on interior branches. GHRL, preproghrelin. MLN, prepromotilin.

The Power of an Evolutionary Perspective in Studies of Endocrinology 15

for gene co-option, involving gene duplication and divergence, should be recognized that creates potential elements which selection can act upon within a biological network to evolve new functions (He et al. 2011; True and Carroll 2002). The growth of gene families allows for more flexible gene expression and/or the evolution of new biochemical specificities (Rubenstein 1990; Sharman and Holland 1996), thus facilitating the evolution of

The evolution of motilin and its specific receptor in rodents provides an illustration of the consequences of gene loss. Motilin is a 22-amino acid peptide synthesized by endocrine cells of the duodeno-jejunal mucosa and has a profound stimulatory effect on gastrointestinal contractility (Poitras and Peeters 2008), indicating that motilin and its specific receptor serve as potent active prokinetic drug target candidates. However, the clinical development of potential therapies is limited as both the mouse and rat, the most frequently used laboratory animals, are natural knockouts for the motilin and its specific receptor, that is these animal lack these genes and functional targets (He et al. 2010). These observations raise a number of intriguing questions – how can these animals survive without motilin? How were the genes lost? Did any other endocrine system compensate? What does this mean for our understanding of the human hormones? While we can't answer all of these questions, our studies revealed that the motilin receptor was pseudogenized specifically on the rodent lineage, while the motilin gene exhibited diverse evolutionary consequences in different rodent species (He et al. 2010). Once an interacting partner is lost, retained partners may be lost, as demonstrated by the independent loss of MLN in mice, rats and in the guinea pig, or serve as raw material in evolution, as suggested by the retention of MLN in the kangaroo rat. Genomic sequence information suggest, that in the the monophyletic Dipodomyinae subfamily, the MLN gene is intact and is under sustained evolutionary constraint, suggesting it has been recruited into a novel function, a function distinct from traditional motilin signaling (our unpublished observations). Intriguingly, studies have suggested that, after the break down of the MLN signaling pathway, the ghrelin signaling pathway was recruited to compensate for this loss in the rat (Dass et al. 2003; Depoortere et al. 2003). Given the ubiquity and its stochastic nature, the simultaneous loss of a hormone and its specific receptor is unlikely. As a dramatic genetic change, a gene loss leads to an immediate loss of specific interactions. The functional redundancy among gene family members could allow a compromise for the deleterious gene loss. Existing genes can be modified, or recruited, into new interactions that yield new functions through mutation and optimization (Jacob 1977; Khersonsky, Roodveldt and Tawfik 2006; Tokuriki and Tawfik 2009). Motilin is not a unique case. As similar events have occurred to leptin, an important adipose derived hormone (Brennan and Mantzoros 2006; Zhang et al. 1994), which does not exist in the chicken, and likely other birds, while a functional leptin receptor has conserved in these species (Horev et al. 2000; Ohkubo, Tanaka and Nakashima 2000; Pitel et al. 2010). It is possible that the lineage specific losses of motilin and leptin during evolution contributed to

greater biological complexity (Duboule and Wilkins1998).

**4.2 Loss of the motilin and its specific receptor genes in rodents** 

the evolution of novel metabolic regulatory mechanisms in these species.

The proglucagon gene illustrates some of these issues. The vertebrate proglucagon gene encodes three glucagonlike sequences (glucagon, glucagon-like peptide-1 [GLP-1], and glucagon-like peptide-2 [GLP-2]) that play distinct roles in mammalian metabolic regulation

**4.3 Evolutionary shifts in existing genes** 

## **4.1.3 Evolution of ghrelin and motilin receptors**

Only a small number of Ghrelin and motilin receptor genes are known and most of these are from mammals, with ortholog from only two species of fish having previously being characterized (Palyha et al. 2000; Chan and Cheng 2004). Our bioinformatic searches of diverse vertebrate genomes resulted in the identification of a great number of potential receptors. The orthology of the different receptors was established using analysis of synteny of the genes. In combination with phylogenetic reconstruction, the monophyly of each receptor type was established, and no more than one copy of each type of receptor was identified in any of the studied species. GHSR and MLNR are more closely related to each other than they are to any other characterized receptor, and the gene duplication that generated them happened more than 450 million years ago, before the divergence of rayfinned fish and tetrapods (He et al. 2011). Through comparative and evolutionary analyses, we found a new type of receptor in fish, which does not have an ortholog in any non-fish vertebrate. The function of this new receptor is unknown. The sequence of this new receptor has some peculiarities, such as possessing long extracellular loops 2 and 3, which are about 100 residues longer than the analogous loops in GHSR and MLNR (He et al. 2011). Residues at both ends of these loops have been shown to be functionally important for hormone binding and action in homologous receptors; however, the function of these residues in the loops of these novel receptors is not clear (Matsuura, Dong and Miller 2002).

Studies have shown that the GHSR orthologs in fish can be activated by growth hormone secretagogues (GHSs), while MLNR orthologs in fish failed to be activated by GHSs or mammalian motilin (Palyha et al. 2000). The identification of the ancient binding state of MLNR prior to the emergence of motilin has proven challenging, however it is reasonable to speculate that GHSR and MLNR experienced functional diversification shortly after their duplication, and the ancient MLNR did not have either ghrelin or motilin binding properties. Evolutionary studies suggest MLNR experienced an episode of rapid evolution on the branch leading to amniotes, which was driven by positive selection, and accumulated amino acid changes in ligand binding cleft. This time period of rapid evolution coincides with the date of the GHRL/ MLN gene duplication event, thus it is reasonable to speculate, that the burst of rapid evolution in MLNR was a consequence of coevolution with its new ligand, and that motilin binding specificity of MLNR only evolved as a result of ligandreceptor coevolution after the motilin gene diverged from the ghrelin gene on the amniote lineage. In contrast, GHSR has evolved under a constant selective constraint throughout vertebrates, with the ghrelin/GHSR system being maintained and functionally conserved from fish to mammals (He et al. 2011).

## **4.1.4 Gene duplication and gene co-option**

The motilin/ghrelin hormone and their receptors were produced by independent duplication events that occurred at different points in time. The discordance of the evolutionary histories for the hormones and receptors indicate that the intimate interacting partners of an endocrine system can be produced by individual duplications, the composition and functions of each part of the endocrine network do not remain static, and that parts of a system can be co-opted for novelties, and that these processes often involve gene duplication and subsequent divergence. Structural and evolutionary relatedness allows promiscuous interaction properties, which can serve as the starting point for accommodation. Divergence of function in different species is accomplished by hormone/receptor coevolution to improve binding affinity and/or specificity. A major role

Only a small number of Ghrelin and motilin receptor genes are known and most of these are from mammals, with ortholog from only two species of fish having previously being characterized (Palyha et al. 2000; Chan and Cheng 2004). Our bioinformatic searches of diverse vertebrate genomes resulted in the identification of a great number of potential receptors. The orthology of the different receptors was established using analysis of synteny of the genes. In combination with phylogenetic reconstruction, the monophyly of each receptor type was established, and no more than one copy of each type of receptor was identified in any of the studied species. GHSR and MLNR are more closely related to each other than they are to any other characterized receptor, and the gene duplication that generated them happened more than 450 million years ago, before the divergence of rayfinned fish and tetrapods (He et al. 2011). Through comparative and evolutionary analyses, we found a new type of receptor in fish, which does not have an ortholog in any non-fish vertebrate. The function of this new receptor is unknown. The sequence of this new receptor has some peculiarities, such as possessing long extracellular loops 2 and 3, which are about 100 residues longer than the analogous loops in GHSR and MLNR (He et al. 2011). Residues at both ends of these loops have been shown to be functionally important for hormone binding and action in homologous receptors; however, the function of these residues in the

loops of these novel receptors is not clear (Matsuura, Dong and Miller 2002).

Studies have shown that the GHSR orthologs in fish can be activated by growth hormone secretagogues (GHSs), while MLNR orthologs in fish failed to be activated by GHSs or mammalian motilin (Palyha et al. 2000). The identification of the ancient binding state of MLNR prior to the emergence of motilin has proven challenging, however it is reasonable to speculate that GHSR and MLNR experienced functional diversification shortly after their duplication, and the ancient MLNR did not have either ghrelin or motilin binding properties. Evolutionary studies suggest MLNR experienced an episode of rapid evolution on the branch leading to amniotes, which was driven by positive selection, and accumulated amino acid changes in ligand binding cleft. This time period of rapid evolution coincides with the date of the GHRL/ MLN gene duplication event, thus it is reasonable to speculate, that the burst of rapid evolution in MLNR was a consequence of coevolution with its new ligand, and that motilin binding specificity of MLNR only evolved as a result of ligandreceptor coevolution after the motilin gene diverged from the ghrelin gene on the amniote lineage. In contrast, GHSR has evolved under a constant selective constraint throughout vertebrates, with the ghrelin/GHSR system being maintained and functionally conserved

The motilin/ghrelin hormone and their receptors were produced by independent duplication events that occurred at different points in time. The discordance of the evolutionary histories for the hormones and receptors indicate that the intimate interacting partners of an endocrine system can be produced by individual duplications, the composition and functions of each part of the endocrine network do not remain static, and that parts of a system can be co-opted for novelties, and that these processes often involve gene duplication and subsequent divergence. Structural and evolutionary relatedness allows promiscuous interaction properties, which can serve as the starting point for accommodation. Divergence of function in different species is accomplished by hormone/receptor coevolution to improve binding affinity and/or specificity. A major role

**4.1.3 Evolution of ghrelin and motilin receptors** 

from fish to mammals (He et al. 2011).

**4.1.4 Gene duplication and gene co-option** 

for gene co-option, involving gene duplication and divergence, should be recognized that creates potential elements which selection can act upon within a biological network to evolve new functions (He et al. 2011; True and Carroll 2002). The growth of gene families allows for more flexible gene expression and/or the evolution of new biochemical specificities (Rubenstein 1990; Sharman and Holland 1996), thus facilitating the evolution of greater biological complexity (Duboule and Wilkins1998).

## **4.2 Loss of the motilin and its specific receptor genes in rodents**

The evolution of motilin and its specific receptor in rodents provides an illustration of the consequences of gene loss. Motilin is a 22-amino acid peptide synthesized by endocrine cells of the duodeno-jejunal mucosa and has a profound stimulatory effect on gastrointestinal contractility (Poitras and Peeters 2008), indicating that motilin and its specific receptor serve as potent active prokinetic drug target candidates. However, the clinical development of potential therapies is limited as both the mouse and rat, the most frequently used laboratory animals, are natural knockouts for the motilin and its specific receptor, that is these animal lack these genes and functional targets (He et al. 2010). These observations raise a number of intriguing questions – how can these animals survive without motilin? How were the genes lost? Did any other endocrine system compensate? What does this mean for our understanding of the human hormones? While we can't answer all of these questions, our studies revealed that the motilin receptor was pseudogenized specifically on the rodent lineage, while the motilin gene exhibited diverse evolutionary consequences in different rodent species (He et al. 2010). Once an interacting partner is lost, retained partners may be lost, as demonstrated by the independent loss of MLN in mice, rats and in the guinea pig, or serve as raw material in evolution, as suggested by the retention of MLN in the kangaroo rat. Genomic sequence information suggest, that in the the monophyletic Dipodomyinae subfamily, the MLN gene is intact and is under sustained evolutionary constraint, suggesting it has been recruited into a novel function, a function distinct from traditional motilin signaling (our unpublished observations). Intriguingly, studies have suggested that, after the break down of the MLN signaling pathway, the ghrelin signaling pathway was recruited to compensate for this loss in the rat (Dass et al. 2003; Depoortere et al. 2003). Given the ubiquity and its stochastic nature, the simultaneous loss of a hormone and its specific receptor is unlikely. As a dramatic genetic change, a gene loss leads to an immediate loss of specific interactions. The functional redundancy among gene family members could allow a compromise for the deleterious gene loss. Existing genes can be modified, or recruited, into new interactions that yield new functions through mutation and optimization (Jacob 1977; Khersonsky, Roodveldt and Tawfik 2006; Tokuriki and Tawfik 2009). Motilin is not a unique case. As similar events have occurred to leptin, an important adipose derived hormone (Brennan and Mantzoros 2006; Zhang et al. 1994), which does not exist in the chicken, and likely other birds, while a functional leptin receptor has conserved in these species (Horev et al. 2000; Ohkubo, Tanaka and Nakashima 2000; Pitel et al. 2010). It is possible that the lineage specific losses of motilin and leptin during evolution contributed to the evolution of novel metabolic regulatory mechanisms in these species.

## **4.3 Evolutionary shifts in existing genes**

The proglucagon gene illustrates some of these issues. The vertebrate proglucagon gene encodes three glucagonlike sequences (glucagon, glucagon-like peptide-1 [GLP-1], and glucagon-like peptide-2 [GLP-2]) that play distinct roles in mammalian metabolic regulation

The Power of an Evolutionary Perspective in Studies of Endocrinology 17

modes of action; one hormone may play roles in different target organs, and conversely, target organs are affected by more than one hormone. Although quite irregular, there are still some formulas that can be followed. Structural and evolutionary relatedness generate promiscuous interaction properties, and provide important clues to interaction capacity exploration. Tracing the origin and studying the molecular evolution of endocrine systems should help us comprehend the main events that have prompted the diversification of these systems. In the light of evolution, through a comparative strategy, integrating information from diverse species helps to enhance our understanding of the assemblage of complex endocrine systems, identifying novel components of endocrine systems, and potential crosstalk between them through evolutionarily related interacting proteins. In addition, knowledge of how elements that underlie cellular functions are evolutionarily and developmentally interact, not only helps in choosing appropriate species to examine function, but also provide genetic makers to probe the evolution of specific traits and characteristics, disclosing the genetic basis that underlie the morphological and behavior changes, and thus helping enhance our understanding of how changing environments led to

This work was supported by grants from the National Basic Research Program of China (973 Program, 2007CB411600), the National Natural Science Foundation of China (30621092,

Avise JC, Ayala FJ. From the Academy: Colloquium Perspective: In the light of evolution I:

Berg J, Lässig M, Wagner A. Structure and evolution of protein interaction networks: a

Brennan AM, Mantzoros CS. Drug Insight: the role of leptin in human physiology and

Carroll SM, Bridgham JT, Thornton JW Evolution of hormone signaling in elasmobranchs by exploitation of promiscuous receptors. Mol Biol Evol. 2008 25:2643–2652. Cegla J, Tan TM, Bloom SR. Gut-brain cross-talk in appetite regulation. Curr Opin Clin Nutr

Chan CB, Cheng CH. Identification and functional characterization of two alternatively

Conant GC. Rapid reorganization of the transcriptional regulatory network after genome

duplication in yeast. Proc Biol Sci. 2010 277:869-876.

Adaptation and complex design. Proc Natl Acad Sci USA. 2007 104 Suppl 1:8563-

statistical model for link dynamics and gene duplications. BMC Evol Biol. 2004

pathophysiology--emerging clinical applications. Nat Clin Pract Endocrinol Metab.

spliced growth hormone secretagogue receptor transcripts from the pituitary of black seabream Acanthopagrus schlegeli. Mol Cell Endocrinol. 2004 214:81-

30623007), and Bureau of Science and Technology of Yunnan Province (O803481101).

biochemical adjustments.

**6. Acknowledgments** 

**7. References** 

8566.

4:51.

95.

2006 2:318-327.

Metab Care. 2010 13:588-593.

(Drucker 2001; Drucker 2002; Jiang and Zhang 2003; Kieffer and Habener 1999). Glucagon, produced by the A cells of the pancreatic islets, counteracts insulin's effect on blood glucose level depression (Jiang and Zhang 2003). GLP-1 functions as an incretin hormone in mammals, potentiating insulin release, and thus regulating glucose metabolism (Drucker 2001, 2002). In contrast, glucagon and GLP-1 have similar physiological functions in fish, and resemble that of mammalian glucagon (Duguay and Mommsen 1994; Plisetskaya and Mommsen 1996). The receptors for glucagon, GLP-1, and GLP-2 have emerged before the divergence of fish and mammals; however, the GLP-1 class of receptors has specifically been lost in fish, and accordingly the incretin action of GLP-1. A fish specific duplication produced a second glucagon receptor-like gene on the ancestral fish lineage. The new glucagon receptor-like gene shifted its binding specificity from glucagon to GLP-1 ensues, meanwhile maintained the ancestral downstream signaling. Thus through receptor loss and gain, existing hormone was recruited into new roles, and undoubtedly enabled evolutionary divergence (Irwin and Wong 2005).

While ghrelin and its specific receptor (GHSR) genes has been maintained and functionally conserved from fish to mammals, there are some significant differences in the function of the ghrelin/GHSR system in birds compared to other vertebrates (Richards 2010). Some of the actions of ghrelin are conserved in birds (e.g., GH release), while others, such as the effect of ghrelin on food intake, are opposite to those found in mammals and other vertebrate species (Hiroyuki et al. 2007; Kaiya et al. 2009; Kaiya et al. 2008). Besides ghrelin, the ghrelin gene has the potential to encode another peptide hormone——obstatin (Zhang et al. 2005). We observed episodic evolution for both the ghrelin and motilin genes during primitive placental mammal evolution, the period when a functional obestatin hormone might have originated (He, Irwin and Zhang 2010). It is possible that some of the lineagespecific physiological adaptations are due to the episodic evolution of the motilin and ghrelin genes.

Gene duplication, pseudonization, and the gain and loss of interactions through mutations in existing genes are major evolutionary processes shaping the specific interaction among biomolecules (Berg, Lässig and Wagner 2004; Wagner 2001; Wagner 2003; He et al. 2010). Thus, once a mutation arises, a stepwise process of elaboration and optimization ensues, which gradually integrates and orders mutations into a coherent pattern. Given the evolutionary dynamics of the genome and the plasticity of biomolecular networks, an evolutionary perspective is necessary to understand many aspects of the molecular basis of endocrinology.

## **5. Conclusion**

Biological evolution is the process of generating biodiversity. Different phenotype corresponds to a given genomic control. New genes, new interactions, and new biochemical processes are essential for the molecular basis of the evolution of biodiversity and complexity. Genetic networks of endocrine systems are developmentally and evolutionarily fictile, elemental compositions within them are prone to be altered through gene gain and loss, and its physiological properties frequently change with mutations in gene coding sequences and/or regulatory systems, and turnover of interacting biomolecules.

The endocrine system consists of several glands in different parts of the body, which secrete hormones directly into the blood. Hormones usually have many different functions and modes of action; one hormone may play roles in different target organs, and conversely, target organs are affected by more than one hormone. Although quite irregular, there are still some formulas that can be followed. Structural and evolutionary relatedness generate promiscuous interaction properties, and provide important clues to interaction capacity exploration. Tracing the origin and studying the molecular evolution of endocrine systems should help us comprehend the main events that have prompted the diversification of these systems. In the light of evolution, through a comparative strategy, integrating information from diverse species helps to enhance our understanding of the assemblage of complex endocrine systems, identifying novel components of endocrine systems, and potential crosstalk between them through evolutionarily related interacting proteins. In addition, knowledge of how elements that underlie cellular functions are evolutionarily and developmentally interact, not only helps in choosing appropriate species to examine function, but also provide genetic makers to probe the evolution of specific traits and characteristics, disclosing the genetic basis that underlie the morphological and behavior changes, and thus helping enhance our understanding of how changing environments led to biochemical adjustments.

## **6. Acknowledgments**

This work was supported by grants from the National Basic Research Program of China (973 Program, 2007CB411600), the National Natural Science Foundation of China (30621092, 30623007), and Bureau of Science and Technology of Yunnan Province (O803481101).

## **7. References**

16 Contemporary Aspects of Endocrinology

(Drucker 2001; Drucker 2002; Jiang and Zhang 2003; Kieffer and Habener 1999). Glucagon, produced by the A cells of the pancreatic islets, counteracts insulin's effect on blood glucose level depression (Jiang and Zhang 2003). GLP-1 functions as an incretin hormone in mammals, potentiating insulin release, and thus regulating glucose metabolism (Drucker 2001, 2002). In contrast, glucagon and GLP-1 have similar physiological functions in fish, and resemble that of mammalian glucagon (Duguay and Mommsen 1994; Plisetskaya and Mommsen 1996). The receptors for glucagon, GLP-1, and GLP-2 have emerged before the divergence of fish and mammals; however, the GLP-1 class of receptors has specifically been lost in fish, and accordingly the incretin action of GLP-1. A fish specific duplication produced a second glucagon receptor-like gene on the ancestral fish lineage. The new glucagon receptor-like gene shifted its binding specificity from glucagon to GLP-1 ensues, meanwhile maintained the ancestral downstream signaling. Thus through receptor loss and gain, existing hormone was recruited into new roles, and undoubtedly enabled evolutionary

While ghrelin and its specific receptor (GHSR) genes has been maintained and functionally conserved from fish to mammals, there are some significant differences in the function of the ghrelin/GHSR system in birds compared to other vertebrates (Richards 2010). Some of the actions of ghrelin are conserved in birds (e.g., GH release), while others, such as the effect of ghrelin on food intake, are opposite to those found in mammals and other vertebrate species (Hiroyuki et al. 2007; Kaiya et al. 2009; Kaiya et al. 2008). Besides ghrelin, the ghrelin gene has the potential to encode another peptide hormone——obstatin (Zhang et al. 2005). We observed episodic evolution for both the ghrelin and motilin genes during primitive placental mammal evolution, the period when a functional obestatin hormone might have originated (He, Irwin and Zhang 2010). It is possible that some of the lineagespecific physiological adaptations are due to the episodic evolution of the motilin and

Gene duplication, pseudonization, and the gain and loss of interactions through mutations in existing genes are major evolutionary processes shaping the specific interaction among biomolecules (Berg, Lässig and Wagner 2004; Wagner 2001; Wagner 2003; He et al. 2010). Thus, once a mutation arises, a stepwise process of elaboration and optimization ensues, which gradually integrates and orders mutations into a coherent pattern. Given the evolutionary dynamics of the genome and the plasticity of biomolecular networks, an evolutionary perspective is necessary to understand many aspects of the molecular basis of

Biological evolution is the process of generating biodiversity. Different phenotype corresponds to a given genomic control. New genes, new interactions, and new biochemical processes are essential for the molecular basis of the evolution of biodiversity and complexity. Genetic networks of endocrine systems are developmentally and evolutionarily fictile, elemental compositions within them are prone to be altered through gene gain and loss, and its physiological properties frequently change with mutations in gene coding

The endocrine system consists of several glands in different parts of the body, which secrete hormones directly into the blood. Hormones usually have many different functions and

sequences and/or regulatory systems, and turnover of interacting biomolecules.

divergence (Irwin and Wong 2005).

ghrelin genes.

endocrinology.

**5. Conclusion** 


The Power of an Evolutionary Perspective in Studies of Endocrinology 19

Holland LZ, Albalat R, Azumi K, et al. The amphioxus genome illuminates vertebrate origins and cephalochordate biology. Genome Res. 2008 18:1100-1111. Holst B, Holliday ND, Bach A, Elling CE, Cox HM, Schwartz TW. Common structural basis

Horev G, Einat P, Aharoni T, Eshdat Y, Friedman-Einat M. Molecular cloning and

Hou Y, Lin S. Distinct gene number-genome size relationships for eukaryotes and non-

Howard AD, Feighner SD, Cully DF, et al. A Receptor in Pituitary and Hypothalamus That

Huminiecki L, Heldin C-H: 2R and modeling of vertebrate signal transduction engine. BMC

Inui A. Ghrelin: an orexigenic and somatotrophic signal from stomach. Nat. Rev. Neurosci.

Irwin DM, Wong K. Evolution of new hormone function: loss and gain of a receptor. J

Irwin DM. Evolution of genes for incretin hormones and their receptors. Vitam Horm. 2010

Jaillon O, Aury J, Brunet F, et al., Genome duplication in the teleost fish Tetraodon

Jiang G, Zhang BB. Glucagon and regulation of glucose metabolism. Am J Physiol. 2003

Kaiya H, Furuse M, Miyazato M, Kangawa K. Current knowledge of the roles of ghrelin in

Kaiya H, Kojima M, Hosoda H, et al. Bullfrog ghrelin is modified by n-octanoic acid at its

Kaiya H, Miyazato M, Kangawa K, Peter RE, Unniappan S. Ghrelin: a multifunctional

Kassahn KS, Dang VT, Wilkins SJ, Perkins AC, Ragan MA. Evolution of gene function and

Kettler GC, Martiny AC, Huang K, et al. Patterns and implications of gene gain and loss in

Khersonsky O, Roodveldt C, Tawfik DS. Enzyme promiscuity: evolutionary and mechanistic

nigroviridis reveals the early vertebrate proto-karyotype. Nature 2004 431:946–

regulating food intake and energy balance in birds. Gen Comp Endocrinol. 2009

hormone in non-mammalian vertebrates. Comp Biochem Physiol A Mol Integr

regulatory control after whole-genome duplication: comparative analyses in

Functions in Growth Hormone Release. Science 1996 273:974-977.

Irwin DM. Molecular evolution of proglucagon. Regul Pept. 2001 98:1-12

third threonine residue. J Biol Chem. 2001 276:40441-40448.

the evolution of Prochlorococcus. PLoS Genet. 2007 312:e231.

Kieffer TJ, Habener JF. The glucagon-like peptides. Endocr Rev. 1999 20:876–913.

Jacob F. Evolution and tinkering. Science 1977, 196: 1161–1166.

vertebrates. Genome Res. 2009 19:1404-1418.

aspects. Curr Opin Chem Biol. 2006 10:498-508.

53817.

4:e6978.

2000 162:95-106.

Biol 2010 8:146.

2001 2:551-560.

284:E671–E678.

Physiol. 2008 149:109-128.

163:33-38.

84:1-20.

957.

Hered. 2005 96:205-11.

for constitutive activity of the ghrelin receptor family. J Biol Chem. 2004 279:53806-

properties of the chicken leptin-receptor (CLEPR) gene. Mol Cell Endocrinol.

eukaryotes: gene content estimation for dinoflagellate genomes. PLoS One. 2009


Danks JA, D'Souza DG, Gunn HJ, Milley KM, Richardson SJ. Evolution of the parathyroid

Dass NB, Munonyara M, Bassil AK, Hervieu GJ, Osbourne S, Corcoran S, Morgan M, Sanger

Dehal P, Boore JL. Two rounds of whole genome duplication in the ancestral vertebrate.

Depoortere I, De Winter B, Thijs T, De Man J, Pelckman P, Peeters T 2003 Comparison of the

Drucker DJ. Biological actions and therapeutic potential of the glucagon-like peptides.

Feighner SD, Tan CP, McKee KK, et al. Receptor for motilin identified in the human

Gagneux P, Varki A. Genetic differences between humans and great apes. Mol Phylogenet

Garcia-Fernández J, Holland PW. Archetypal organization of the amphioxus Hox gene

Gout JF, Duret L, Kahn D. Differential Retention of Metabolic Genes Following Whole-

Gregory TR. Synergy between sequence and size in large-scale genomics. Nature Rev Genet.

Hahn MW, Demuth JP, Han SG. Accelerated rate of gene gain and loss in primates. Genetics

He J, Irwin DM, Chen R, Zhang YP. Stepwise loss of motilin and its specific receptor genes

He J, Irwin DM, Zhang YP. Motilin and ghrelin gene experienced episodic evolution

He J, Irwin DM, Zhang YP. Gene duplication plays a major role in gene co-option: Studies

Hiroyuki Kaiya, Veerle M Darras, Kenji Kangawa. Ghrelin in Birds: Its Structure,

Hodson DJ, Molino F, Fontanaud P, Bonnefont X, Mollard P. Investigating and

Hoffmann FG, Opazo JC. Evolution of the relaxin/insulin-like gene family in placental mammals: implications for its early evolution. J Mol Evol. 2011 72:72-79.

Distribution and Function. Journal of Poultry Science 2007 44:1–18.

during primitive placental mammal evolution. Sci China Life Sci. 2010 53:677-

into the evolution of the motilin/ghrelin family and their receptors. Chinese

modelling pituitary endocrine network function. J Neuroendocrinol. 2010

Duboule D and Wilkins A S. The evolution of 'bricolage'. Trends Genet. 1998 14:54–59. Duguay SJ, Mommsen TP. Molecular aspects of pancreatic peptides. In: Fish

and the effects of ghrelin. Neuroscience 120:443–453.

Drucker DJ. The glucagon-like peptides. Endocrinology 2001 142:521–527

gastrointestinal system. Science 1999 284:2184-2188.

Genome Duplication. Mol Biol Evol. 2009 265:1067-1072.

170:79-91.

PLoS Biol. 2005 3:e314.

Gastroenterology 124:580.

1994 225–271.

Evol. 2001 18:2–13.

2005 6:699–708.

22:1217-1225.

682.

2007 177:1941-1949.

Gastroenterology 2002 122:531–544.

cluster. Nature 1994 370:563–566.

in rodents. J Mol Endocrinol. 2010 44:37-44.

Science Bulletin. 2011 56: 2690-2697.

hormone family and skeletal formation pathways. Gen Comp Endocrinol. 2011

GJ. Growth hormone secretagogue receptors in rat and human gastrointestinal tract

prokinetic effects of ghrelin, GHRP-6 and motilin in rats in vivo and in vitro.

physiology, vol. 13 (Hoar WS and Randall DJ, eds). Academic Press, San Diego.


The Power of an Evolutionary Perspective in Studies of Endocrinology 21

Pitel F, Faraut T, Bruneau G, Monget P. Is there a leptin gene in the chicken genome?

Plisetskaya EM, Mommsen TP. Glucagon and glucagon-like peptides in fishes. Int Rev

Poitras P, Peeters TL. Motilin. Current Opinion in Endocrinology, Diabetes and Obesity 2008

Poitras P: Motilin. In: Gut Peptides: Biochemistry and Physiology. Edited by Walsh JH,

Powell AJ, Conant GC, Brown DE, Carbone I, Dean RA. Altered patterns of gene

Samson WK, Lumpkin MD, Nilaver G, McCann SM. Motilin: a novel growth hormone

Semyonov J, Park JI, Chang CL, Hsu SY. GPCR genes are preferentially retained after whole

Sharman A C and Holland P W H. Conservation duplication and divergence of

Sun Y, Ahmed S, Smith RG. Deletion of ghrelin impairs neither growth nor appetite. Mol

Sundström G, Dreborg S, Larhammar D. Concomitant duplications of opioid peptide

Ting CT, Tsaur SC, Sun S, Browne WE, Chen YC, Patel NH, Wu CI. Gene duplication and

van Hoek MJ, Hogeweg P. Metabolic adaptation after whole genome duplication. Mol Biol

Wagner A. How the global structure of protein interaction networks evolves. Proc Biol Sci.

Wagner A. The yeast protein interaction network evolves rapidly and contains few

Wallis M. Episodic evolution of protein hormones in mammals. J Mol Evol. 2001 53:10-

Wang X, Grus WE, Zhang J. Gene losses during human origins. PLoS Biology. 2006

Wilkins AS. Between "design" and "bricolage": genetic networks, levels of selection, and adaptive evolution. Proc Natl Acad Sci USA. 2007 104 Suppl 1:8590-8596.

redundant duplicate genes. Mol Biol Evol. 2001 18:1283-1292.

Tokuriki N, Tawfik DS. Protein dynamism and evolvability. Science 2009 324:203-207. True JR, Carroll SB. Gene co-option in physiological and morphological evolution. Annu

developmental genes during chordate evolution. Netherlands Journal of Zoology

and receptor genes before the origin of jawed vertebrates. PLoS One. 2010

speciation in Drosophila: evidence from the Odysseus locus. Proc Natl Acad Sci

Richards MP, McMurtry JP. The avian proghrelin system. Int J Pept. 2010. pii: 749401. Rubenstein PA. The functional importance of multiple actin isoforms. Bioessays 1990 12:309-

duplication and differential gene gain and loss in fungal pathogens. BMC

Dockaray GJ. Raven Press. New York, 1993. 261–303.

releasing agent. Brain Res Bull. 1984 12:57-62.

genome duplication. PLoS One. 2008 3:e1903.

2010 167:1-5.

15:54–57.

315.

1996 46:47-67.

5:e10512.

Cell Biol. 2003 23:7973-7981.

USA. 2004 101:12232-12235.

Evol. 2009 26:2441-2453.

2003 270:457-466.

18.

4:e52.

Rev Cell Dev Biol. 2002 18:53-80.

Cytol, 1996 168:187–257.

Genomics. 2008 9:147.

Lessons from phylogenetics, bioinformatics and genomics. Gen Comp Endocrinol.


Kim DK, Cho EB, Moon MJ, Park S, Hwang JI, Kah O, Sower SA, Vaudry H, Seong JY.

Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K. Ghrelin is a

Konstantinidis KT, Tiedje JM. Trends between gene content and genome size in prokaryotic species with larger genomes. Proc Natl Acad Sci USA. 2004 101:3160–3165.

Liu JC, Makova KD, Adkins RM, Gibson S, Li WH. Episodic evolution of growth hormone

Macqueen DJ, Delbridge ML, Manthri S, Johnston IA. A newly classified vertebrate

Manning G, Scheeff E. How the vertebrates were made: selective pruning of a double-

Markov GV, Paris M, Bertrand S, Laudet V. The evolution of the ligand/receptor couple: a

Matsuura B, Dong M and Miller LJ. Differential determinants for peptide and non-peptidyl

Moyle WR, Campbell RK, Myers RV, Bernard MP, Han Y, Wang X. Co-evolution of ligand-

Murray C D, Kamm M A, Bloom S R, et al. Ghrelin for the gastroenterologist: history and

Nakazato M, Murakami N, Date Y, Kojima M, Matsuo H, Kangawa K, Matsukura S. A role for ghrelin in the central regulation of feeding. Nature 2001 409:194-198. Ohkubo T, Tanaka M, Nakashima K. Structure and tissue distribution of chicken leptin

Palyha OC, Feighner SD, Tan CP, et al. Ligand activation domain of human orphan growth

Peeters T L. Ghrelin: a new player in the control of gastrointestinal functions. Gut 2005

Peter EC, Vallo T. Recent advances Advances in endocrinology. Arch Dis Child. 1998 78:278-

hormone (GH) secretagogue receptor (GHS-R) conserved from Pufferfish to

receptor (cOb-R) mRNA. Biochim Biophys Acta. 2000 1491:303-308.

peptide binding and action. J Biol Chem. 2002 277:9834-9839.

Kojima M, Kangawa K. Ghrelin: structure and function. Physiol Rev. 2005 85:495-52.

Lynch M, Conery JS. The origins of genome complexity. Science 2003 302:1401–1404.

Li WH. Molecular Evolution. Sinauer Associates, Sunderland, 1997.

receptor. Mol Biol Evol. 2001 18:945-953.

duplicated genome. BMC Biol. 2010 8:144.

receptor pairs. Nature 1994 368:251-255.

potential. Gastroenterology 2003 125:1492-1502.

Ohno S. Evolution by gene duplication. Springer-Verlag. Berlin, 1970.

humans. Mol Endocrinol. 2000 14:160-169.

2011 170:68-78.

402:656-60.

27:1886-1902.

54:1638-649.

284.

Endocrinol. 2008 293:5-16.

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

growth-hormone-releasing acylated peptide from stomach. Nature 1999

in primates and emergence of the species specificity of human growth hormone

calpain protease, directly ancestral to CAPN1 and 2, episodically evolved a restricted physiological function in placental mammals. Mol Biol Evol. 2010

long road from comparative endocrinology to comparative genomics. Mol Cell

ligand binding to the motilin receptor. Critical role of second extracellular loop for


**Part 2** 

**Pituitary Adenomas** 

