Evolution of peptide hormones and their receptors

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by Graeme Roch

BSc, University of Victoria, 2003

A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of

DOCTOR OF PHILOSOPHY in the Department of Biology

 Graeme Roch, 2011 University of Victoria

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Supervisory Committee

Evolution of Peptide Hormones and Their Receptors by

Graeme Roch

BSc, University of Victoria, 2003

Supervisory Committee

Nancy M. Sherwood (Department of Biology) Co-Supervisor

Ben F. Koop (Department of Biology) Co-Supervisor

Robert L. Chow (Department of Biology) Departmental Member

Juan Ausio (Department of Biochemistry and Microbiology) Outside Member

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Abstract

Supervisory Committee

Nancy M. Sherwood (Department of Biology) Co- Supervisor

Ben F. Koop (Department of Biology) Co-Supervisor

Robert L. Chow (Department of Biology) Departmental Member

Juan Ausio (Department of Biochemistry and Microbiology) Outside Member

Peptide hormones are critical modulators of physiology and development in humans and have been well characterized for their effects on humans and other mammals. The question of the origin of the many families of peptide hormones in mammals is pressing, as it gives us a window into the evolution of important systems in all extant animals and their common ancestors. The focus of this thesis was to examine the origin of a select group of peptide hormone families including the secretin superfamily, reproductive neuropeptides, insulin and the insulin-like peptides, and stanniocalcin. The evolution of the secretin superfamily was found to have originated with the vertebrates, and new information from the genomes of basal vertebrates like the lamprey Petromyon marinus and elephant shark Callorhinchus milii allows us to better piece together the gene duplications that produced the current hormone family in humans and fish. The reproductive hormones, including gonadotropin-releasing hormone (GnRH),

vasopressin/oxytocin, and kisspeptin were examined, with a focus on the evolution of their G protein-coupled receptors. GnRH was found to have originated in the early

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bilaterians, and its receptors clearly belong to a superfamily also containing receptors of the related neuropeptides adipokinetic hormone and corazonin, which have only been found in protostome invertebrates. Vasopressin/oxytocin receptors share a common ancestor with the GnRH receptors, although their peptides are not structurally related, and evolved at a similar time. Kisspeptin evolved later, within the vertebrates, however its receptors are closely related to an orphan receptor in protostome invertebrates, GPR54, with an unknown ligand. Insulin family members from the tunicate Ciona intestinalis and the amphioxus Branchiostoma floridae were identified, isolated and characterized to determine the nature of the insulin superfamily at the origin of the chordates, and it appears this family was well-developed already. Finally, the calcium-regulator

stanniocalcin was identified, isolated and characterized in C. intestinalis and compared with the vertebrate and amphioxus stanniocalcins. A group of stanniocalcins were also discovered in a wide range of both protostomes and unicellular eukaryotes, indicating this ancient group of neurohormones appeared early in eukaryotic evolution.

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List of Tables

Table 2.1. Secretin superfamily sequence identities and locations for zebrafish (Zf), fugu

(Fu), medaka (Md), stickleback (St), elephant shark (Es) and lamprey (Lp). ... 52

Table 4.1. Sequences used for phylogenetic analysis. ... 110

Table 5.1. Stanniocalcin amino acid identity matrix ... 136

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List of Figures

Figure 1.1. Secretin superfamily peptides from a variety of vertebrates. ... 7 Figure 1.2. Two-domain binding model of Family 2 (secretin-like) GPCR activation ... 10 Figure 1.3. Mature GnRH peptides from a variety of vertebrates and invertebrates ... 14 Figure 1.4. Insulin/relaxin and insulin-like growth factor (IGF) processing ... 23 Figure 1.5. Insulin/IGF receptor structure ... 24 Figure 2.1. Secretin superfamily members in humans, zebrafish (Zf), fugu pufferfish

(Fu), elephant shark (Es) and lamprey (Lp), displaying the presence of duplicates in different species ... 49

Figure 2.2. Maximum likelihood tree of secretin superfamily peptides ... 56 Figure 2.3. Maximum likelihood tree of secretin superfamily receptors, either

uncompressed (A) or compressed (B)... 59

Figure 2.4. A) Diagram of the human secretin superfamily peptide genes. B) Proposed

evolutionary course of secretin superfamily peptides ... 62

Figure 3.1. Maximum likelihood tree of GnRH receptors from vertebrates and

invertebrates ... 83

Figure 3.2. Maximum likelihood tree of GnRH, AKH (adipokinetic hormone), ACP

(adipokinetic hormone/corazonin-related peptide) and corazonin (Crz) receptors from vertebrates and invertebrates ... 87

Figure 3.3. Maximum likelihood tree of the GnRHR superfamily and

vasopressin/oxytocin receptor (VPR/OTR) superfamily from vertebrates and

invertebrates ... 91

Figure 3.4. Maximum likelihood tree of kisspeptin, GPR54, galanin and allatostatin

receptors from vertebrates and invertebrates ... 95

Figure 4.1. qPCR of Ciona intestinalis INS2 and INSR ... 109 Figure 4.2. CLUSTALW alignment of amphioxus, tunicate, and human insulin

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Figure 4.3. Comparison of invariant receptor binding sites found in vertebrate insulins

with human IGF-I and protochordate insulin-like molecules ... 116

Figure 4.4. Possible relaxin receptor binding motifs in human and B. floridae B-domains

... 116

Figure 4.5. Maximum-likelihood tree of insulin superfamily peptides from vertebrates

and invertebrates ... 118

Figure 5.1. A) ClustalW alignment of putative stanniocalcin peptides from tunicate

(Ciona intestinalis), amphioxus (Branchiostoma floridae) as well as representative sequences for STC1 and STC2 from human and zebrafish. B) Gene diagrams for human and protochordate stanniocalcins ... 134

Figure 5.2. Phylogenetic tree of stanniocalcin sequences ... 138 Figure 5.3. RT-PCR and qPCR of Ciona intestinalis stanniocalcin ... 140 Figure 5.4. Hormones related to calcium homeostasis in amphioxus (B. floridae),

tunicate (C. intestinalis) and human ... 147

Figure 6.1. Alignment of stanniocalcin homologs from a variety of eukaryotes ... 163 Figure 6.2. Amino acid identity matrix of stanniocalcin homologs ... 166 Figure 6.3. Phylogenetic tree of stanniocalcin homologs from multicellular and

unicellular eukaryotes ... 168

Figure 6.4. Diagram of the evolution of several proteins related to calcium control in

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1.1 The Evolution of Peptide Hormones and their Receptors

Hormones within the endocrine system form one of the major intercellular signalling networks within animals, along with neurotransmitters and cytokines. Hormones are expressed by every tissue of the vertebrate body and are involved in a variety of critical functions, including development and growth, the reproductive cycle, metabolic

regulation, stress response, and immune system activation. They are derived from a variety of structural forms: the aromatic amino acid-based monoamines (thyroid

hormones), the cholesterol originating steroids, and the peptide hormones which include a diverse group of peptides from a few amino acids to several hundred. In every case, a common theme is present among all endocrine factors. A hormone is secreted by a specific group of cells to actuate its message by binding and activating a cognate receptor, either at the plasma membrane of a recipient cell or within its cytoplasm or nucleus.

This process, as well as the respective mechanisms employed by a variety of hormone families, is well conserved in animals that share a common ancestor with vertebrates very early in animal evolution. The cnidarians, which include jellyfish, hydras, corals and sea anenomes, are simple marine animals that evolved before the bilaterian animals, which include protostomes (molluscs, arthropods, annelids and nematodes) and deuterostomes (vertebrates, protochordates, hemichordates and echinoderms). Cnidarians have a very limited range of tissues compared with the bilaterians, lacking true digestive organs and possessing a primordial, decentralized nervous system. They are the most anciently diverged group of animals with significant tissue organization. Given this, it is interesting that the recently sequenced genome of the sea anemone Nematostella vectensis was found

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to contain gene orthologs for a plethora of peptide hormones and receptors, including neuropeptides related to the RFamides, galanin, tachykinin, GnRH/vasopressin, α-MSH and insulin (Anctil, 2009). The evolution of the endocrine system now appears to be ancient within the metazoan (animal) lineage and was already well-developed in animals with simple tissue differentiation.

My interests, the objectives of this dissertation, lie within the evolution of several groups of peptide hormone families and their receptors that arose at different times during the radiation of animals. The secretin superfamily includes glucagon, pituitary adenylate cyclase-activating peptide (PACAP), growth hormone-releasing hormone (GHRH), secretin, and others. This group of peptides is predicted to have arisen at or just before the early evolution of vertebrates. These hormones perform a variety of metabolic and growth-related functions. Gonadotropin-releasing hormone (GnRH) is the

hypothalamic factor responsible for the release of the pituitary gonadotropins. Until recently, it was thought to have appeared sometime before the protostomes diverged from the deuterostomes, however, my work examines an alternative theory for the evolution of vertebrate GnRH in the context of a larger superfamily. The insulin superfamily,

including the familiar glucose modulator insulin, the insulin-like growth factors (IGFs) and pleiotrophic relaxins, have thus far been characterized in vertebrates and insects. My research focussed on studying the origin of this family at the base of the chordates, using the invertebrate chordate (protochordate) models tunicate Ciona intestinalis and

amphioxus Branchiostoma floridae. Additionally, an investigation into the earliest ancestor of this hormone superfamily was conducted. Finally, the calcium modulator stanniocalcin was previously characterized within fishes and mammals. I initially set out

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to investigate stanniocalcin in the protochordates, and followed this research with a study on the origins of this peptide hormone within the metazoans and unicellular eukaryotes. Advances in genomics and phylogenetic analyses have provided the tools to examine the origin of hormone-receptor systems in evolution.

1.2 The Secretin Superfamily

1.2.1 Physiology and Distribution of the Secretin Superfamily Hormones

The secretin superfamily includes several well-known peptide hormones that have been extensively characterized in humans, other mammals and fish. Secretin itself was the first hormone ever characterized, a feat conducted by Bayliss and Starling at the turn of the 20th century. Their investigation on the secretion of a blood-borne substance from the duodenum (the initial segment of the small intestine) to stimulate the pancreas to release chyme resulted in the isolation of a substance they called secretin (Bayliss and Starling, 1902), and the subsequent coining of the term ‘hormone’. In the following decades a multitude of further effects mediated by secretin were identified, including a variety of effects on the liver, intestine, kidney and brain (reviewed in Lam et al., 2008). Secretin’s role as a pancreatic modulator has expanded to include a much larger portfolio and the hormone is now considered a pleiotrophic factor. This is also the case for the homologous pituitary adenylate cyclase-activating peptide (PACAP), a hormone characterized

predominantly in the nervous systems of several mammalian and fish species. As indicated by its name, PACAP stimulates the production of cyclic adenosine

monophosphate (cAMP) in a variety of target cells in several physiological systems including the pituitary and other endocrine tissues, and additionally the gastrointestinal,

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neural, immune and circulatory systems (see Sherwood et al., 2000). PACAP-related peptide (PRP), known as growth hormone releasing hormone-like peptide (GHRH-LP) in fish, is encoded on the same gene as the PACAP peptide. Its functions are unknown in mammals; in zebrafish its location in the brain suggests it is associated with feeding (Castro et al., 2009). A separate gene encodes growth hormone-releasing hormone

(GHRH) in tetrapods and fish (Guillemin et al., 1982; Lee et al., 2007; Rivier et al., 1982; Roch et al., 2009). Vasoactive intestinal peptide (VIP) shares the common VPAC1 and VPAC2 receptors with PACAP in vertebrates and is responsible for vasodilation in several tissues including the gut and brain, amongst other roles (Sherwood et al., 2000). Peptide histidine-isoleucine (PHI), or peptide histidine-methionine (PHM) in humans, exerts similar effects to VIP and is encoded on the same gene (Tatemoto and Mutt, 1981).

Another group of the secretin superfamily hormones includes glucagon, glucagon-like peptide 1 and 2 (GLP1 and 2) and glucose-dependent insulinotropic peptide (GIP). Glucagon, GLP1 and GLP2 are encoded on the same gene in mammals. Glucagon is another classical hormone that was first described by Kimball and Murlin in 1923 as a hyperglycemic factor released from the pancreas that counteracted the effect of insulin (Kimball and Murlin, 1923). Other functions have been characterized for hormones expressed at alternate sites in the intestine and brain. GLP1 is released from the intestine and regulates feeding behaviour and stimulates insulin expression and release in

mammals. In contrast, GLP1 in salmonids appears to exert a similar effect to glucagon (Plisetskaya et al., 1989). GLP2 has mitogenic and cytoprotective effects in the

gastrointestinal tract of mammals (summarized in Estall and Drucker, 2006). Similar to GLP1 in mammals, GIP is released from the intestine in response to elevated glucose

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levels from food and increases insulin release from the pancreas. The secretin

superfamily hormones have evolved to encompass a variety of critical roles regulating a huge diversity of physiological functions in vertebrates.

As noted earlier, several members are encoded on the same gene, including PACAP and PRP/GHRH-LP, VIP and PHI/PHM, and glucagon, GLP1 and GLP2. As these gene organizations are partially preserved in the jawless lamprey Petromyzon marinus, it is likely the secretin superfamily evolved at or just previous to the evolution of vertebrates from a single gene that duplicated into a PACAP/GHRH-LP/VIP/PHI-type and a

glucagon/GLP1/GLP2-type gene; further exon and gene duplications produced GHRH, secretin and GIP, and exon loss (Cardoso et al., 2010). Within the teleost fish, the presence of duplicate copies of many of these hormones has diversified these roles further, a topic that will be discussed in the next chapter.

1.2.2 Mechanisms of the Secretin Superfamily Hormones and their Receptors

The ten secretin superfamily hormones share a homologous core structure comprising the first 27 amino acids of mature hormones. Figure 1.1 displays an alignment of

superfamily peptides from a variety of species. Given the tight conservation of a few critical N-terminal residues in the primary structure of the superfamily peptides, and the loss of potency if these residues are removed or replaced, the peptides probably all bind and activate their cognate receptors by a similar mechanism (Cardoso et al., 2010). All of the secretin superfamily peptides share the same homologous superfamily of receptors, known as the family B (or type 2, secretin-like) G protein-coupled receptors (GPCRs).

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Figure 1.1. Secretin superfamily peptides from a variety of vertebrates. The conserved

27-amino acid region of each hormone is aligned, and identical residues are bolded. Consensus amino acids are indicated at the bottom. PACAP – pituitary adenylate cyclase- activating peptide, VIP – vasoactive intestinal peptide, PRP – PACAP-related peptide, PH – peptide histidine, GHRH – growth hormone-releasing hormone, SCT – secretin, GCG – glucagon, GLP1 – glucagon-like peptide 1, GLP2 – glucagon-like peptide 2, GIP – glucose-dependent insulinotropic peptide. From Cardoso et al., 2010.

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These receptors retain the classical GPCR structure, with an extracellular N-terminus, seven transmembrane regions, and a cytoplasmic C-terminus. This superfamily includes a group of hormone receptors that bind the secretin superfamily peptides, and additionally corticotrophin-releasing hormone, calcitonin, calcitonin gene-related peptide and

parathyroid hormone (Cardoso et al., 2006; Sherwood et al., 2006). These hormones are not homologous to the secretin superfamily peptides. As there are family B receptors found within the protochordates and protostomes, members of this group appear to have been co-opted by the secretin superfamily hormones when they evolved.

A common mechanism for the binding and activation of family B receptors has

emerged with specific examples from the secretin superfamily. Family B receptors retain an extracellular N-terminus of 100-160 amino acids that is glycosylated and stabilized by three disulphide bonds (Hoare, 2005). Chimeric secretin/VPAC receptors showed that the region including the N-terminus and first extracellular loop were critical for ligand binding (Holtmann et al., 1995; 1996b; Vilardaga et al., 1995), as well as the second extracellular loop of the secretin receptor (Holtmann et al., 1996a). Supporting these experiments, the N-terminal domain of the GLP1 receptor was found to bind a peptide agonist with high affinity (Lopez de Maturana et al., 2003). These experiments and others comprising non-secretin superfamily receptors have generated a generalized

‘two-domain’ model of peptide binding, where the peptide first binds the receptor’s N-terminus and then binds the seven-transmembrane region including extracellular loops, triggering activation (Hoare, 2005). A representation of this is displayed in Fig. 1.2. Family B receptors typically signal through Gs (adenylate cyclase stimulation) although many bind Gq/11 and Gi as well depending on the context (Rashid et al., 2004).

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Figure 1.2. Two-domain binding model of Family 2 (secretin-like) GPCR activation. The

C-terminal region of the peptide first binds the extracellular N-terminus of the receptor, bringing the N-terminal region of the peptide into close proximity with the

juxtamembrane (J-domain) or the receptor. Interaction of this region of the peptide and receptor results in a conformational shift within the receptor, stimulating G-protein activation. From Hoare, 2005.

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1.3 Reproductive Neurohormones: GnRH and Kisspeptin

1.3.1 Physiology and Distribution of GnRHs

Gonadotropin-releasing hormone is the neurohormone primarily responsible for the release of the major gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), from the anterior pituitary of vertebrates. Guillemin and Schally

separately isolated the mature decapeptide in the early 1970s, part of the work that led to both receiving the Nobel Prize in Physiology or Medicine in 1977. In mammals, the primary site of expression for GnRH is the preoptic-hypothalamic region, and together with the pituitary gonadotropins and their downstream effectors, the gonadal steroids, these hormones comprise the major constituents of the hypothalamic-pituitary-gonadal axis in vertebrates that controls sexual maturation and the reproductive cycle.

All mammals produce the GnRH peptide known as GnRH1 (also referred to as mammalian, or mGnRH) as do amphibians (Wang et al., 2001) and some fishes (Lescheid et al., 1995). In some mammals there is a second functional GnRH2 (also known as chicken, or cGnRH), encoded on a separate gene (Millar, 2005). Upon the advent of puberty, GnRH1 is released from nerve axons in the median eminence of the hypothalamus where it travels through the portal bloodstream to act on gonadotrope cells in the pituitary. This results in the expression and secretion of LH and FSH, which travel to the gonads and trigger their development, as well as the expression of steroids. FSH stimulates follicular development in the ovaries and estrogen secretion, whereas FSH in males promotes testicular development and induces Sertoli cells to secrete inhibin. LH promotes ovulation in females and testosterone secretion (resulting in spermatogenesis) in males. The frequency of the pulsatile secretion of GnRH on the gonadotropes

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determines which gonadotropin is stimulated (Millar, 2005). The steroids provide positive and negative feedback on the hypothalamus and pituitary to regulate the production of GnRH and the gonadotropins.

GnRH2 was characterized more recently in avians (Mikami et al., 1988; Miyamoto et al., 1982), reptiles (Lovejoy et al., 1991a), fishes and humans (White and Fernald, 1998), and is consistently found in distinct populations of neurons in the midbrain. The function of GnRH2 is less clear, although it appears to play a more neuromodulatory than

pituitary-regulating role (Kah et al., 2007). A third GnRH hormone, GnRH3 (also known as salmon, or sGnRH), is exclusive to teleost fish (Sherwood et al., 1983). The

localization of GnRH3 within the fish brain overlaps with GnRH1 (Okubo et al., 2006). The functions of GnRH3 are related to reproduction; teleost species that have lost GnRH1 but retain GnRH3 do not appear to suffer reproductive detriments, suggesting a compensatory function (Kah et al., 2007). In addition to these major GnRH lineages, lampreys have three GnRH peptides (lamprey GnRH-I, -II and –III); GnRH-II is similar to GnRH2 (Kavanaugh et al., 2008), but GnRH-I and –III (Sherwood et al., 1986) are more distinct from any vertebrate homologs. A recent study that used syntenic analysis to reconstruct paralagous regions corresponding to GnRHs in jawed vertebrates suggested both GnRH3 and 4 were lost in different lineages after diverging from a common ancestor of jawed vertebrates (Tostivint, 2011).

Outside of the vertebrates, examples of unique GnRH peptides have been identified in protochordates (tunicates) and protostomes. In the tunicate C. intestinalis, six forms of GnRH were encoded on two separate genes in triplet (Adams et al., 2003). Synthesized tunicate peptides induced the release of gametes when injected into adult tunicates.

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Within the protostomes, unique 11 or 12-amino acid GnRH-like peptides have been isolated from the octopus O. vulgaris (Iwakoshi et al., 2002) and the sea hare Aplysia

californica (Zhang et al., 2008b). Additionally, homologous gene models were identified

in the owl limpet Lottia gigantea and the annelid marine worm Capitella teleta (Tsai and Zhang, 2008). The physiological function of these peptides are not well understood; one report demonstrated steroidogenesis in octopus gonads after exposure to O. vulgaris GnRH (Kanda et al., 2006), whereas a more recent study found no link to activation of reproduction in A. californica after injection with species-specific synthetic hormone (Tsai et al., 2010). It should be noted that none of these invertebrates have an anterior pituitary or defined hypothalamic-pituitary-gonadal axis (see Roch et al., 2011).

1.3.2 Mechanisms of GnRHs and their Receptors

GnRHs are small (10-12 amino acids) hormones with moderate primary structure conservation at a few residues (Fig. 1.3). All peptides characterized to date have an N-terminal pyroglutamate and an amidated C-terminus. The first three amino acids of GnRH1 (pQHW) are critical for receptor binding and activation; the last three (RPG-amide) are important for receptor binding based on studies of peptide analogs for the GnRH1 receptor (Sealfon et al., 1997). The glycine at position 6 of GnRH1, 2 and 3 also appears to confer a structural bend important to bring the N- and C-termini of the peptide together for receptor binding. As displayed in Fig. 1.3, the arginine at position 8 is not conserved outside of mammalian GnRH1 and the glycine at position 6 is not conserved in most tunicate sequences, indicating some flexibility in the requirements for successful receptor coupling.

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Figure 1.3. Mature GnRH peptides from a variety of vertebrates and invertebrates.

Residues are numbered according to vertebrate GnRH positions. Identical residues are shaded in colour and similar residues are shaded in gray. From Roch et al., 2011.

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The GnRH receptors are GPCRs from family A (or type 1, rhodopsin-like). In contrast with the aforementioned family B receptors, the GnRH receptors have a much shorter extracellular N-terminus and the mammalian GnRH1 receptors lack an intracellular C-terminal tail. Almost all other GnRH receptors possess a long C-C-terminal tail. Numerous mutational studies on the GnRH1 receptor have identified residues important for ligand interaction and helped establish a generalized binding model. Two disulfide bridges, one between extracellular loops 1 and 2 and the other between the N-terminus and

extracellular loop 2, are thought to stabilize the transmembrane pockets together in a tight bundle (Cook and Eidne, 1997). The extracellular loops and transmembrane domains form a hydrophilic binding pocket, with residues presumed to be critical to ligand binding found at the juxtamembrane region of the extracellular surface (see Millar et al., 2004). After ligand binding, GnRH receptors in the pituitary preferentially activate Gq/11 and the phospholipase C pathway, although they can activate Gs or Gi as well (Arora et al., 1998; Cheng and Leung, 2000; 2005). Intriguingly, the stimulation of adenylate cyclase through Gs was the only pathway activated by 3 out of 4 tunicate GnRH receptors characterized (Tello et al., 2005).

Mammalian GnRH1R is most sensitive to GnRH1 whereas all other GnRH receptors are most sensitive to GnRH2, indicating its paramount importance to GnRH systems outside of the mammals (Kah et al., 2007). Within the mammals, only a few species characterized (4 of 22) had a functional GnRH2 system (Stewart et al., 2009). This indicates that while GnRH2 remains an essential regulator in non-mammalian

vertebrates, within the mammals it is either compensated for by GnRH1, or the function of GnRH2 is no longer necessary.

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1.3.3 Relationship of Vasopressin/Oxytocin Receptors to GnRHRs

In mammals, oxytocin has a role in reproduction including lactation, uterine

contractility, vas deferens ejaculation, prolactin secretion and reproductive behaviour. Likewise, vasopressin/oxytocin (VP/OT) peptides in earthworm, leech and octopus have been shown to induce egg laying behaviour and oviduct contraction (Fujino et al., 1999; Oumi et al., 1996). However, in this thesis my interest is not in the VP/OT peptides, but rather in the relationship of the VP/OT receptors with GnRHRs, as they are related in structure during metazoan evolution.

In humans and other mammals, at least three types of vasopressin receptors (V1aR, V1bR, V2R) and one oxytocin receptor (OTR) have been characterized (for reviews see (Gimpl and Fahrenholz, 2001; Lolait et al., 1995). The VP/OT receptors are GPCRs from family A (rhodopsin-like), like GnRHR. The signaling pathways are also similar:

IP3/Ca++ is dominant for GnRHR, V1aR, V1bR and OTR, but cAMP stimulation is the signaling path for V2R and a minor path for GnRHR. In 1992 it was reported that the mouse GnRH receptor was most closely related in structure to that of the

vasopressin/oxytocin (VP/OT) receptors (Reinhart et al., 1992). Subsequently, more VP/OT and GnRH receptors have been identified supporting the close structural relationship. However, the GnRH and VP/OT peptides in these two superfamilies are very different. Although the length of GnRH (10 amino acids) and VP/OT (9 amino acids) in vertebrates is similar, GnRH is a linear peptide whereas VP/OT peptides have a disulfide bond creating a ring structure that exists in all the peptides from annelids to humans. Also of interest throughout vertebrate genomes is the arrangement of GnRH2 and VP/OT genes as adjacent neighbors on the chromosome (Gwee et al., 2009).

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1.3.4 Physiology and Distribution of Kisspeptins

Also known as metastatin for its role in metastasis suppression in tumours, kisspeptin 1 (Kiss1) was found to bind the previously orphaned GPR54 receptor by several groups in 2001 (Kotani et al., 2001; Muir et al., 2001; Ohtaki et al., 2001). Although the function of this hormone/receptor pairing was initially obscured, null mutations in the receptor (now referred to as Kiss1R) resulted in hypothalamic hypogonadism for both humans and mice (de Roux et al., 2003; Funes et al., 2003; Seminara et al., 2003), indicating a crucial role in reproduction. Conversely, an activating mutation of Kiss1R results in precocious puberty (Navarro et al., 2004; Teles et al., 2008).

A connection between Kiss1 and GnRH was soon made, as administration of the peptide to rats (Irwig et al., 2004; Thompson et al., 2004) and monkeys (Plant et al., 2006) resulted in the release of LH and FSH. This stimulatory effect was ablated by the concurrent administration of a GnRH antagonist. Additionally, Kiss1R expression was localized to GnRH neurons in rodent brains (Han et al., 2005; Irwig et al., 2004; Messager et al., 2005) and kisspeptin immunoreactive fibres were found in close proximity (Clarkson and Herbison, 2006). Kisspeptin can thus act directly to stimulate GnRH neurons, opposing the actions of another neuropeptide, GnRH inhibiting hormone (GnIH).

Kisspeptin also provides a link between GnRH-mediated stimulation of the sex steroids and their positive and negative feedback on the reproductive axis. Kiss1 neurons in the rodent brain are found in both the arcuate nucleus of the hypothalamus and the

anteroventral periventricular nucleus (AVPV) of the third ventricle (Oakley et al., 2009). In both populations, Kiss1 neurons express the estrogen receptor (ERα) and in the arcuate

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nucleus the androgen receptor (AR) is expressed as well. In castrated mice, Kiss1

expression increased in the arcuate nucleus, indicating negative feedback by sex steroids (Smith et al., 2005a; 2005b). The opposite was true for Kiss1 expression in the AVPV, suggesting positive feedback. Other factors may also influence kisspeptin expression, including the metabolic regulator leptin, whose receptors can be found on Kiss1 neurons in the arcuate nucleus (Smith et al., 2006). Additionally, both Kiss1 and its receptor are expressed in the pituitary and there is debate as to whether they directly stimulate the gonadotropins (Oakley et al., 2009).

1.3.5 Mechanisms of Kisspeptins and their Receptors

Human Kiss1 is comprised of a 145 amino acid propeptide that is cleaved into a mature peptide of 54 amino acids which may be further processed to shorter peptides of 14, 13 or 10 amino acids; they bind and activate the human Kiss1R equally (Kotani et al., 2001). Kisspeptins belong to the RFamide group of peptide neurohormones, indicating the amino acid motif at the C-terminus of the mature peptide. Other members of this group include gonadotropin-inhibiting hormone, neuropeptide FF, prolactin-releasing peptide and QRFP in the vertebrates, and a host of unique peptides in the invertebrates including the well-known molluscan FMRFamide. Similar to the other RFamides, kisspeptins bind specific GPCRs from the rhodopsin-like (family A) group. The kisspeptin receptor, formerly referred to as the orphan GPR54, is structurally most closely related to receptors of the unrelated neuropeptides galanin and the invertebrate-specific allatostatin. Given this phylogenetic relationship, Kiss1R is specific for its cognate ligand and does not bind galanin (Lee et al., 1999). Upon binding Kiss1, the activated receptor triggers the Gq/11-mediated phospholipase C pathway, resulting in the release of intracellular calcium stores

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and arachidonic acid, and the activation of ERK1/2 and p38 kinases (Castellano et al., 2006; Kotani et al., 2001; Stafford et al., 2002). This signaling cascade is hypothesized to mediate GnRH release through the depolarization of GnRH neurons via several types of cation channels (Liu et al., 2008; Zhang et al., 2008a).

1.4 The Insulin Superfamily

1.4.1 Physiology and Distribution of the Insulin Superfamily Hormones

Insulin is one of the most familiar hormones due to its central role in glucose

metabolism and association with type 1 and 2 diabetes mellitus. The isolation of insulin took place in a famous series of experiments by Banting and Best in the early 1920s and resulted in the first viable therapy for the previously incurable, terminal illness. For this accomplishment Banting received the 1923 Nobel Prize in Physiology or Medicine, and triggered a wave of research into endocrinology and protein chemistry that culminated in another insulin-related Nobel Prize in 1958, this time in chemistry for the determination of its primary sequence, discovered by Frederick Sanger. Additionally, Rosalyn Yalow received the 1977 Nobel in Physiology or Medicine (shared with Guillemin and Schally) for development of the radioimmunoassay for insulin. Due to these achievements and others, research on insulin and its structurally related superfamily has remained a hot topic for almost a century.

Insulin is the most notable of the superfamily members, which also include the IGFs and relaxins. In mammals, insulin is expressed in the beta cells of the pancreatic islets of Langerhans, the endocrine secreting tissue that also produces glucagon, amylin,

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blood glucose levels from a meal stimulate insulin release from the pancreas; insulin, in turn, activates its receptors throughout the body. The physiological response triggered by insulin differs depending on the tissue affected, including uptake of glucose from blood into cells, increased glycogen synthesis in liver and muscle, increased fatty acid synthesis and esterification in adipose tissue, and decreased proteolysis, lipolysis and

gluconeogenesis. A lack of insulin or acquired insulin resistance leads to abnormally high blood glucose levels (hyperglycemia) and the corresponding effects of type 1 or type 2 diabetes, respectively.

The IGFs, on the other hand, do not play a significant role in the regulation of glucose metabolism. IGF1, released from the liver after activation by growth hormone, stimulates growth in many tissues. IGF2 is similar, however, its effects are thought to occur

primarily during prenatal growth as demonstrated by mouse knockout models for each hormone (DeChiara et al., 1991; Liu et al., 1993). The relaxins are a diverse group of seven insulin-like hormones (in humans) with a wide variety of functions including parturition and other reproductive roles, cardiovascular growth and response, and wound healing (Hsu, 2003; Lu et al., 2005). As most of these hormones have only been

characterized recently, an increasing portfolio of functions is likely to develop. The insulin superfamily is an important regulator not only in vertebrates, but invertebrates as well. Protochordate homologs have been isolated from the tunicate

Chelyosoma productum, where both an insulin- and IGF-like cDNA were isolated

(McRory and Sherwood, 1997) and in C. intestinalis where three homologs were identified . In the amphioxus Branchiostoma californiensis, a single homolog characterized as a ‘hybrid’ insulin/IGF (ILP) was isolated (Chan et al., 1990). Many

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examples can be found in the protostomes as well, including bombyxin from silkworm (Bombyx mori) (Nagasawa et al., 1984; 1986), molluscan insulin-related peptides from pond snail (Lymnaea stagnalis) (Smit et al., 1988), insulin-like proteins from nematode (Caenorhabditis elegans) (Duret et al., 1998) and insulin-like peptides from fruit fly (Drosophila melanogaster) (Brogiolo et al., 2001; Vanden Broeck, 2001).

The functions of these peptides appear to focus less on metabolic control and more on growth and aging. Disruptions of the insulin signalling system within fruit flies by mutation of the insulin receptor or its substrate chico resulted in a dwarf phenotype (Bohni et al., 1999; Brogiolo et al., 2001), whereas overexpression of an insulin-like gene resulted in gigantism (Brogiolo et al., 2001). Additionally, invertebrate insulin

superfamily hormones may influence reproduction as bombyxin has been localized to silkworm ovaries (Iwami et al., 1996) and female fruit flies with the chico mutation displayed severely reduced fertility (Bohni et al., 1999). The most prominent function studied in invertebrate insulin biology has been the effect of these hormones on the aging process. In C. elegans, disruption of insulin signalling components including the receptor (DAF2) resulted in individuals with much longer lifespan (Dorman et al., 1995; Kenyon et al., 1993). This was corroborated by fruit fly receptor and chico mutants (Clancy et al., 2001; Tatar et al., 2001).

1.4.2 Mechanisms of the Insulin Superfamily Hormones and their Receptors

The tertiary structure of the insulin peptides has been well conserved throughout the evolution of the superfamily. The basic structure is composed of two domains: a B-chain of approximately thirty amino acids coupled to a twenty amino acid A-chain through two disulfide bonds (Fig. 1.4). Within the A-chain an internal disulfide bridging is also

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present and this pattern of bonding with six conserved cysteine residues is strictly

retained throughout the superfamily, with the exception of highly divergent peptides such as some of those found in C. elegans (Duret et al., 1998). In addition to the highly

conserved B- and A-chains, a poorly conserved connecting C-domain is also present in the prohormone which is cleaved at dibasic sites in the mature insulins and relaxins but retained by the IGFs (Fig. 1.4). The IGFs retain two additional, poorly conserved

domains downstream of the A-chain, the D- and E-domains; the latter of which is cleaved in the mature peptide.

The insulin/IGF receptors, members of the receptor tyrosine kinase (RTK) family, are also well conserved throughout the lineages bearing insulin-like peptides. The basic structure of these receptors is a single-pass heterotetramer with a membrane bound β-subunit, which forms a disulfide bridge to an extracellular ligand-binding α-β-subunit, two of which are also bridged together by disulfide bonds to dimerize the two monomers (Fig. 1.5). The extracellular α-subunit retains the binding domains for ligands while the β-subunit retains the activation domains including the tyrosine kinase domain. Upon ligand binding, a conformational shift causes these domains to trans-phosphorylate their dimeric partner and then phosphorylate a number of cytoplasmic effectors. These downstream activators include the insulin receptor substrates (IRSs) and Shc (Src-homology) proteins, which activate two major signaling pathways. The predominant signal is mediated

through the protein kinase B (PKB) pathway. The second pathway stimulates growth factor receptor bound protein-2 (Grb2) which results in stimulation of the mitogen-activated protein kinase (MAPK) cascade (see Siddle et al., 2001 for a review).

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Figure 1.4. Insulin/relaxin and insulin-like growth factor (IGF) processing. After

translation of the initial propeptide, insulin and relaxin B- and A-chains are bound by two disulfide bridges and the connecting C-domain is removed at dibasic cleavage sites. IGF, on the other hand, is similarly bridged by disulfide bonds but the C-domain is not cleaved and an additional D-domain is also retained in the mature hormone.

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Figure 1.5. Insulin/IGF receptor structure. The α- and β-subunits are initially bound by

disulfide bridges as a heterodimer, and upon ligand binding are heterotetramized with another dimer. Important regions of each subunit are indicated. L-domain indicates the ligand-binding domain.

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Single receptors, homologous to the vertebrate insulin and IGF receptors, are found in a variety of protostomes including D. melanogaster (Ruan et al., 1995) and C. elegans (DAF-2) (Kimura et al., 1997). As well, a single receptor for the hybrid ILP has been isolated from amphioxus (Pashmforoush et al., 1996). The signaling pathways for these ancient receptors appear to be homologous to their vertebrate counterparts and have been well characterized in fruit flies (DIR) and C. elegans (DAF2) (Claeys et al., 2002). Interestingly, one of the DIR variants and DAF-2 both have an extended cytoplasmic C-terminal tail that possesses homology to the insulin receptor substrates (Fernandez et al., 1995; Kimura et al., 1997).

The relaxins bind a family of receptors unrelated to either the insulin/IGF or IGF-II receptors. The relaxin receptors belong to a superfamily known as the leucine-rich repeat GPCRs (LGRs), a group which also includes receptors for follicle-stimulating hormone, luteinizing hormone and thyroid-stimulating hormone. The relaxin LGRs are classified as family C (or type 3, glutamate-like) receptors, distinguished by a large cysteine-rich extracellular N-terminus (Hsu, 2003). Two receptors have been identified in a variety of mammals, and are designated LGR7 and LGR8. In addition, two orphan G-protein coupled receptors, GPCR142 and GPCR135, have demonstrated binding and activation by relaxin peptides (Liu et al., 2003a; 2003b; 2005).

1.5 Stanniocalcin

The peptide hormone stanniocalcin (STC) was originally isolated from specialized glands adjacent to the kidneys of teleost fishes, known as the corpuscles of Stannius. Stanniocalcin (originally teleocalcin or hypocalcin) was first isolated from the sockeye

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salmon (Oncorhynchus nerka) and it was shown to reduce calcium uptake in the gills of a related salmonid (Wagner et al., 1986). Identification of the hormone in several related teleosts followed, including other salmonids (Lafeber et al., 1988b; Wagner et al., 1988) eel (Anguilla australis) (Butkus et al., 1987), flounder (Pleuronectes americanus), goldfish (Carassius auratus) (Wendelaar Bonga et al., 1989) and a host of other fish species. Stanniocalcin isolated from these teleosts retained a conserved structure, present in their active forms as a glycosylated dimer approximately 50 kDa in size

(approximately 250 amino acids per monomer).

A number of homologs have been characterized from basal teleosts including the silver arawana (Osteoglossum bicirrhosum) (Amemiya et al., 2002), elephantfish

(Gnathonemus petersii) and the butterflyfish (Pantadon buchholzi) (Amemiya et al., 2006). These fish have an STC protein sequence homologous to that of the other teleosts, but only have 10 of the 11 conserved cysteine residues found in the prototypical

structure. The lack of the 11th cysteine used to form a bond between two STCs results in a monomeric circulating variant of stanniocalcin (Amemiya et al., 2002). Stanniocalcins were also isolated from non-teleost, basal ray-finned fishes, including the gar

(Lepisosteus osseus) and the bowfin (Amia calva) (Amemiya and Youson, 2004). These species retain the conserved dimerizing cysteine, suggesting the monomeric form of stanniocalcin is a lineage-specific derivation unique to some basal teleosts.

Orthologs of STC with all 11 conserved cysteine residues were identified in humans (Chang et al., 1995; Olsen et al., 1996) and later rodents (Chang et al., 1996; Haddad et al., 1996). Additionally, several groups identified a second stanniocalcin gene in humans and mice (Chang and Reddel, 1998; DiMattia et al., 1998; Ishibashi et al., 1998). This

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peptide, STC2, was longer (~300 amino acids) than the previously characterized stanniocalcin, now referred to as STC1. STC2 orthologs in fish were identified more recently (Luo et al., 2005; Shin and Sohn, 2009).

The most prominent function of stanniocalcin described in teleosts is to downregulate calcium uptake from environmental water. Early experiments established that in response to rising calcium levels stanniocalcin is released into the circulatory system to prevent calcium influx into gill cells . In addition to this primary effect, addition of stanniocalcin improved phosphate reabsorption in cultured flounder kidney cells (Lu et al., 1994).

After the isolation of STC1 from humans, a host of mammalian-specific functions were uncovered. Mammals utilize parathyroid hormone as their major calcium regulator and do not live in a hypercalcemic environment, unlike fish. As well, human STC1 and STC2 are expressed in a wide variety of tissues, rather than specific glands like the corpuscles of Stannius (Chang et al., 1995; Chang and Reddel, 1998; DiMattia et al., 1998; Ishibashi et al., 1998). This suggested that the mammalian hormones might not function as

endocrine effectors but rather in a paracrine or autocrine fashion, a hypothesis that has been supported by several reports detailing the influence of stanniocalcin on cell differentiation in the ovary (Luo et al., 2004) and bone formation (Wu et al., 2006; Yoshiko et al., 2002).

Mammalian stanniocalcin may possess a pleiotropic quality that extends beyond simple mineral metabolism. Mice expressing a human STC1 transgene displayed significantly reduced postnatal growth suggesting a negative mitogenic effect, unrelated to bone mineralization, caused by stanniocalcin over-expression (Filvaroff et al., 2002; Varghese et al., 2002). Conversely, STC1 overexpression stimulated osteoblast growth and

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differentiation in fetal rat cell cultures (Yoshiko et al., 2003) and STC1 knockout-mice displayed no detrimental phenotype whatsoever (Chang et al., 2005). A number of other roles have been implied by the expression pattern of STC1 in various tissues and it has even been used as a molecular marker for the detection of various cancers (Tohmiya et al., 2004; Wascher et al., 2003).

Significantly less is known about the functions of STC2 but so far they seem to be related to phosphate metabolism. Transgenic mice over-expressing STC2 displayed a dwarf phenotype like those over-expressing STC1 (Gagliardi et al., 2005). The lack of an identified receptor for STC1 or 2 presents a unique problem that prevents analysis of the molecular mechanism responsible for its varied functions. One key piece of the puzzle that must be uncovered is the structure of the receptor, which has been localized to the membranes of mitochondria but remains uncharacterized (McCudden et al., 2002).

1.6 Objectives

The objectives of characterizing the hormone systems detailed in the following

chapters differed between each system, however, one fundamental theme applies to all of the work in this dissertation. The primary goal in each project was to identify the

evolutionary origin of each hormone family in question, and try to chart the course of their expansion and loss throughout various lineages. This was especially true for hormone families that might have appeared as single genes earlier in their evolutionary history and then expanded into larger, more complex families. As well, a comparison of the evolution of receptor families to their cognate ligands was conducted in certain

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instances to determine the scope of hormone-receptor co-evolution. An overview of each chapter follows below.

Chapter 2 will detail my analysis of the secretin superfamily in several fish to determine the extent of gene duplication amongst the peptides and receptors resulting from the teleost genome duplication, as well as the ultimate origin of superfamily members within the vertebrates. Chapter 3 will show my investigation into the roots of the reproductive neurohormone receptors, including the GnRHR superfamily and the KISSR/GPR54 family. I argue that a new superfamily of peptides and receptors exists including GnRH, adipokinetic hormone (AKH), and corazonin. As well, the relationship between the GnRH receptor superfamily and the vasopressin/oxytocin receptor family will be shown. Chapter 4 will include work I performed on the protochordate insulin superfamilies, including the isolation of hormone cDNAs in order to estimate the composition of the superfamily at the origin of the chordates. As well, a project I completed to determine the extent of the insulin superfamily’s distribution across the metazoans will be shown in brief. Chapter 5 will present the work I conducted on protochordate stanniocalcins, including expression profiles within the tunicate Ciona

intestinalis. This is the first invertebrate species in which STC was cloned. In Chapter 6, I

will present the work I completed analyzing the distribution of stanniocalcin homologs across the eukaryotic spectrum. STC is present not only in muticellular organisms, but also in single-celled eukaryotes.

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