Variant GnRH ligand and receptor subtypes have been identified in a number of non-mammalian vertebrate species, however research into avian species GnRH systems is lacking. Two isoforms of GnRH are present in the domestic chicken, the evolutionarily conserved GnRH-II and diverged cGnRH-I. The expression of two GnRH ligands parallels the expression of two chicken GnRH receptor subtypes; cGnRH-R-I and the novel cGnRH-R-III. The occurrence of two isoforms of the receptor in the chicken raises questions about their specific biological functions and interactions with the two ligands. Differential roles for these molecules in regulating gonadotrophin secretion or other functions are currently unclear. To investigate this, cGnRH-R-III cDNA was cloned from a broiler chicken anterior pituitary gland and its structure and expression was compared with cGnRH-R-I. Expression profiling of cGnRH-R-III cDNA showed that it is predominantly expressed in the anterior pituitary, approximately 1400 times more abundantly than cGnRH-R-I suggesting that cGnRH-R-III is the predominant regulator of chicken gonadotrophin synthesis and secretion. Additionally, pronounced sex and age differences existed, with higher pituitary cGnRH-R-III mRNA levels in sexually mature females versus juvenile females. In contrast, higher mRNA expression levels occurred in juvenile males compared to sexually mature males. Determination of ligand-binding selectivity and the level of cGnRH-R-III activation in response to the endogenous ligands, cGnRH-I and GnRH-II, was anticipated as facilitating the elucidation of the physiological roles of the receptor subtypes. Additionally, the development of analogs that differentially promote or inhibit activation of the receptor subtypes may be valuable tools for determining the role of receptor types in the regulation of gonadotrophin production. To investigate this, pharmacological profiling of cGnRH-R-III in terms of ligand-binding selectivity and inositol phosphate production in response to GnRH analogs was determined in comparison with the pharmacological profile of cGnRH-R-I. Functional studies in COS-7 cells indicated that cGnRH-R-III has a higher binding affinity for GnRH-II than cGnRH-I (IC50: 0.57 v 19.8 nM) and more potent stimulation of inositol phosphate production (EC50: 0.8 v 4.38 nM). Similar results were found for cGnRH-R-I, (IC50: 0.51 v 10.8 nM) and (EC50: 0.7 v 2.8 nM). Mammalian receptor antagonist 27 distinguished between cGnRH-R-I and cGnRH-R-III (IC50: 2.3 v 351 nM), and application of this synthetic peptide may facilitate delineation of receptor subtype function either in-vitro or in-vivo. The length of the C-terminal tail of cGnRH-R-III is 8 residues longer than that of cGnRH-R-I and this observation stimulated investigation of differences in ligand-induced internalisation between the two receptor subtypes. The initial rate of receptor internalisation was faster for cGnRH-R-III than for cGnRH-R-I (26%.min-1 v 15.8%.min-1). Although proteins encoded by cGnRH-R-III splice variants do not bind GnRH ligands independently and mRNAs were not detectable by Northern blot analysis, cGnRH-R-III_SV2 significantly reduced maximum ligand-binding of cGnRH-R-III, suggesting that it may impair the function of the full-length type III cGnRH receptor. It was anticipated that the two cGnRH-R subtypes may have differential roles in the regulation of luteinising hormone (LH) and follicle stimulating hormone (FSH) gene transcription through the activation of differential second messenger pathways. Three putative Src homology domain 3 (SH3) binding motifs were identified in the type III cGnRH receptor cytoplasmic C-terminal tail domain which are not present in the type I cGnRH-R and suggested the potential for differential coupling to the Mitogen Activated Protein Kinase (MAPK) cascade. To investigate this possibility, activation of the MAPK cascade via cGnRH-R-III and cGnRH-R-I was determined by quantifying elevation of phosphorylated ERK (pERK 1/2) in response to GnRH. Studies performed in COS-7 cells showed a 4-6 fold increase in ERK 1/2 phosphorylation via the type I and type III receptors within 10 minutes of GnRH-I or GnRH-II stimulation, indicating that both receptors signal through the ERK 1/2 pathway in response to cGnRH-I or GnRH-II. The responses were dose-dependent at cGnRH-R-I and cGnRH-R-III. Effects of pre-treatment with PLC and c-Src inhibitors showed that both cGnRH-Rs may activate pERK 1/2 independently of PLC but dependently upon c-Src. However, it must be noted that 100% of the PLC activity was not inhibited by PLC inhibitor as measured by inositol phosphate production at 60 minutes, and the PLC inhibitor has not been shown to inhibit PLC in the same time frame used for the pERK experiments. Mutagenesis of the individual SH3 binding motifs of cGnRH-R-III were performed and the effects on pERK 1/2 levels quantified. The results indicated that the SH3 binding motifs of cGnRH-R-III do not contribute to additional MAPK activation when compared to the native cGnRH-R-III. Both cGnRH-R-I and cGnRH-R-III were HA epitope-tagged (HA-cGnRH-R-I and HA-cGnRH-R-III) and the methodology was optimised for HA-cGnRH-R-III immuno-precipitation. Several size forms of HA-cGnRH-R-III were detectable by immuno-precipitation, facilitating characterisation of the composition of the receptor protein-protein complexes formed using a western blot approach. In summary, the abundance of cGnRH-R-III expression compared to cGnRH-R-I suggests it is probably the major mediator of pituitary gonadotroph function, and that tissue-specific recruitment of cGnRH-R-isoforms has occurred in the avian pituitary during evolution. Pharmacological profiling demonstrated that cGnRH-R-III, like cGnRH-R-I, has a higher ligand-binding selectivity and induction of inositol phosphate production in response to GnRH-II than with cGnRH-I, although cGnRH-I is established as the physiological regulator of gonadotroph function. These results suggest that evolutionary recruitment of ligand-receptor pairing for particular physiological processes does not correlate with in-vitro properties such as highest ligand-binding affinity or efficacy of inositol phosphate production. Therefore evolutionary plasticity has occurred in the tissue-specific adoption of GnRH ligand and receptor subtypes for regulation of particular physiological functions in birds.

<p>Olfaction is one of the most primitive of our senses, and the olfactory receptors that mediate this very important chemical sense comprise the largest family of genes in the mammalian genome. It is therefore surprising that we understand so little of how olfactory receptors work. In particular we have a poor idea of what odorous chemicals are detected by most of the olfactory receptors in the genome, and for those receptors which we have paired with ligands, we know relatively little about how the structure of these ligands can either activate or inhibit the activation of these receptors. Furthermore the large repertoire of olfactory receptors, which belong to the G protein coupled receptor (GPCR) superfamily, can serve as a model to contribute to our broader understanding of GPCR- ligand binding, especially since GPCRs are important pharmaceutical targets.</p><p>In this dissertation, I explore the relationship between olfactory receptors and their ligands, both by manipulating the ligands presented to the olfactory receptors, as well as by altering the structure of the receptor itself by mutagenesis. Here we report the probable requirement of a hydrated germinal-diol form of octanal for activation of the rodent OR-I7 receptor by ligand manipulation, and the successful in vitro modeling and manipulation of ketamine binding to MOR136-1. We also report the results of a large-scale screen of 1190 human and mouse olfactory receptors for receptors activated by volatile general anesthetics, which has lead to the identification of 32 olfactory receptor-volatile general anesthetic pairs.</p> / Dissertation

G protein-coupled receptors (GPCRs) are seven-transmembrane domain receptors that sense extracellular signal and activate intracellular signaling pathways. Metabotropic glutamate receptor 2 (mGluR2) is one of the GPCRs coupled to Gi/o proteins whose Gβγ subunits stimulate G protein-gated inwardly rectifying K+ channels (GIRKs). Previous experiments demonstrated that in planar lipid bilayer both active forms of G proteins [Gα (GTPγS-stimulated) and Gβγ subunits] were required to activate GIRK channels in the absence of the receptor, but surprisingly, the Gβγ subunit alone could activate GIRK channel in the presence of GPCR. Currently, it is not clear whether GPCRs play a role beyond catalyzing the dissociation of Gα and Gβγ subunits in the presence of extracellular agonist and intracellular GTP. Here we compare the G protein-stimulated GIRK currents in the presence and absence of mGluR2 by performing whole-cell patch clamp recordings on two types of cells: a HEK293 cell line stably expressing GIRK channels (HEK/GIRK) and HEK/GIRK cells with mGluR2 expressed transiently. Our experiments revealed that mGluR2 affects the behavior of G proteins even in the absence of the agonist. We show that intracellular application of GTP activated GIRK currents, and the GTP-induced GIRK currents became greater in the presence mGluR2. We also show that desensitization kinetics of the GTP-stimulated GIRK currents became greater and faster in the presence of mGluR2.

G-protein coupled receptors (GPCRs) implicated in disease are the predominant pharmaceutical targets. Growing evidence suggests that GPCRs form homo- and heteromeric complexes, resulting in allosteric functional changes. Ligands targeting one receptor can alter the function of the other receptor or receptors. Knowledge of these functional changes will provide unique opportunities to treat diseases. We examined two GPCR heteromers implicated in psychosis: mGlu2R-5HT2AR and D2R-5HT2AR. Using whole-cell patch clamp, we studied HEK-293 cells stably transfected with mGlu2R and 5HT2AR. Maximal heteromer formation allows inverse agonists to increase the G-protein activity of the opposite receptor, while sub-maximal heteromer formation does not. However, similar results are obtained in sub-maximal heteromer cells by applying a combination of a mGlu2R synthetic agonist with a 5HT2AR anti-psychotic drug. These results confirm our oocyte results, now in a mammalian cell line. Using two-electrode voltage clamp, we also investigated the allosteric changes upon heteromerization of D2R-5HT2AR in oocytes injected with appropriate cRNAs. Heteromer formation in the presence of dopamine or serotonin results in an increase in G-protein activity of each receptor while the simultaneous presence of both neurotransmitters further increases the G-protein activity. The addition of synthetic agonists or anti-psychotics decreases the G-protein activity of the opposite receptor while agonizing or antagonizing its target receptor, respectively. Maximal allosteric effects upon D2R-5HT2AR formation only occur at a specific cRNA injection ratio, but partial effects exist at other ratios. Our data suggest that allosteric functional changes upon heteromerization are physiologically relevant and are mostly different when comparing mGlu2R-5HT2AR to D2R-5HT2AR.

Calcitonin gene-related peptide (CGRP) and adrenomedullin (ADM) receptors are heterodimeric complexes composed of the calcitonin receptor-like receptor (CLR) and a receptor activity-modifying protein (RAMP). Association with RAMP1 gives a high affinity CGRP receptor, whereas association with RAMP2 or RAMP3 gives high affinity ADM receptors. CGRP and ADM are widely distributed throughout the body and play important roles and are implicated in many diseases including migraine, heart failure and sepsis. Recently, CGRP has been shown to promote nitric oxide (NO) production and inducible NO synthase (iNOS) expression in trigeminal ganglion glial cells via ERK activation. CGRP is known to induce iNOS/NO production in thoracic artery smooth muscle cells (TA-SMC) pretreated with interleukin-1b. However, the molecular mechanism of CGRP-induced iNOS/NO production in TA-SMC is unknown. Therefore, in order to determine if CGRP induces iNOS/NO production via ERK activation, I first investigated the exact mechanisms through which CGRP activates ERK1-2 in HEK cells. By using different inhibitors I showed that CGRPinduced ERK activation is mainly activated through two major pathways. I showed for the first time that CGRP induces ERK activation through transactivation of ErbB1 and as expected through the cAMP/PKA pathway. Then, in order to characterise a suitable model to study CGRP-induced iNOS expression, I used primary TA-SMC and I showed that CGRP induces iNOS upregulation, which is reduced when cells are incubated with U0126, a MEK inhibitor. Thus, these results suggest that CGRP induces iNOS expression via ERK activation in TA-SMC. However, further experimentation is required to determine the exact ERK pathway responsible for iNOS induction. Compared to CLR•RAMP1 and CLR•RAMP3, little is known about the postendocytic sorting of CLR•RAMP2. Using HEK cells stably expressing CLR•RAMP2, I investigated the molecular mechanisms regulating the ADM receptor. I first showed that, unlike CLR•RAMP1, even transient stimulation of CLR•RAMP2 with ADM promotes degradation of both CLR and RAMP2, indicating that this ADM receptor does not recycle to the cell-surface. Moreover, I showed that CLR, not RAMP2, is constitutively ubiquitinated, which was further enhanced upon ADM stimulation. In order to elucidate the role of ADM-mediated ubiquitination of CLR, I made a lysine-less mutant of CLR, named CLRD9KR. I showed that ubiquitination of CLR did not affect ADM-induced trafficking of CLR•RAMP2 to lysosomes, nor did it affect the degradation or the ERK signalling of CLR•RAMP2. However, I showed that ubiquitination of CLR regulated the rate of degradation of the receptor. Together, these results indicate that CLR•RAMP2 does not recycle and is degraded via a molecular mechanism that is accelerated by ADM-induced ubiquitination of CLR.

Membrane proteins are important targets that represent more than 50% of current drug targets. However, characterisation of membrane proteins falls behind compared to their soluble counterparts. The most challenging part of membrane protein research is finding a suitable membrane mimetic that stabilises them in solution and maintains their native structure and function. The recently developed saposin-A (SapA) based lipid nanoparticle system seems to be advantageous over existing membrane mimetic system. It provides a native-like lipid bilayer, high incorporation yield and more importantly size adaptability. SapA lipid nanoparticles have been applied to structural studies and two high-resolution structures of membrane proteins were previously obtained using cryo-electron microscopy. This thesis aimed to study small-to-medium sized membrane proteins in SapA lipid nanoparticles using NMR spectroscopy. We first explore the mechanism of SapA lipid nanoparticle formation for the purpose of establishing an incorporation protocol that can be applied to most membrane proteins. The effect of pH and the presence of detergents on the opening of SapA was investigated in Chapter 2. A proposed energy diagram describing the mechanism of SapA opening is reported with which we were able to develop a protocol that can generate different sizes of SapA-1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) nanoparticles. In addition, we also showed that SapA can form lipid nanoparticles with various lipid compositions, showing the versatility of the system. In Chapter 3, we validated the ability of SapA lipid nanoparticles to be used as a membrane mimetic. A -barrel model protein, bacterial outer membrane protein X (OmpX), was incorporated into SapA-DMPC nanoparticles and a 2D 15N-1H correlation NMR spectrum was recorded. Our result was compared to the NMR parameters of the same protein in MSP nanodiscs from the literature, and it was concluded that SapA lipid nanoparticles indeed provide a lipid bilayer environment similar to MSP nanodiscs. Because of high incorporation yield, we were able to incorporate OmpX into different lipid compositions to investigate the effect of lipid head groups and aliphatic chains on the membrane protein's chemical environment. Next, the applicability of SapA lipid nanoparticles was expanded to -helical transmembrane proteins in Chapter 4. Two microbial rhodopsins, Anabaena sensory rhodopsin (ASR) and Natronomonas pharaonis sensory rhodopsin II (pSRII), were tested. The parameters for expression and purification of ASR were first screened for the optimal yield. Although incorporation of ASR resulted in inhomogeneous particles due to imperfect experimental procedure, pSRII in SapA-DMPC nanoparticles showed high sample quality. The 2D NMR spectrum of pSRII in SapA-DMPC nanoparticles shows distinct differences to pSRII in detergent micelles, suggesting substantial effects from the membrane mimetic on the conformation of the membrane protein. Despite the good NMR spectral quality considering the large particle size, perdeuteration of pSRII and the lipids will be necessary for further investigation. With the SapA lipid nanoparticles established, we aimed to use it for the study of a biologically important G protein-coupled receptor, 1-adrenergic receptor (1AR), discussed in Chapter 5. The possibility of expressing 1AR using a cell-free expression system was explored first. Although a good amount of the protein was obtained, only a fraction of it was functional. Therefore, a conventional baculovirus-insect cell expression system was used to produce selective isotope labelled 1AR for NMR studies. NMR spectra of 1AR in SapA-DMPC nanoparticles with activating ligands and an intracellular binding partner were recorded and compared to the spectra of the same protein in detergents. This revealed a more active-like conformation of ligand-bound 1AR in the lipid bilayer, suggesting that certain parts of the protein are sensitive to the membrane mimetic used. This emphasises the importance of using a native-like membrane mimetic to capture the full properties of membrane proteins. In conclusion, I demonstrate in this thesis that SapA lipid nanoparticles are a versatile membrane mimetic system that can accommodate membrane proteins with different sizes and folds. This system is also compatible with solution NMR spectroscopy enabling structure and dynamics studies of biologically important membrane proteins. We believe SapA lipid nanoparticles will have a significant impact on membrane protein research in the future.

Regulators of G-protein signaling (RGS) proteins are a family of proteins that act as GTPase accelerating proteins (GAPs) through their interaction with GΑ subunits, including GΑo, GΑi, and GΑq but not GΑs. This increased rate of hydrolysis of GTP to GDP temporally regulates G-protein coupled receptor (GPCR) signaling. A member of this family, RGS4, has been implicated in several neurological disorders including Parkinson's Disease (PD). A hallmark of PD is the induction of oxidative stress within dopaminergic neurons. In this thesis, we evaluate the role of oxidative stress, including lipid peroxidation products with 4-hydroxy-2-nonenal (4HNE) as a model, in regulating RGS4 activity within neurons. Utilizing transfected RGS4, we evaluated whether RGS4 is readily modified by physiologically relevant concentrations of 4HNE by immuonoprecipitation of RGS4 from 4HNE treated cells. Further examination of recombinant RGS4 by mass spectrometry, revealed that RGS4 is readily modified at several cysteine residues by 4HNE, including C148. Modification at this residue has been shown to be a critical site for allosteric regulation of RGS4. This is confirmed through a malachite green based phosphate generation assay we developed to observe the GAP activity of RGS4 on its native binding partner GΑi. This malachite green based assay was then adapted for high throughput screening. The assay was successfully miniaturized to a 1536-well format. In a screen of 2300 compounds, 4 were identified as hits. The development of this simple and cheap assay can be adapted for usage with a variety of RGS proteins with little work to interrogate other pathways and identify novel RGS modulators. Finally, expansive study of PD has linked oxidative stress to the pathology of both diseases. What has not been discerned is the potential relationship between oxidative stress and the induction of RGS4. In support of the hypothesis, we evaluated the potential relationship between oxidative stress and RGS4 expression. This was accomplished by evaluating two striatal neuron like cell lines, SH-SY5Y and HCN-1A. After treatment with hydrogen peroxide, both cell lines showed increased RGS4 in response to oxidative stress. This response is not however related to mRNA expression, indicating this change is most likely an adjustment of proteasomal regulation of RGS4. This phenomenon may explain the rapid onset of Parkinsonian motor symptoms in reserpine treated animal models of PD, as excess dopamine in the cytoplasm may be rapidly metabolized in reactive products.

Substance abuse and mental health disorders are a leading source of years lost to disability from medical causes worldwide. Unfortunately, for most neurological disorders it is unclear how underlying genetic predispositions govern behavioral response to environmental stressors. Owing to their convenience, genetic tractability, and small brains, the fruit fly, Drosophila melanogaster, has become an invaluable model in which to dissect the neurological basis of conserved complex behaviors. Here, I characterized the respective roles of two genes in alcohol response and sleep behavior. Steroid hormones profoundly influence behavioral response to alcohol, yet the role of unconventional non-genomic steroid signaling in this process is unknown. I discovered that Drosophila DopEcR, a G-protein coupled receptor (GPCR) activated by dopamine or the major insect steroid hormone ecdysone, plays a critical role in ethanol-induced behaviors. DopEcR mutants took longer to sedate when exposed to ethanol vapor, and post-eclosion expression of DopEcR-RNAi phenocopied mutant resistance. DopEcR was necessary in particular neuronal subsets, including cholinergic and peptidergic neurons, and promoted ethanol sedation by suppressing epidermal growth factor/extracellular signal-regulated kinase signaling. In adult flies, ecdysone negatively regulated DopEcR-mediated ethanol-induced sedation. We also found that DopEcR inhibits ethanol-induced locomotion, a conserved dopaminergic behavior. Together, these findings provide novel insight into how an unconventional steroid GPCR interacts with multiple downstream signaling cascades to fine tune behavioral response to alcohol. Despite an established link between epilepsy and sleep behavior, it remains unclear how epileptogenic mutations affect sleep and seizure susceptibility. To address this, I examined the rest/wake behavior of two fly models of epilepsy with paralytic voltage-gated sodium channel mutations known to cause human generalized epilepsy with febrile seizures plus (GEFS+) and Dravet syndrome (DS). GEFS+ and DS flies display heat-induced seizure susceptibility, but at normal temperatures I found that both mutants had exaggerated nighttime sleep behavior. GEFS+ sleep was more resistant to pharmacologic and genetic reduction of GABA transmission as compared to control’s response. This finding is consistent with augmented GABAergic suppression of wake-promoting pigment-dispersing factor (PDF) neurons in GEFS+ mutants. Contrastingly, DS sleep was almost completely resistant to pharmacologic GABA reduction, suggesting that PDF neurons are incapable of functioning despite disinhibition. The sleep of both GEFS+ and DS flies was largely suppressed, but not eliminated, by scotophase light, highlighting the importance of light stimulus and circadian signals in the manifestation of their phenotypes. Following sleep deprivation, GEFS+ and DS mutants failed show to homeostatic rebound. Sleep loss also unexpectedly reduced the seizure susceptibility of GEFS+ flies. This study revealed the sleep architecture of Drosophila voltage-gated sodium channel mutants and provides a unique platform in which to further study the sleep/epilepsy relationship.

The Rho family of GTPases plays a crucial role in the regulation of diverse cellular processes, including proliferation and actin cytoskeletal rearrangement to promote cell migration. However, dysregulation of RhoGTPases has been associated with disease, particularly cancers such as leukemia. Despite this, RhoGTPases are rarely mutated in cancer. Rather, dysregulation of their regulatory proteins through mutation or overexpression contributes to disease pathogenesis. RhoGTPases are activated through Rho guanine nucleotide exchange factors (GEFs). Although over eighty RhoGEFs have been identified that activate the 25 RhoGTPases, the pathological role of the majority of these proteins remains unclear. Further, whereas the majority of RhoGEFs are activated through tyrosine phosphorylation, a small subset can be activated through heterotrimeric G proteins, including through GΒ;Γ; subunits. However, the mechanism by which GΒ;Γ; induces RhoGEF activation remains unclear. PLEKHG2 is a Dbl family RhoGEF that was originally identified as a gene upregulated in a leukemia mouse model, and later shown to be activated by heterotrimeric G protein Β;Γ; subunits. However, its function and activation mechanisms remain elusive. Here we show that, as compared to primary human T cells, the expression of PLEKHG2 is upregulated in leukemia cell lines. Downregulation of PLEKHG2 by siRNAs specifically inhibited GΒ;Γ;-stimulated Rac and Cdc42, but not RhoA activation. Consequently, inhibition of PLEKHG2 blocked actin polymerization, protrusion formation, and leukemia cell migration in response to SDF1alpha;. Additional studies indicate that GΒ;Γ; likely activates PLEKHG2 by binding the N-terminus of PLEKHG2. This interaction results in the release of autoinhibition imposed by the C-terminus within a region encompassing the catalytic DH domain. As a result, overexpressing either the N-terminus of PLEKHG2 that binds GΒ;Γ; or the C-terminus that autoinhibits PLEKHG2 blocked GΒ;Γ;-stimulated Rac and Cdc42 activation and the ability of leukemia cell to form membrane protrusions and to migrate. Together, our results have demonstrated that PLEKHG2 functions as a novel GΒ;Γ; -stimulated RhoGEF that could contribute to chemokine-induced leukemia cell dissemination and leukemia pathogenesis.

<p>The superfamily of G protein-coupled receptors (GPCRs) is one of the largest protein families of mammalian genomes and can be divided into five main families; <i>Glutamate</i>, <i>Rhodopsin</i>, <i>Adhesion</i>, <i>Frizzled</i>, and <i>Secretin</i>. GPCRs participate in most major physiological functions, contributing to the fact that they are important targets in drug discovery. In paper I we mined the human and mouse genomes for new <i>Adhesion</i> GPCR genes. We found two new human genes (GPR133 and GPR144) and 17 mouse <i>Adhesion</i> genes, bringing the number up to 33 human and 31 mouse genes. In paper II we describe 53 new splice variants for human <i>Adhesion</i> receptors supported by expressed sequence tags (EST) data. 29 of these variants seem to code for functional proteins, several of which lack one or more functional domains in the N-termini. Lack of certain domains is likely to affect ligand binding or interaction with other proteins. Paper III describes the <i>Glutamate</i> GPCR in human, mouse, <i>Fugu</i>, and zebrafish. We gathered a total of 22 human, 79 mouse, 30 <i>Fugu</i>, and 32 zebrafish sequences and grouped these into eight clans using phylogenetic methods. The report provides an overview of the expansion or deletions among the different branches of the <i>Glutamate</i> receptor family. Paper IV focuses on the trace amine (TA) clan of <i>Rhodopsin</i> GPCRs. We identified 18 new rodent genes, 57 zebrafish genes, and eight <i>Fugu</i> genes belonging to the clan. Chromosomal mapping together with phylogenetic relationships suggests that the family arose through several mechanisms involving tetraploidisation, block duplications, and local duplication events. Paper V provides a comprehensive dataset of the GPCR superfamily of human and mouse containing 495 mouse and 400 human non-olfactory GPCRs. Phylogenetic analyses showed that 329 of the receptors are found in one-to-one orthologous pairs, whereas other receptors may have originated from species-specific expansions.</p>