A Dissertation
Entitled
Are C. elegans receptors useful targets for drug discovery:
Identification of genes encoding seven potential biogenic amine
receptors in the parasitic nematode Brugia malayi and pharmacological comparison of tyramine receptor homologues from
Caenorhabditis elegans (TYRA-2) and B. malayi (Bm4)
By
Katherine Ann Smith
Submitted as partial fulfilment of the requirements for
The Doctor of Philosophy in Biology
______
Advisor: Dr Richard Komuniecki
______
College of Graduate Studies
The University of Toledo
May 2007 An Abstract of
Are C. elegans receptors useful targets for drug discovery: Identification
of genes encoding seven potential biogenic amine receptors in the parasitic
nematode Brugia malayi and pharmacological comparison of tyramine receptor
homologues from Caenorhabditis elegans (TYRA-2) and B. malayi (Bm4)
by
Katherine Ann Smith
Submitted as partial fulfilment of the requirements for
The Doctor of Philosophy in Biology
The University of Toledo
May 2007
Filarial nematodes, such as Brugia malayi, cause major health problems worldwide. At present, no vaccine against B. malayi is available and current chemotherapy is ineffective against adult parasites. Biogenic amines (BAs) regulate a number of key processes in nematodes, suggesting that nematode BA receptors may be useful targets for drug discovery. Therefore, we have used a bioinformatics approach to identify genes encoding putative B. malayi BA receptors, using BA receptors recently identified in the free-living nematode, Caenorhabditis elegans.
Surprisingly, the B. malayi genome appears to contain less than half of the genes predicted to encode BA receptors in the genomes of C. elegans or C. briggsae; however, all of the predicted B. malayi receptors have clear orthologues in C. elegans.
ii The B. malayi genes encode each of the major BA receptor subclasses, including three serotonin, two dopamine and two tyramine (TA) receptors. There is little overall synteny between the B. malayi and C. elegans genomes surrounding the genes encoding the predicted BA receptors; however, most of the intron/exon borders of orthologous BA receptor genes are conserved among the three species. Multiple BA receptor alignment and phylogenetic analysis suggests that potential ligand specificity and G-protein coupling of the individual receptors can be predicted via this bioinformatics approach. Further study of the seven B. malayi receptors identified a putative TA receptor (Bm4) and compared its pharmacology to its putative C. elegans orthologue, TYRA-2, under identical expression and assay conditions. In the present study, membranes from HEK-293 cells stably expressing Bm4 exhibited specific,
3 3 saturable, high-affinity [ H]LSD and [ H]TA binding with Kds of 18 ± 0.9 nM and 15
± 0.2 nM, respectively. More importantly, both TYRA-2 and Bm4 receptors exhibited similar rank orders of potencies for a number of potential tyraminergic ligands.
However, some significant differences were noted. For example, chloropromazine exhibited an order of magnitude higher affinity for Bm4 than TYRA-2 (pKis of 7.6 ±
0.2 and 6.49 ± 0.1, respectively). In contrast, TYRA-2 had significantly higher affinity for phentolamine than Bm4. These results highlight the utility of the nearly completed B. malayi genome and the importance of using receptors from individual parasitic nematodes for drug discovery.
iii DEDICATION
For my family:
Your love, support, encouragement and belief in me through this entire journey,
Thank you. This thesis is dedicated to them.
iv ACKNOWLEDGEMENTS
Many people at the University of Toledo have had a big impact upon my life and career and I would like to acknowledge them and say thank you. My advisor, Dr
Richard Komuniecki inspired me to take this challenging path and has guided me throughout my graduate career and helped me grow not just as a scientist but as a person. Thank you for never crossing the line, despite moving it so often! Dr. Patricia
Komunecki was responsible for introducing me to the Komuniecki lab, and under the initial guidance of Dr Emilio Duran, my enthusiasm for research was ignited. Both
Rick and Patsy have also helped me through a tough event here in Toledo, and I can never repay the kindness you both bestowed upon me, thank you both.
I would also like to acknowledge my committee members, Dr. J. Gray, Dr. W.
Messer, Dr. J. Plenefish and Dr. M. Funk. In particular, I would like to express gratitude to Dr. John Gray for his insight into bioinformatics. I would also like to thank Dr. Scott Leisner and Dr. Debera Vestal for the use of their computers.
Finally I would like to thank the Komuniecki lab members both past and present who provided a pleasant working environment. Dr. Emilio Duran and Dr.
Sally Harmych for their initial guidance and helping me become settled in the
Komuniecki lab. Dr. Hong Xiao for her advice and lastly Dr Elizabeth Rex, for her advice, training of culture assays, but mostly for her invaluable friendship.
v TABLE OF CONTENTS
ABSTRACT…………………………………………………………………………...ii
DEDICATION...... iv
ACKNOWLEDGEMENTS...... v
TABLE OF CONTENTS……………………………………………………...……...vi
LIST OF TABLES ………………………………………………………………….....x
LIST OF FIGURES …………………………………………………………………..xi
ABBREVIATIONS………………………………………………………………...... xii
CHAPTER I
OVERVIEW/SIGNIFICANCE...... …………………..………………………2
Brugia malayi………………………………………………………………….2
Caenorhabditis elegans as a model for parasitic nematodes………………….4
Biogenic amines as potential targets for anti-nematodal drug discovery……..5
G-protein coupled receptors………………………………………………….11
C. elegans BA GPCRs……………………………………………………….17
Bioinformatics………………………………………………………………..17
The Caenorhabditis and B. malayi genomes………………………………...20
HYPOTHESIS……………………………………………………………………….23
Hypothesis and Experimental Approach……………………………………..23
vi CHAPTER II
Genes encoding putative biogenic amine receptors in the parasitic nematode
Brugia malayi..……………………………………………….………24
ABSTRACT………………………………………………………………………….25
INTRODUCTION…………………………………………………………………...26
MATERIALS AND METHODS
Mining The Institute for Genomic Research (TIGR) B. malayi database for
GPCR homologous genes……………………………………...... 31
Mining the C. briggsae and C. elegans databases……………………………32
Screening of 5’ and 3’ RACE libraries from B. malayi………………………32
Gene Maps……………………………………………………………………34
Protein alignments……………………………………………………………34
Phylogenetic tree……………………………………………………………...35
RESULTS
Identification of genes encoding potential G-protein coupled biogenic amine
receptors in C. elegans, C. briggsae and B. malayi ………………….37
Identification of serotonin receptor homologs in B. malayi………………….38
Identification of tyramine receptor homologs in B. malayi……...... 42
Identification of dopamine receptor homologs in B. malayi………………….44
Phylogenetic relationships of biogenic amine receptors predict G-protein
coupling and ligand binding………………………………………….46
Identification of highly conserved residues amongst diverse GPCR
receptors………………………………………………………………51
vii Determining amino acids responsible for divergence in the phylogenetic
tree……………………………………………………………………52
DISCUSSION………………………………………………………………………..58
CHAPTER III
Are C. elegans receptors useful targets for drug discovery: Pharmacological
comparison of tyramine receptor homologues from Caenorhabditis elegans
(TYRA-2) and Brugia malayi (Bm4) ………………………………………...66
ABSTRACT…………………………………………………………………………..67
INTRODUCTION……………………………………………………………………68
METHODS AND MATERIALS
Materials……………………………………………………………………...71
Methods………………………………………………………………………71
Mining the TIGR B. malayi database for C. elegans TYRA-2 homologues…71
Screening of 5’ and 3’ RACE libraries from B. malayi………………………72
Gene Maps……………………………………………………………………73
Protein alignments……………………………………………………………73
Phylogenetic tree.……………………………………………………………..74
Cloning and sequencing Bm4 cDNA…………………………………………74
Expression of Bm4 in COS-7 and HEK-293 cell lines.………………………75
Immunoflorescence of cells stably expressing Bm4……………………….....76
Membrane preparation………………………………………………………..76
Radioligand binding assay……………………………………………………77
viii RESULTS
Cloning and sequence analysis of Bm4………………………………………78
Identification of Bm4, a putative C. elegans TYRA-2 homologue in
B. malayi...... 78
Characterization of Bm4 after heterologous expression in mammalian
cells…………………………………………………………………...81
Comparison of the pharmacological profiles of Bm4 and TYRA-2 ………...87
DISCUSSION………………………………………………………………………..91
REFERENCES……………………………………………………………………….96
ix LIST OF TABLES
Table 1: Comparison of C. elegans and putative B. malayi biogenic amine
receptor genes…………………………………………………...... ……...41
Table 2: Location of amino acid residues with greater than 50% conservation
amongst 98 BA GPCRs………………………………………………57
Table 3: Pharmacological profile of Bm4 and TYRA-2…………………………...…90
Table 4: Comparison of pharmacological profiles of Bm4 and TYRA-2……………90
x LIST OF FIGURES
Figure 1: Biosynthesis pathway of biogenic amines in C. elegans……………………6
Figure 2: Receptor activation states…………………………………………………..14
Figure 3: Gene maps and alignments of serotonin receptors…………………………39
Figure 4: Gene maps and alignments of tyramine and octopamine receptors………..43
Figure 5: Gene maps and alignments of dopamine receptors………………………...45
Figure 6: Unrooted phylogram of aligned biogenic amine G-protein coupled
receptors………………………………………………………………47
Figure 7: Alignment of the second intracellular loop of characterized G-protein
coupled biogenic amine receptors…………………………………….54
Figure 8: GPCR model of conserved amino acids………………………………...... 56
Figure 9. Gene maps and protein alignment for TYRA-2 and Bm4 ……...... ….80
Figure 10. Unrooted phylogenetic tree of TA and OA
receptors………………………...... 82
Figure 11. Alignment of characterized TA, OA and TAAR receptors……………….84
Figure 12. Saturation binding with membranes expressing Bm4 of [3H]LSD and
[3H]TA…………………………………….………………………….85
Figure 13. Pharmacological profile of Bm4………………………………………….88
Figure 14. Pharmacological profile of TYRA-2……………………………………...89
Figure 15. Chemical structure of chlorpromazine and phentolamine………………...95
xi ABBREVIATIONS
Aa Amino acids
Abs Antibodies
AC Adenyly cyclase
Ach Acetylcholine
β2-AR β2-adrenergic receptor
BA Biogenic amine
BAC Bacterial artificial chromosome
Bmax Maximal binding capacity
BSA Bovine serum albumin cAMP 3’5’-cyclic adenosine monophosphate cDNA Complimentary DNA
DA Dopamine
DAG Diacylglycerol
DGK Diaclglycerol kinase
DMEM Dulbeccos Modified Eagles Medium
EDTA Ethylenediamine tetraacetic acid
FBS Fetal bovine serum
FITC Fluorescein isothiocyanate
FlcDNA Full length cDNA
FLIPR Fluorometric imaging plate reader
GABA γ-aminobutyric acid
xii GFP Green fluorescent protein
G-protein Guanine nucleotide binding protein
Glu Glutamate
GPCRs G-protein coupled receptors
5-HT 5-hydroxytyramine
HEK Human Embryonic Kidney
IBMX Isobutylmethylaxanthine
IC50 50% inhibition concentration
IPs Inositol phosphates
Kd Dissociation constant
Ki Inhibition constant
KH buffer Krebs-Henseleit buffer
LSD Lysergic acid diethylamide
MSA Multiple sequence alignment
MSC Multiple cloning site nACh Nicotinic acetylcholine
NCBI National instute for health
NLS nuclear localization site
Nts Nucleotides
OA Octopamine
OTU Operational taxonomic unit
ORF Open reading frame
PBS Phosphate buffered saline
xiii PKA cAMP-dependant protein kinase
PKC Protein kinase C
PMSF Phenylmethylsulphonyl fluoride
PLC Phospholipase C
RT Room temperature
RT-PCR Reverse transcription-polymerase chain reaction
SEM Standard error of the mean
TA Tyramine
TAAR Trace amine associated receptor
TM Transmembrane domain
Tris 2-amino-2 (hydroxymethyl)-1,3-propandiol
WGS Whole genome shotgun
xiv CHAPTER I
Overview and Significance
1 Overview and Significance
Parasitic nematodes have a dramatic impact on many aspects of the human condition. For example, lymphatic filariasis is a major health problem striking people of all ages, inflicting both economic and social burdens on many tropical and subtropical countries (Anosike, Nwoke et al. 2005). Wuchereria bancrofti, Brugia timori and B. malayi infect 150 million people in 73 different countries, with 1 billion living in endemic areas at risk of infection (Williams, Lizotte-Waniewski et al. 2000;
Hoerauf 2006). The visual manifestations of the infection can be severe and lead to profound physical and psychological disabilities for the individual and for society
(Wamae 1994). Nematode infections are a major cause of human morbidity and contribute to a significant increase in Disability Adjusted Life Years (DALY, a summary measure that combines the impact of illness, disability and mortality on population health) with lymphatic filariasis having a 5.55 million DALY burden
(Molyneux 2003). Effective chemotherapy is still needed to control and potentially eliminate filarial infections, especially lymphatic filariasis caused by W. bancrofti, and river blindness caused by Onchocerca volvulus.
Brugia malayi
B. malayi is a parasitic filarial nematode that cycles between mosquito and humans. The study of human filariae is limited for obvious ethical reasons; therefore much of their life cycle in humans is unknown. For example, the proportion of inoculated larvae that develop, the route of migration, pairing of the sexes, control of
2 egg-laying and microfilarial migration have yet to be determined. What we do know about B. malayi life cycle is as follows; L3 move forward into the proboscis of the mosquitoes and, upon penetration of the human host skin, the L3 enter the lymphatic system and take from three to fifteen months to reach adulthood. Upon reaching sexual maturity, B. malayi mate and the female produces sheathed microfilariae (mf) that migrate into the blood. A second mosquito ingests mf from the blood of an infected host and once inside the mosquito gut, the mf shed their sheaths. Then the mf penetrate the mid-gut region, and migrate the wall of the proventriculus and cardiac portion of the mid-gut to the thoracic muscles, where they develop from L1 to L3. L3 ultimately migrate through the hemocoel to the proboscis in the head, ready to infect another host taking a blood meal. Therefore, in contrast to free-living nematodes that face a myriad of environmental variables, the entire filarial life cycle takes place within the relatively homeostatic environments of different hosts.
No vaccine against B. malayi is yet available and current chemotherapy cannot kill adults. Attempts to control filariasis have taken two approaches: 1) minimizing vector contact and 2) using chemotherapy to decrease the number of microfilaria and prevent transmission (Mak 1987). Unfortunately, current anthelmintics have adverse effects (diethylcarbamazine) and are showing levels of high resistance (ivermectin and albendazole) (de Silva, Guyatt et al. 1997). Since it is nearly impossible to work directly with B. malayi, the free living nematode, Caenorhabditis elegans, has been used as a model to characterize key proteins identified by the recent completion of the
B. malayi genome sequencing.
3 Caenorhabditis elegans as a model for parasitic nematodes
C. elegans is a soil-based, free-living, nematode that has been used extensively as a model to study signal transduction and the mode of action of many commercially available anthelmintics. C. elegans is useful because of its completely sequenced genome, its defined molecular genetics and the ready availability of signaling mutants.
Mutant phenotypes can often be rescued by the cell-specific expression of promoter::gfp translational fusions, permitting the detailed dissection of individual signaling pathways. Most importantly, from the perspective of drug development, all of the commercially available anthelminthics are also active in C. elegans, making it a useful model for the identification of additional targets for chemotherapy. However, nematodes exhibit enormous diversity and significant physiological/ biochemical/ molecular differences between nematode species have been clearly demonstrated
(Gomez-Escobar, Lewis et al. 1998; Murray, Manoury et al. 2005). Indeed, because of the enormous diversity among the nematodes, there is no guarantee that processes in C. elegans will be exactly duplicated in parasitic nematodes, highlighting the importance of the comparative bioinformatics and receptor characterization described below. In truth, C. elegans proteins are probably not good targets for high throughput screening for anthelminthic development, as binding constants and regulation can differ significantly among orthologues of different nematode proteins (Murray,
Manoury et al. 2005). However, it should be noted that all of the currently available anthelminthics are effective against C. elegans (Lochnit, Bongaarts et al. 2005).
4
Biogenic amines as potential targets for anti-nematodal drug discovery
In vertebrates, biogenic amines (BAs) such as dopamine (DA), 5- hydroxytryptamine (serotonin; 5-HT) and histamine, act physiologically as neurotransmitters, neuromodulators and neurohormones and regulate many key processes, including circadian rhythms, endocrine secretion, blood pressure, cardiovascular events, sexual behaviour, learning, thermoregulation, memory and pain
(Wolfgang, Hoskote et al. 2001). Biogenic amines also regulate a variety of key processes in invertebrates. Interestingly, invertebrates in general, and nematodes in particular, appear to lack an autonomic nervous system and adrenergic receptors. In fact, norepinephrine and epinephrine do not appear to be present in nematodes, as they lack the enzyme dopamine β-hydroxylase that converts DA to norepinephrine. In fact, the human dopamine β-hydroxylase precursor protein (NP_000778.3) is most identical to C. elegans TBH-1 (tyramine β-hydoxylase-1), an enzyme that converts tyramine (TA) to octopamine (OA) (Protein BLAST searches, NCBI). Instead, nematodes appear to use these BAs, TA and OA, to oppose the action of 5-HT in modulating many of the same pathways regulated by the autonomic nervous system in vertebrates.
5
O Tyrosine O L-tryptophan A B OH
OH
NH2 Tyrosine hydroxylase + NH2 HO TH cofactor Tyrosine decarboxylase 6-tetrahydrobiopterin Tryptophan hydroxylase (tdc-1) (tdc-1) (tph-1) 5-hydroxy- O Tyramine L-Dopa O tryptophan OH NH2 HO HO OH
NH2 NH2 HO HO Tyramine-hydroxylase L-dopa decarboxylase 5-hydroxy- tryptamine Aromatic L-amino acid (tbh-1) (bas-1) decarboxylase (5-HT or Serotonin) (bas-1) Octopamine Dopamine OH NH2 HO NH2 HO NH2
HO HO
Figure 1: Biosynthesis of biogenic amines utilized by nematodes.
Tyrosine decarboxylase (tdc-1) converts tyrosine to TA and then tyramine β- hydroxylase (tbh-1) hydroxylates TA to form OA. In addition, tyrosine hydroxylase
(cat-2) converts tyrosine into L-DOPA with the help of TH cofactor 6- tetrahydrobiopterin then aromatic L-amino acid decarboxylase converts L-DOPA into
DA. 5-HT is synthesized from L-tryptophan (Figure 1A). L-tryptophan is hydroxylated by tryptophan hydroxylase (tph-1) to 5-hydroxytryptophan which, in turn, is decarboxylated by aromatic L-amino acid decarboxylase (bas-1) to 5-HT
(Figure 1B).
TA and OA are major neurotransmitters in invertebrates and recently TA- and
OA-specific signaling pathways have been described in both insects and C. elegans
(Saudou, Amlaiky et al. 1990; Rex and Komuniecki 2002; Alkema, Hunter-Ensor et al. 2005). OA is synthesized in the RIC interneurons and gonadal sheath cells, based
6 on the expression of tbh-1 and, in many cases, its effects are opposite to those observed for 5-HT (Alkema, Hunter-Ensor et al. 2005). For example, OA enhances locomotion, suppresses egg-laying on bacteria or 5-HT and inhibits pharyngeal pumping (Horvitz, Chalfie et al. 1982; Segalat, Elkes et al. 1995; Rogers, Franks et al.
2001; Niacaris and Avery 2003). Specifically, OA appears to increase the activity of the inhibitory M3 pharyngeal motor neuron and thus the frequency of the action potentials inhibiting pharyngeal pumping (Niacaris and Avery 2003). TA is an OA precursor, but recent studies have indicated that TA is a neuromodulator in its own right. For example, G-protein coupled receptors with high affinity for TA have been identified in both nematodes and insects (Saudou, Amlaiky et al. 1990; Rex and
Komuniecki 2002). In addition, cells (RIM and UV1) that appear to express tdc-1, but not tbh-1, have been identified in C. elegans. These cells cannot convert TA to OA and are presumably tyraminergic (Alkema, Hunter-Ensor et al. 2005). The unique physiological role of TA in C. elegans has been uncovered by examining tdc-1 and tbh-1 null mutants that are unable to synthesize TA/OA and TA, respectively. tdc-1, but not tbh-1, mutants do not suppress head oscillations in response to anterior touch, suggesting a specific role for TA in this behaviour (Alkema, Hunter-Ensor et al.
2005). TA also appears to specifically inhibit egg-laying and modulate reversal behaviour (Alkema, Hunter-Ensor et al. 2005). However, the phenotypes of these null mutants are difficult to interpret, as the tbh-1 null animals also have dramatically elevated TA levels, so that additional studies using animals lacking individual TA and
OA receptors will be necessary to confirm these observations (Alkema, Hunter-Ensor et al. 2005).
7 TA and OA also appear to function independently in insects. For example, in
Apis mellifera (the honeybee) TA or manserin (an OA receptor antagonist) decreases flight activity, while OA increases flight activity (Fussnecker, Smith et al. 2006).
Interestingly, OA levels are higher in forager bees than in bees engaged in brood care, and increased levels of TBH-1, the enzyme responsible for OA synthesis, are associated with social behaviour (Lehman, Schulz et al. 2006). OA levels are higher in the antennal lobes of foragers where OA receptors also have been identified
(Wagener-Hulme, Kuehn et al. 1999; Grohmann, Blenau et al. 2003; Farooqui,
Vaessin et al. 2004). OA causes bees to forage precociously and to be more responsive to foraging-related stimuli, such as learning and memory (Barron, Schulz et al. 2002; Farooqui, Robinson et al. 2003; Barron and Robinson 2005; Lehman,
Schulz et al. 2006). In Drosophila melanogaster (the fruit fly), TA and OA receptors have opposite effects on adenyl cyclase activity, with OA stimulating and TA deceasing cAMP levels, respectively (Arakawa, Gocayne et al. 1990; Saudou,
Amlaiky et al. 1990; Han, Millar et al. 1998). Flies lacking tbh-1 are egg-laying defective and, as predicted, this phenotype is partially rescued by feeding OA
(Monastirioti, Linn et al. 1996). OA also plays a role in flight, including the activation of flight motor patterns and the upregulation of glycolysis for energy generation
(Mentel, Duch et al. 2003). TA but not OA appears to be involved in sensitization to cocaine (McClung and Hirsh 1998; McClung and Hirsh 1999). For example, iav mutants have low TDC activity and TA levels in the brain and fail to become sensitized to cocaine. Again, as predicted, this phenotype can be rescued by feeding
TA, but not by feeding other biogenic amines (McClung and Hirsh 1998; McClung
8 and Hirsh 1999). Importantly, OA receptors have been useful targets for pesticide development. For example, the formamidine pesticides, such as amitraz, mimic the action of OA in octopaminergic systems, suggesting that TA and OA receptors may make potentially useful drug targets in nematodes (Hashemzadeh et al. 1987; Baxter and Barker 1999).
Serotonin modulates many essential functions in invertebrates, such as aggression in lobsters and learning in snails. In C. elegans, 5-HT modulates locomotion, feeding, egg-laying, mating and a number of more complex behaviours, including aversive learning and olfaction. Serotonin is found in nine hermaphrodite neurons: the NSMs, HSNs, ADFs, AIMs, RIHs (Sawin, Ranganathan et al. 2000; Sze,
Victor et al. 2000; Hare and Loer 2004; Zeng, Yu et al. 2005). The NSMs are the only serotonergic neurons in the pharynx and appear to have sensory endings in the lumen capable of sensing bacteria and signaling food. These sensory endings are also capable of releasing 5-HT into the lumen, facilitating wider 5-HT effects (analogous to the release of epinephrine from the adrenal medulla?). However, ablation of the
NSMs has only subtle effects on pharyngeal pumping, suggesting that other serotonergic neurons also are involved (Albertson and Thomson 1976; Avery 1993).
The HSNs synapse onto the vm2 muscles of the vulva and play a key role in regulating egg-laying. Interestingly, the HSNs release acetylcholine as well as at least one peptide, in addition to 5-HT. In fact, almost all neurons in C. elegans release multiple neurotransmitters.
Serotonin levels in C. elegans are predicted to increase in the presence of bacteria and serve as the “food is at hand” signal, modulating most food-related
9 behaviours. For example, 5-HT induces an enhanced slowing response, observed when C. elegans encounter a bacterial lawn (Horvitz, Chalfie et al. 1982). In addition,
5-HT increases the rate of pharyngeal pumping through the stimulation of the MC and
M3 pharyngeal motor neurons, allowing rapid cycles of contraction and relaxation, which increase the overall rate of pumping (Niacaris and Avery 2003). 5-HT also stimulates the M4 pharyngeal motor neuron and co-ordinately increases isthmus peristalsis facilitating feeding and insuring the bacteria make their way to the intestine
(Croll 1975; Horvitz, Chalfie et al. 1982; Avery 1993; Niacaris and Avery 2003). 5-
HT stimulates egg-laying by switching vulval muscle from an inactive phase and increases the contraction frequency of vulval muscle (Horvitz, Chalfie et al. 1982;
Trent, Tsuing et al. 1983; Desai, Garriga et al. 1988; Waggoner, Zhou et al. 1998; Sze,
Victor et al. 2000; Waggoner, Hardaker et al. 2000; Kindt, Tam et al. 2002; Sze,
Zhang et al. 2002; Shyn, Kerr et al. 2003). Serotonin is also involved in mating. For example, 5-HT increases male tail curling and posterior body bends (Loer and Kenyon
1993; Duerr, Frisby et al. 1999). Serotonin also modulates synaptic transmission at neuromuscular junctions and inhibits the release of acetylcholine through Gαo dependent pathway (Horvitz, Chalfie et al. 1982; Segalat, Elkes et al. 1995;
Weinshenker, Garriga et al. 1995; Nurrish, Segalat et al. 1999). Serotonin also has been implicated in a number of other behaviours, including foraging, carbohydrate metabolism, neuroendocrine signaling in metabolic control, fat storage, entrance into the diapause-like dauer stage; aversive learning, touch avoidance and defecation
(Segalat, Elkes et al. 1995).
10 Dopamine is synthesized in eight sensory neurons in the hermaphrodite, two anterior deirid neurons (ADEs), two posterior deirid neurons (PDEs) and four cephalic neurons (CEPs) and an additional six neurons in the male, three pairs of sex- specific dopaminergic neurons exist in the tail R5A, R7A and R9A (Sulston, Dew et al. 1975;
Wintle and Van Tol 2001; Suo, Ishiura et al. 2004). DA inhibits locomotion, egg- laying defecation and feeding in the wild type (WT) C. elegans (Weinshenker, Garriga et al. 1995; Schafer 1999). DA is responsible for regulating locomotion in well-fed C. elegans, by slowing their movement when they encounter a bacterial lawn (Chase,
Pepper et al. 2004). Dopamine also plays a role in foraging behaviour (Hare and Loer
2004).
G-protein coupled receptors
G-protein coupled receptors (GPCRs) are activated by a wide variety of ligands, including BAs, and are the largest family of membrane receptors (Moro,
Deflorian et al. 2006; Li, Scarselli et al. 2007). Upon ligand binding, GPCRs activate specific heterotrimeric guanine nucleotide-binding proteins (G-proteins) and initiate a variety of downstream second messenger-mediated signaling cascades. GPCRs share common structural motifs, including seven transmembrane (TM) hydrophobic helical regions with an extracellular N-terminus, often N-glycosylated, three extracellular and three intracellular loops (IL) with the posttranslational palmitoylation of cysteines in the C-terminus generating a fourth intracellular loop and an intracellular C-terminus.
GPCRs are located in the plasma membrane with the TM regions forming a ligand- binding pocket from tight bundles among TM’s III, V, VI and VII. Conventional
11 numeration of the amino acid (aa) positions in TM helical domains uses conserved residues in TMs I-VII. The aas within the 5-HT receptor family have been studied extensively. 5-HT activates both GPCRs and ligand-gated ion channels. For example, in humans 5-HT receptors are divided into seven families: six GPCRs and one, 5-HT3, a Cl- gated anion channel (Kroeze, Kristiansen et al. 2002). Human 5-HT receptors have 33 aa that are 100% conserved and 27 that are 80% conserved. The 33 aa that are 100% conserved are involved in ligand binding and G-protein coupling, desensitization and receptor trafficking (Kroeze, Kristiansen et al. 2002). Amino acids at the ends of individual TM helixes are either positively charged or a W. A P at
1.30 and 1.31 in all 5-HT receptors initiates the first α-helix.
• TM I has 2 conserved aa: N1.50 and L1.63.
• The first intracellular loop is between 6-7 aa in length and a P2.38 or 2.39
initiates the helix of TM II.
• TM II has 4 conserved aa: S2.45, D2.50, V2.57 and P2.59, with L2.46, 95%
conserved.
• The first extracellular loop is 6-7 aa in length and has a conserved W at 2.70.
• TM III has conserved residues: C3.25, D3.32, S3.39, I3.40, I3.46, D3.49
R3.50, Y3.51, the DRY motif at the end of TM III and at the beginning of
intracellular loop two is responsible for binding G-protein α subunits. The
four hydrophobic aa from 3.51 to 3.54 suggest that TM III protrudes into the
cytoplasmic space.
• The second intracellular loop is between 9-10 aa, residue 3.58 is hydrophobic,
67% of 3.57 is a P and T/S4.38 is the capping of TM IV helix.
12 • In TM IV only W4.50 is conserved.
• The second extracellular loop is between 19 to 33 aa and C5.33 is conserved.
• In TM V, P/G5.37, F5.47, P5.50 and Y5.58 are conserved with 24%K 16%Q
and 60% R at position 5.66.
• The third intracellular loop varies greatly between 23 and 106 aa and has a
span of positively charged aa in the lipid head group environment close to TM
VI, 6.29, 6.31, 6.32, 6.35 with 11 of the 12 human 5-HT receptors containing
E6.30.
• TM VI and TM VII are the most highly conserved with F6.44, W6.48, P6.50,
F6.51, and F6.52 conserved in TMVI and W7.40, G7.42, Y7.43, S7.46, N7.49,
P7.50 and Y7.53 in TM VII. The NPxxY motif in TMVII is responsible for
sequestration and desensitization of the receptor.
• The C-terminal varies in length and a putative palmitoylation site may be
present at 7.70.
Amino acids essential for ligand-binding also have been identified previously. For example, mutations in TM III and TM VII of the Drosophila OA/TA receptor (S194A and S576A, respectively) reduce coupling to the inhibition of adenyl cyclase without altering ligand affinity (Chatwin, Rudling et al. 2003). In addition, D3.32 and
SS5.42/5.43 are involved in DA binding to rat D2 receptor and that D3.32, S5.42 and
F6.52 are responsible for 5-HT binding in 5-HT1A, 5-HT2A, 5-HT2B 5-HT4, 5-HT6, 5-
HT1A/B and 5-HT1B, respectively (Livingstone, Strange et al. 1992; Pollock, Manelli et al. 1992; Kroeze, Kristiansen et al. 2002). A D2.50A mutation abolishes 5-HT binding in the human 5HT1A receptor and is thought to interfere with hydrogen
13 bonding between N7.49, since a double mutation D2.50A/N7.49D restores 5-HT binding. This observation also suggests that D2.50 and N7.49 are adjacent within the plasma membrane and aid in the formation of the ligand-binding pocket (Collins
1993; Schwinn 1993; Bouvier, Moffett et al. 1995). Mutational analysis of TA receptors indicates that a conserved motif, S1S/AxxS5, within TM V of the B. mori TA receptor is essential for [3H] TA-specific binding and the attenuation of cAMP levels
(Ohta, Utsumi et al. 2004).
GPCRs are activated by ligand binding, resulting in the activation of G- proteins. GPCRs exist in an equilibrium between two states, an inactive (R) and a spontaneous active conformation (R*) that couples to G-proteins in the absence of ligand (Figure 2).
R R*
RL R*L
Figure 2: Receptor activation states. R, inactive receptor;
R*, active receptor; L, ligand.
Agonists and antagonists have different affinities for GPCRs that are dependent upon the activation state of the receptor. Antagonist binding is independent of G-protein coupling and antagonist affinity is not affected by receptor state. High affinity agonists bind to receptors in the active state (coupled to G-proteins; R*) and
14 low affinity agonists bind to inactive receptors (R) (Bond, Leff et al. 1995; Tate and
Grisshammer 1996).
Ligand binding and signal transduction can be dramatically affected by cell type, receptor density and the presence of additional signaling elements (Tate and
Grisshammer 1996; Gudermann, Schoneberg et al. 1997). In addition, over 100
GPCRs exhibit a variety of additional modifications, including N-linked glycosylation, disulfide formation and phosphorylation, which are essential for correct protein folding, expression and coupling. Thus, heterologous expression systems used to characterize GPCRs need to faithfully reflect the receptor’s physiological milieu and contain not only the appropriate G-proteins and accessory signaling elements, but also the ability to catalyze the appropriate protein modifications to insure that ligand- binding and downstream signaling truly reflect the properties of the native receptor
(Tate and Grisshammer 1996). Finally, the concentrations of the receptor and associated signaling elements (for example, G-proteins, adenyl cyclase, scaffold proteins) may also affect ligand affinity and coupling by altering functional receptor expression and/or the proportion of the receptors in an active conformation.
GPCRs function physiologically as dimers (Devi 2000; Elmhurst, Xie et al.
2000; Prinster, Hague et al. 2005). The concept of receptor dimerization was first proposed by Agnati et al. in 1982, but was not widely accepted until Kaupmann et al. demonstrated the functional heterodimerization of GABAB isoforms, GABABR1 and
GABABR2, both indispensable for the formation of functional GABAB receptor binding sites (Jones, Borowsky et al. 1998; Kaupmann, Malitschek et al. 1998; White,
Wise et al. 1998). Many different GPCRs have the ability to form either homo- or
15 heterodimers between different receptors. For example, mammalian 5-HT-1B and 5-
HT1D receptors form homodimers when expressed individually, but when co- expressed also form heterodimers. More importantly, when expressed alone, 5-HT-1B and 5-HT1D form homodimers and initiate separate signaling cascades. However, the co-expression of 5-HT-1B and 5-HT1D eliminates homodimer formation and the receptors form heterodimers exclusively, activating a third unique signaling cascade.
Regulation of receptor function and specific signaling therefore can be controlled not only via homo- and hetero-dimerization, but also by cell specific expression, as 5-
HT1D receptors expressed alone or co-expressed with 5-HT1B are found in different regions of the brain (Salim, Fenton et al. 2002). Other receptors that form functional heterodimers with 5-HT receptors include 5-HT1A, 5-HT1B and 5-HT1D (Salim, Fenton et al. 2002), D2 dopamine receptor (Lee, O'Dowd et al. 2003), neuropeptide Y receptor (Dinger and Beck-Sickinger 2002) and the 5-TH2C receptor (Herrick-Davis,
Grinde et al. 2004), or as heterodimers between the mammalian 5-HT1B and the 5-
HT1D receptors (Xie, Lee et al. 1999), 5-HT1A with 5-HT1B, 5-HT1D, EDG3, GPR26 and
GABAB2 receptors, 5-HT1D with 5-HT1B and EDG1 receptors (Salim, Fenton et al.
2002) and adenosine A2A and dopamine D2 receptors (Canals, Marcellino et al. 2003).
Agonist stimulation can increase the homodimerization of the β2-adrenergic (Herbert and Flugge 1995) and the TRH receptors (Kroeger, Hanyaloglu et al. 2001), decrease homodimerization in the δ-opioid receptor (Cvejic and Devi 1997) or have no apparent effect, as in the M3 muscarinic receptor (Zeng, Soldner et al. 1999; Zeng and
Wess 1999). Receptor dimerization appears to be constitutive (activity in the absence of ligand), occurring early in biosynthesis within the endoplasmic reticulum (ER) and
16 could play a pivotal role in ER export and quality control (Issafras, Angers et al.
2002). 5-HT2c receptors form dimers in the ER and truncated or mutant receptors can be trapped within the ER (Grosse, Schoneberg et al. 1997; Zhu and Wess 1998; Karpa,
Lin et al. 2000; Lee, O'Dowd et al. 2000).
C. elegans BA GPCRs
At least 15 putative BA receptors have been identified in the C. elegans genome [see Figure 6 (Olde and McCombie 1997; Hamdan, Ungrin et al. 1999;
Hobson, Geng et al. 2003) ]. More importantly, many C. elegans BA receptors have been pharmacologically characterized and their individual roles at least partially described through the use of null mutants prepared by different knockout consortia.
C. elegans contains three 5-HT receptors SER-1, SER-4 and SER-7 each coupling to different G-proteins Gαq, Gαi/o and Gαs, respectively (Olde and McCombie 1997;
Hamdan, Ungrin et al. 1999; Hobson, Geng et al. 2003). C. elegans have four DA receptors: DOP-1, DOP-2, DOP-3 and DOP-4, with DOP-1 and DOP-2 coupling to
Gαs and DOP-3 and DOP-4 coupling to Gα i/o (Sanyal, Wintle et al. 2004; Suo,
Ishiura et al. 2004). Two Gαi/o coupled TA receptors SER-2 and TYRA-2 have also been characterized (Rex and Komuniecki 2002; Rex, Hapiak et al. 2005). In addition, insects contain Gαs-coupled OA receptors (Gerhardt, Bakker et al. 1997) and candidate OA receptors have been identified in C. elegans SER-3 and F14D12.6 (Suo,
Kimura et al. 2006).
Bioinformatics
17 Bioinformatics is essential for the processing and interpretation of the wealth of sequence data generated with the explosion of the “genome era.” The creation of
BAC (bacterial artificial chromosome) vectors with the ability to hold up to 350 Kb of
DNA greatly facilitated the sequencing of many genomes. More recently, with the development of automated sequencing methods and upgraded analysis software, a whole genome shotgun (WGS) approach has been developed. The entire genome is broken into fragments, sequenced at random and then overlaps in the sequences are used to assemble the genome. Two libraries of different sizes, 2000 bp and 10,000 bp, are often constructed using this approach to provide complementary/overlapping data.
In fact, to date, over 180 genomes have been fully sequenced and more are on the way. In turn, bioinformatic analyses of the rapidly expanding sequence database have also expanded to keep pace. For example, the number of gene sequences entered into
Genbank increased from 1.6 x109 in December of 1997 to 30 x109 in April of 2004
(Mount et al. 2004).
The primary task for bioinformatics is the analysis and handling of DNA sequences and can be divided into two areas: functional genomics and comparative genomics. Functional genomics examines the role of a particular sequence within a cell (e.g. promotor or regulatory element) or the role of its predicted protein.
Comparative genomics compares sequences from different organisms or individuals to determine ancestries and/or correlations with specific variables. With the growing number of genomic databases, covering DNA, EST and protein sequences, the ability to compare unknown sequences is relatively straightforward. However, these analysis systems come with specific caveats. For example, the function of many predicted
18 genes still remains to be determined, in spite of the diversity of the genomes already sequenced. About a third of new sequences have no significant similarity to known sequences within existing databases (Ramsden, 2004). More importantly, although
180 genomes have been sequenced and DNA, EST and protein sequences loaded into
GenBank, many have yet to be fully annotated. Annotation is still a lengthy process.
Much of the initial annotation can be done via computer and recently programs have been developed to predict species-specific intron/exon borders. However, these programs often lead to the misidentification of intron/exon borders or the identification of pseudogenes. Also, errors can occur in depositing sequences within the databases, either via sequencing errors in the amplification of the genome or through misentry via the keyboard. Full annotation is only possible through the expression and characterization of each individual gene. That said, as long as these caveats are understood, sequence databases and bioinformatics can be invaluable tools in predicting function.
The comparison of “like” protein sequences via alignments enables the identification of domains with high identity and also for a prediction of divergence
(the better the alignments, the better the divergence predictions). However, it must be remembered that these are only predictions and even though two proteins may have a high identity, they can still differ functionally. For example the B. malayi cystatin,
Bm-CPI-2, has 21% identity to a C. elegans cystatin, Ce-CPI-2, and 18% to H. sapiens cystatin C. However, Bm-CPI-2 inhibits AEP (C13 legumain-like asparapinyl endopeptidase) like its human counterpart, but Ce-CPI-2 does not (Murray, Manoury et al. 2005). Taken together, these observations suggest that bioinformatics alone can
19 only predict function and modern science is now moving into an era where predictive bioinformatics is being coupled to effective bench science.
The Caenorhabditis and B. malayi genomes
The genealogy of the nematode phylum, based on cytochrome c and globin amino acid sequence, suggests that the primordial nematode ancestor diverged from a line leading to mammals one billion years ago, and that the most recent common ancestor of extant species lived about 550 million years ago. The rhabditids
(Rhabditina, clade V: C. elegans; Spirurina, clade III: B. malayi) and strongylids diverged as one branch from this nematode ancestor and the ascarids as another branch (Vanfleteren, Van de Peer et al. 1994). The Rhabditina and Spirurina appear to have diverged 450 million years ago (Vanfleteren, Van de Peer et al. 1994; Ghedin,
Wang et al. 2004)
C. elegans was one of the first eukaryotes to have its genome fully sequenced
(The C. elegans Sequencing Consortium, 1998). The complete genome sequence spans 100.3 Mb and has been extensively annotated using both bioinformatics and functional genomic approaches (Stein 2001). Within the nematodes, comparative genomics has been limited to Caenorhabditis, (with only C. elegans and C. briggsae having completed, annotated genomes), yet nematodes exhibit enormous diversity. B. malayi is a parasitic nematode whose genome is almost completely sequenced and is currently under annotation. Initially thought to be of a comparable size to both the C. elegans and C. briggase genomes (100.3 and 104 Mb, respectively), the B. malayi genome is now estimated to be between 85 and 95 Mb (Ghedin, Wang et al. 2004). B.
20 malayi has five chromosomal pairs, one less than C. elegans, four autosomal pairs and one X-Y sex determining pair (Sakaguchi, Tada et al. 1983). It is hypothesized that the different chromosome number in B. malayi and C. elegans may result from the fusion of two ancestral chromosomes (Whitton, Daub et al. 2004). Comparison of B. malayi BAC end sequences with predicted C. elegans proteins was used to determine whether the C. elegans and B. malayi genes were on the same chromosomes.
Interestingly, for instance when the matching C. elegans sequences were on separate chromosomes, they did not have a random distribution. For example, 39% of the unlinked matches at one end of chromosome I had their match at the end of chromosome III, and 38% of the unlinked matches on chromosome III had their partner at the end of chromosome I. This unlinked match-pair pattern indicates that either a rare translocation transferred some of the genes between two ancestral chromosomes or the retention of a chromosome that has now split in C. elegans, explaining the smaller chromosomal number in B. malayi (Whitton, Daub et al. 2004).
The genomes of C. elegans and B. malayi have been compared previously for the presence of specific genes, (Streit, Li et al. 1999; Murray, Manoury et al. 2005), or for overall synteny (Blaxter, Aslett et al. 1999; Guiliano, Hall et al. 2002; Lee,
O'Dowd et al. 2003; Stein, Bao et al. 2003). Surprisingly, comparison of B. malayi
BAC sequences with the C. elegans and C. briggsae genomes identified 2789 new B. malayi protein-coding genes, not present in either C. elegans or C. briggsae, highlighting the diversity within the nematodes (Whitton, Daub et al. 2004). The 26,
000 B. malayi ESTs have been clustered into about 8,000 genes. Many of these genes have few apparent homologues in the database, preventing any functional predictions,
21 but strongly suggesting that many of these genes are unique to B. malayi (Ghedin,
Wang et al. 2004). For example, it appears that 459 genes have been lost from the caenorhabditid lineage since the two free-living nematodes diverged from their last shared common ancestor with B. malayi (Ghedin, Wang et al. 2004).
At least 75 Mb of the B. malayi nuclear genome have been assembled into large regions known as scaffolds, the largest scaffolds ranging from 1 to 3 Mb
(Ghedin, Wang et al. 2004). Scaffolds and long contiguous fragments have been used to study the overall arrangement of the B. malayi genome and to compare it to that of
C. elegans. Initial investigations into potential genomic synteny between the two species indicate that adjacent genes in B. malayi are commonly 2-10 Mb apart in C. elegans (Ghedin, Wang et al. 2004). Similar observations have previously been made through mapping B. malayi BAC ends to C. elegans chromosomes (Guiliano, Hall et al. 2002; Whitton, Daub et al. 2004). These results suggest that, surprisingly, the two genomes appear to exhibit little overall synteny. In contrast, comparison of a 65 kb B. malayi genomic fragment with C. elegans found that 6-7 predicted genes in this region were conserved in both species, suggesting that the organization of smaller regions of the two genomes may be more similar (Blaxter, Aslett et al. 1999). Not surprisingly, intrachromosomal rearrangements appear to be common, even among more closely related nematodes, such C. elegans, C. briggsae and Pristionchus pacificus (Lee,
Eizinger et al. 2003; Stein, Bao et al. 2003).
22 Hypothesis and Experimental Approach
As noted above, nematode BA receptors appear to be potentially useful targets for anthelminthic drug discovery. Therefore, we sought to identify genes encoding putative BA receptors in the recently completed B. malayi genome using a bioinformatics approach. In addition, with the availability of completely sequenced genomes from two caenorhabditid species and the near completion of the sequencing of the B. malayi genome, we propose to compare the structures and conservation of genes encoding BA receptors in these genomes. Most importantly, since the pharmacology and coupling of BA receptors from B. malayi and other parasitic nematodes are likely to differ from those recently characterized from C. elegans, we also propose to heterologously express a B. malayi BA receptor and its predicted C. elegans orthologue to examine their pharmacologies under identical expression and assay conditions to estimate how useful C. elegans BA receptors might be as models for drug discovery.
23 CHAPTER II
Genes encoding putative biogenic amine receptors in the parasitic nematode
Brugia malayi
24
Abstract
Filarial nematodes, such as Brugia malayi, cause major health problems worldwide. At present, no vaccine against B. malayi is available and current chemotherapy is ineffective against the adult. Biogenic amines (BAs) regulate a number of key processes in nematodes, suggesting that nematode BA receptors may be useful targets for drug discovery. Therefore, we have used a bioinformatics approach to identify genes encoding putative B. malayi BA receptors. Surprisingly, the B. malayi genome appears to contain less than half of the genes predicted to encode BA receptors than in the genomes of the free-living nematodes,
Caenorhabditis elegans or C. briggsae; however, all of the predicted B. malayi receptors have clear orthologues in C. elegans. The B. malayi genes encode each of the major BA receptor subclasses, including three serotonin, two dopamine and two tyramine receptors. The B. malayi and C. elegans genomes exhibit little synteny in regions surrounding the predicted BA receptor genes; however, the structure of orthologous BA receptor genes is conserved among the three species. Multiple sequence alignments of BA receptors, coupled with phylogenetic analyses, suggest that potential G-protein coupling and ligand-specificity of the individual receptors can be predicted via this bioinformatics approach. Our results provide a framework for the analysis of G-protein coupled receptors that may be targeted for drug development in medically-important parasitic nematodes.
25 Introduction
Parasitic nematodes have a dramatic impact on many aspects of the human condition. For example, lymphatic filariasis is a major health problem, imposing both economic and social burdens on many tropical and subtropical countries (Anosike,
Nwoke et al. 2005). Wuchereria bancrofti, Brugia timori and B. malayi infect 150 million people in 73 different countries, with 1 billion more living in endemic areas at risk of infection (Williams, Lizotte-Waniewski et al. 2000; Hoerauf 2006). Nematode infections are a major cause of human morbidity and contribute to a significant increase in Disability Adjusted Life Years (DALY, a summary measure that combines the impact of illness, disability and mortality on population health) with lymphatic filariasis having a 5.55 million DALY burden (Molyneux 2003). Effective chemotherapy is still needed to control and potentially eliminate filarial infections, especially lymphatic filariasis and river blindness. B. malayi cycles between mosquitoes and humans. No vaccine against B. malayi is available and chemotherapy is ineffective in controlling the adult parasite. In addition, anthelmintics directed against larval stages have adverse side effects (diethylcarbamazine) or developing resistance (ivermectin and albendazole)(de Silva, Guyatt et al. 1997).
Since B. malayi is difficult to culture and resistant to molecular genetics approaches, the free-living nematode Caenorhabditis elegans has been proposed as a useful model to characterize potential drug targets identified in the recently completed
B. malayi genome (Ghedin, Wang et al. 2004). C. elegans has been used extensively as a model to study signal transduction and the mode of action of many commercially
26 available anthelminthics. C. elegans is especially useful because of its completed genome, its defined molecular genetics, and the ready availability of signaling mutants. Mutant phenotypes can often be rescued by the cell-specific expression of promoter::gfp translational fusions, permitting the detailed dissection of individual signaling pathways. Most importantly, from the perspective of drug development, all of the commercially available anthelminthics are also active in C. elegans. However, nematodes exhibit enormous diversity and significant physiological, biochemical, and molecular differences between nematode species have been clearly demonstrated
(Gomez-Escobar, Lewis et al. 1998; Murray, Manoury et al. 2005). The nematode ancestor appears to have diverged from a line leading to mammals about 1 billion years ago, and the most recent common ancestor of all extant nematode species lived about 550 million years ago. The rhabditids (including Rhabditina, clade V: C. elegans Spirurina, clade III: B. malayi) and strongylids diverged as one branch from this ancestor and the ascarids, including B. malayi as another (Vanfleteren, Van de
Peer et al. 1994). Indeed, given the significant time for divergence between extant nematodes, processes in C. elegans may not be exactly duplicated in parasitic nematodes, highlighting the importance of the comparative bioinformatic studies reported in this study.
The genomes of C. elegans and B. malayi have been compared previously for the presence of specific genes, from EST databases, and BAC, (Streit, Li et al. 1999;
Murray, Manoury et al. 2005), or for overall synteny (Blaxter, Aslett et al. 1999;
Blaxter, Daub et al. 2002; Guiliano, Hall et al. 2002; Lee, Eizinger et al. 2003; Stein,
Bao et al. 2003). Surprisingly, a comparison of B. malayi BAC sequences with the C.
27 elegans and C. briggsae genomes identified 2789 new B. malayi protein-coding genes, not present in either C. elegans or C. briggsae, highlighting the diversity within the nematodes (Whitton, Daub et al. 2004). The 26,000 B. malayi ESTs have been clustered into about 8,000 genes. Many of these genes do not have clear homologues in the database, preventing any functional predictions, but strongly suggesting that many of these genes are unique to B. malayi (Ghedin, Wang et al. 2004). For example, 459 genes appear to have been lost from the caenorhabditid lineage since the two free-living nematodes diverged from their last shared common ancestor with B. malayi 550 million years ago (Ghedin, Wang et al. 2004).
In vertebrates, biogenic amines (BAs) such as dopamine (DA), serotonin (5-
HT), epinephrine, norepinephrine and histamine act physiologically as neurotransmitters, neuromodulators and neurohormones to regulate many key processes. In contrast, norepinephrine and epinephrine do not appear to be present in nematodes, as they lack dopamine β-hydroxylase that converts DA to norepinephrine.
In fact, the human dopamine β-hydroxylase precursor protein (NP_000778.3) is most identical to C. elegans TBH-1, an enzyme that converts tyramine to octopamine
(BLAST searches, NCBI). Instead, nematodes appear to use the biogenic amines, tyramine (TA) and octopamine (OA), to oppose the action of 5-HT in modulating many of the same pathways regulated by the autonomic nervous system in vertebrates
(Rogers, Franks et al. 2001; Niacaris and Avery 2003; Alkema, Hunter-Ensor et al.
2005; Roeder 2005), suggesting that nematode BA receptors may be useful targets for anthelminthic drug discovery. Therefore, the present study focused on using comparative genomics to identify the genes encoding putative BA receptors in C.
28 elegans, C. briggsae and the parasitic filarial nematode, B. malayi. B. malayi is an excellent choice for these studies since it exhibits significant phylogenetic distance from C. elegans and its genome has recently been sequenced. Can bioinformatics be used to effectively identify BA receptors in the three genomes and predict their ligand- binding and G-protein coupling? Is C. elegans a useful model for the identification of key receptors for parasitic nematodes?
Previously, at least 15 putative BA receptors have been identified in the C. elegans genome (Figure 6 and (Olde and McCombie 1997; Hamdan, Ungrin et al.
1999; Rex and Komuniecki 2002; Hobson, Geng et al. 2003; Tsalik, Niacaris et al.
2003; Chase, Pepper et al. 2004; Sanyal, Wintle et al. 2004; Suo, Ishiura et al. 2004;
Rex, Hapiak et al. 2005; Carre-Pierrat, Baillie et al. 2006). More importantly, many
C. elegans BA receptors have been pharmacologically characterized and their individual roles at least partially described through the use of null mutants prepared by different knockout consortia. C. elegans contains Gαs, Gα i/o and Gαq-coupled 5-HT receptors, (Olde and McCombie 1997; Hamdan, Ungrin et al. 1999; Hobson, Geng et al. 2003), Gαs-and Gα i/o-coupled DA receptors (Sanyal, Wintle et al. 2004; Suo,
Ishiura et al. 2004) and Gα i/o-coupled TA receptors (Rex and Komuniecki 2002; Rex,
Hapiak et al. 2005). In addition, insects contain Gαs-coupled OA receptors (Gerhardt,
Bakker et al. 1997) and candidate OA receptors have been identified in C. elegans
(Suo, Kimura et al. 2006). With the availability of complete genomes from two caenorhabditid species and the B. malayi, we have undertaken to survey the conservation of BA receptor genes in these genomes. Our results provide a
29 framework for the analysis of G-protein coupled receptors (GPCRs) that may be targeted for drug development in this medically important parasitic nematode.
30 Materials and Methods
Mining the institute for genomic research (TIGR) B. malayi database for GPCR homologous genes
C. elegans protein sequences were used to identify genes encoding potential
G-protein coupled biogenic amine receptors in B. malayi. TBLASTN searches of the
B. malayi WGS_database were performed (http://tigrblast.tigr.org/er- blast/index.cgi?project=bma1) using C. elegans protein sequences obtained from
Wormbase (http://www.wormbase.org). TBLASTN searches were performed for each previously characterized C. elegans BA receptor. Contigs with high matches to C. elegans proteins were run through a modified gene prediction program, Softberry
(http://sun1.softberry.com/berry/phtml?topic=fgenesh
&group=program&subgroup=gfind) (Goff, Ricke et al. 2002) designed to be specific for B. malayi intron/exon splice patterns. Individual B. malayi genes, from the original contig hit, were translated into protein sequences and motifs characteristic to
GPCR were searched for by eye, e.g., the presence of a DRY motif at the end of TM
III and an NPxxY motif within TM VII. Proteins with either of these motifs were then compared with the C. elegans protein database (www.ncbi.nih.gov/BLAST) to confirm whether the original C. elegans query had the highest identity to the B. malayi sequence. In general, highest identity scores ranged from 149 to 375 (with expect (E) values between 2e -37and 1e-76) where the higher identity scores and lower e values, indicate stronger alignment. ExPasy translate (http://ca.expasy.org/) was used to read through predicted exon splice sites of predicted protein sequences lacking either of the
31 conserved DRY or NPxxY motif, to try and find potential exon sequence coding for these motifs within predicted intronic regions.
Mining the C. briggsae and C. elegans databases
We conducted a similar scan of the completed C. briggsae and C. elegans genomes to ensure that we had identified all GPCR gene family members in these two species. A similar approach was used to scan the C. briggsae database within wormbase.org as described for B. malayi above.
Screening of 5’ and 3’ RACE libraries from B. malayi
In order to identify full length GPCR transcripts from B. malayi, RACE libraries were constructed using total RNA isolated from male and female specimens.
Libraries were constructed using the BD SMARTTM RACE cDNA Amplification kit
(BD Biosciences, CA), according to the manufacturer’s protocol. Two sets of gene- specific primers were designed, primary and nested, (Integrated DNA Technologies,
IA) for both 5’ and 3’ RACE for 6 of the 7 B. malayi genes, (bm1 5’race 1º 5’
GTTGGCGGTAAGGCACATCGTTTGCCGC 3’, bm1 5’race nested 5’-
CATTGGTGAGTTCTGTGCTGCGCTTTG- 3’, bm1 3’race 1º 5’-
GGACTGCTACCAACAGTCACACTGAAGTGC -3’, bm1 3’race nested 5’-
GGCATGACAAAAGGCCCGCTGAAGACTAC-3’, bm2 5’race 1º 5’-
TGTCCAACAGGCTACAAATGTTCC, bm2 5’ race nested 5’-
GCGTACGCCATGCTTTACGTTCACG, bm2 3’ race 1º 5’-
CGTGAACGTAAAGCATGGCGTACGC, bm2 3’race nested 5’-
32 GAACATTTGTAGCCTGTTGGC, bm3 5’race 1º 5’-
GACGAGTACGCTGAGCGGCATAGCG-3’, bm3 5’race nested 5’-
CGTAACGGTTGACTAACGGCGCAATAGCGG-3’, bm3 3’ race 1º 5’-
CCAGCTAGCCTAAAATGAGCCGTCAAGTCGG-3’, bm4 5’ race 1º 5’-
GTAAATGATTGGATTCATTGCTG-3’, bm4 5’ race nested
CGGTGTCCAACAAATTGC-3’, bm4 3’race 1º 3’-
GAACCATTGCTGCCAATATAC-3’, bm4 3’race nested 5’-
GCCAATCTTGCCTTCAGTG-3’, bm5 5’race 1º 5’-
GCCTAGCCATAGGAAAACTTCTTGTGTGGC-3’, bm5 5’race nested 5’-
GGGCTGCTCACTAGCACTTCGATCAGG-3’, bm5 3’race 1º 5’-
CCTGATCGAAGTGCTAGTGAGCAGCCC-3’, bm5 3’ race nested 5’-
GCCACACAAGAAGTTTTCCTATGGCTAGGC-3’, bm6 5’race 1º 5’-
ATTGATGCATGAGTTGATGTAGCCAAGCC-3’, bm6 5’ race nested 5’-
CGACAACAACACCGAGTGTTTTTGTAGC-3’, bm6 3’ race 1º 5’-
CAGTCATTAGTTTTGATAGGTACCGAGCAG-3’, bm6 3’ race nested 5’-
GGGAAGGCTTCAGGAGGCG-3’. The specificities of initial PCR products were assessed using nested vector and gene-specific primers. Amplified PCR products were cloned into the TOPO 2.1 vector (Invitrogen, CA) and the DNA sequence determined by MWG (High point, NC). About 20 to 30 clones were sequenced for each RACE product. Sequence data from both 5’ and 3’ RACE was compiled and the full length cDNA (flcDNA) sequence compiled (Seqman Program, DNASTAR,
Madison WI) and aligned with the corresponding genomic sequence to confirm gene structure (Megalign, DNASTAR, Madison WI).
33
Gene Maps
C. elegans gene maps were created from the EST wormbase database
(www.wormbase.org). C. briggsae gene maps were compiled using Genefinder prediction from Wormbase. B. malayi gene maps were compiled from 5’ and 3’
RACE data and from Softberry gene predictions, when RACE was incomplete or unavailable.
Protein alignments
Protein alignments were carried out using the ClustalW and Lipman Pearson
Algorithms within the Megalign program (DNASTAR, Madison WI). For optimal protein alignment, the operational taxonomic unit (OTU) that includes only the areas of high identity was defined; specifically, 17 amino acids prior to the first conserved aas in TM I (N1.50), 10 aas after TM V and before TM VI and 15 aas after TM VII were removed from the full length protein. Abridged protein sequences were initially aligned with ClustalW using default parameters and some minor manual adjustments were made to the final alignment (these adjustments did not affect the alignment of the
TM regions). For presentation purposes, multiple sequence alignments were saved in a Pileup GCG format and uploaded into the BOXSHADE program
(http://bioweb.pasteur.fr/seqanal/interfaces/boxshade.html). Output BOXSHADE.ps files were imported into the Canvas X program (ACD Systems Victoria, BC) for figure construction.
34 Phylogenetic tree
BA receptor protein sequences from different animal species were downloaded from NCBI and abridged to the OTU comprising the seven transmembrane regions; specifically the N-termini were deleted to include 19 amino acids before the first conserved amino acids in TM I (N1.50). The third intracellular loop was deleted 10 aas after TM V and 10 aas before TM VI. The C-termini were also truncated to 15 aas after TM VII. Annotated sequences were initially aligned using the Clustal W algorithm within MegAlign (DNAStar, Madison WI) using default parameters and finalized using manual adjustment (all alignments available on request). The statistical reliability of tree branching was assessed using bootstrap analysis (1000 replicates with random seed) and trees were compiled using the Phylogenetic
Alignment Utility program (PAUP, Version 10.3). Caenorhabditis briggsae (Cb) and
C. elegans (Ce) protein sequences were downloaded from Wormbase
(www.wormbase.org), accession numbers for B. malayi are in the process of deposition. Accession numbers of each sequence were as follows. Serotonin receptors: Aedes aegypti (Aa5HT7: AAG49292), Ascaris suum (As5HT2c:
AAC78396), Bombyx mori (Bmo5HT: Q17239), Drosophila melanogaster (Dm5HT2:
NP_730859.1, Dm5HT2a: CAA77571, Dm5HT2b: NP_725849, Dm5HT7:
AAM49860), Haemonchus contortus (Hc5HT1e: AAO45883), Heliothis virescens
(Hv5HT: Q25190), Homo sapiens (Hs5HT1a: CAA40962, Hs5HT1b: P28222,
Hs5HT1d: P28221, Hs5HT1e: CAA77558, Hs5HT1f: AAA36605, Hs5HT2a:
CAA40963, Hs5HT2b: CAA54513, Hs5HT2c: AAF35842, Hs5HT4: Q13639,
Hs5HT5a: CAA57168, Hs5HT6: AAA92622, Hs5HT7: CAH69965).
35 Tyramine/octopamine receptors: Anopheles gambiae (AgOA: EAA06361), Aplysia californica (AcOA: AAF37686), Aplysia kurdai (AkOA: AAF28802), Apis mellifera
(AmOA: NP_001011565), Ascaris suum (AsTA/OA: AAS59268), Bophilus microplus
(BmiOA: CAA09335), Bombyx mori (BmoOA: BAD11157), Drosophila melanogaster (DmOA: NP_524669, DmTA: ABE73326), Heliothis virescens (HvOA:
CAA64864), Locusta migratoria (LmOA: CAA49269), Lymnaea stagnalis (LsOA1:
O77408, LsOA2: O01670), Periplaneta americana (PaOA: AAP93817). Dopamine receptors: Apis mellifera (AmD2: NP_001014983), Drosophila melanogaster
(DmD2b: NP_524548, DmDOP1: NP_477007, DmDOP2: AAN15957), Homo sapiens (HsD1: CAG46720, HsD2: AAC78779, HsD3: AAH95510, HsD4:
AAB59386, HsD5: CAA41360). Mus musculus (MmD1: NP_034206, MmD2:
NP_034207, MmD3: NP_031903, MmD4: AAB50730, MmD5: NP_038531).
Panulirus interruptus (PiD1: ABB87183, PiD1a: ABB87182), Renilla koellikeri
(RkD1: AAL25619), Xenopus laevis (XlD1a: P42289, XlD1b: P42290, XlD1c:
P42291). Trace amine-associated receptors: Homo sapiens (HsTAAR1: AAI01826),
Mus musculus (MmTAAR1: AAK71238), Macaca mulatta (MmuTAAR1:
XP_001102243.1). C. elegans GAR-1 and C. briggsae CbP01300 sequences were used as outliers for tree analysis.
36 Results
Identification of genes encoding potential G-protein coupled biogenic amine receptors in C. elegans, C. briggsae and B. malayi
To identify genes encoding putative BA receptors in the B. malayi genome, the
B. malayi WGS_database was searched using TBLASTN and C. elegans protein sequences obtained from Wormbase as search queries. A similar approach was used to identify putative BA receptors in the C. briggsae genome. Searches were performed using nine previously characterized C. elegans BA receptors: 5-HT (SER-
1, SER-4, SER-7), DA (DOP-1, DOP-2, DOP-3, DOP-4), TA (SER-2, TYRA-2).
Four putative receptors also were used as search queries: OA (SER-3), TA (M03F4.3) and DA (T02E9.3 and C24A8.1). Individual B. malayi genes were predicted by a modified SoftBerry program specific for B. malayi intron/exon splice patterns
(Solovyev, Kosarev et al. 2006). Predicted protein sequences of the B. malayi receptors were compared with the C. elegans protein database and aligned with the C. elegans sequence that exhibited most identity (Figures 3-5). BLAST alignment scores ranged from 149 to 375 with low expect (E) values from 2e -37 to 1e-76. These searches and comparisons have identified seven genes encoding putative BA receptors in the B. malayi genome, compared to 15 in C. elegans and C. briggsae. The B. malayi genome contains potential homologues of the C. elegans ser-1 (Bm1), ser-4
(Bm2) and ser-7 (Bm3) 5-HT receptors the tyra-2 (Bm4) TA receptor and the dop-1
(Bm6) and dop-2 (Bm7) DA receptors (Figure 5). The remaining receptor, (Bm5), has
37 most identity to M03F4.3 and appears to be a TA receptor, based on TA inhibition of
[H3]LSD binding to membranes prepared after the heterologous expression of the m03f4.3 cDNA in COS-7 cells (Hapiak and Komuniecki unpublished). In fact, m03f4.3 has recently been renamed tyra-3 in the C. elegans database. A fourth potential 5-HT receptor, F16D3.7, also was identified via BLAST analysis of the C. elegans genome and verified by RACE (Smith and Komuniecki, unpublished).
F16D3.7 has a C. briggsae homolog, CbP14966.
Identification of serotonin receptor homologs in B. malayi
To examine the conservation of the three 5-HT receptors previously characterized in C. elegans (ser-1, ser-4, and ser-7) with potential homologues in B. malayi and C. briggsae, gene maps were constructed for each gene using genomic and
EST sequence data for the C. elegans and C. briggsae genes. Predicted B. malayi genes were compiled from genomic database sequences and also from experimental sequence data, generated by amplifying and sequencing the 5’ and 3’ transcript ends
(RACE) from B. malayi cDNA libraries (see Materials and Methods). C. elegans and
C. briggsae genes contain identical numbers of exons, but intron sizes differ from 1 up to 2885 nt.
• C. elegans ser-1 shares all 10 intron/exon boundaries with the C. briggsae
CbG16350 (Figure 3A). The potential B. malayi ser-1 homologue (Bm1) has 3 more introns (8) than either C. elegans or C. briggsae. Bm1 shares only 3 intron/exon borders, but when predicted Bm1 coding regions were compared to other C. elegans genes encoding 5-HT receptors, Bm1 had greatest identity (29%) to SER-1, 64% in
38 A Ce ser-1 64 294 716 341 160 477 Cb G16350 64 294 713 314 160 477 Bm1-a 263 116 358 100 126 188 108 180 64 Scale Bm1-b 100 nt Exon 263 11 6 10 35 1kB Intron B Ce ser-4 Transmembrane region 150 150 293 49 258 223 105 110 Premature Cb G15112 150 150 293 49 258 223 105 110 stop codon Bm2 Untranslated cDNA 230 114 Predicted cDNA region C Ce ser-7 332 256 330 173 138 39 40 Cb G02068 335 256 330 173 138 39 40 Bm3-a 40 332 237 361 14193 144 145 Bm3-b 40 332 237 361 141
D III CeSER-1 56 NGV ALF LLP VLC LI GLIGNFLVCVAI ATD RRL HNV TN- YFL FSL ALA DLL VCCIVMPLSIVVEVRHG V WTW CbP03892 8 NGF ALF LLP VLC LI GLVGNFLVCVAI ATD RRL HNV TN- YFL FSL ALA DLL VCCIVMPLSIVVEVK H G V WTW Bm1 25 NIL IIV LLS LLC AV GLLGNLLVCMAI KLN RKL HNI TN- YFL FSL TLT DLL VCGVVMPLSLIVELN Q - M WTW CeSER-4 51 TVI LAS VLL VLI LS CFIGNLFVILAI IME RDL RGR PQY YLI FSL AVA DLL VGMIVTPLGAWFTVT G - T WNL CbP03707 54 TVI LAS VLL LLI LS CFIGNLFVILAI IME RDL RGR PQY YLI FSL AVA DLI VGMIVTPLGAWFTVT G - S WNL Bm2 ------CeSER-7 41 KAL LAI AIL AMI IM TTVGNALVCLAV LLV RKL K-H PQN FLL VSL AVA DFF VGLVVMPLALIDLLF D - K WPL CbP00480 42 KAV LTL IIL AMM MM TIVGNALVFLAV LIV RKL K-T PQN FLL VSL AVA DFF VGLVVMPLALIDLLF D - K WPL Bm3 54 VAI ICL LIG MII VA TLVGNSLVIMAV LLV RKL KIQ PAN YLF VSM AVA DFC VGLFVMPIALIDLLT D - R WIL III IV CeSER-1 S-VSMC LLY VYS DVF LCS ASI VHM SVI SLDRYLGISQPL R-T RNR SKT LIFI KIA IVW VVT LL VS- C PIAVLA CbP03892 S-MSLC LLY IYS DVF LCS ASI VHM SVI SLDRYLGISQPL R-S RNR SRT LIVL KIT FVW VAT LL VS- C PIAALA Bm1 N-FLMC LLY IYA DVF LCT ASI VHM SII SIDRYLGISKPL K-A RNK SKT LMKV KLA SVW IAT IL IS- C PIVIMA CeSER-4 G-VVVC DFW ISV DVL VCT ASI LHL VAI ALDRYWSITDIC -YV QNR TPK RITL MLA VIW FTS LL ISL A PFAGWK CbP03707 G-VVVC DFW ISV DVL VCT ASI LHL VAI ALDRYWSITDIC -YV QNR TPK RITC MLA IIW FVS LL ISL A PFAGWK Bm2 ------CeSER-7 G-STMC SVY TTS DLT LCT ASI VNL CAI SVDRYLVISSPL RYS AKR TTK RIMM YIA CVW IIA AI VSI S SHIIAN CbP00480 G-STMC SVY TTA DLT LCT ASI VNL CAI SVDRYLVISRPL QYS AIR TTR RIGW YIA CVW ITA AV VSI S SHIIAR Bm3 GGVVVC RFW TSA DLT LCT ASI VNL CMI SVDRYCAVSQPL RYA AQR TRQ RIFC YVI IVW ILS LI VSI S PLVIWP V CeSER-1 MHDTAN-I LRN NQ CMIFSR Y-Y IIY GST MTF LIP LCIMG VTY AKT TQL LNKQ 196 361 LA NE H KAT RVL AVV CbP03892 VIDQNN-I LQD NQ CMIFSR Y-Y IIY GST MTF LIP LGIMS ITY TKT TKL LKKQ 244 363 LA NE H KAT RVL AVV Bm1 LIDSRN-V FNG NT CRITNR Y-Y MIY GSI LAF LIP FLIMV VTY IRT TNL LKRQ 212 316 LT NE H KAT RVL AIV CeSER-4 DEGFSDRV LKS HV CLISQQ ISY QVF STA TAF YIP LIAII CVY WKI MRA AKKR 244 360 MK RE R KAW RTL AII CbP03707 DEGFSDRV LKQ HV CLISQQ ISY QVF STA TAF YIP LIAIV VIY WKI MRA AKKR 234 315 MK RE R KAW RTL AII Bm2 ------1 MK RE R KAW RTL AII CeSER-7 LLNDGTYV DDT GT CQVIPH FIY QSY ATI ISF YAP TFIMV ILN IKI WRA AKRL 233 311 EK SE C KAR KTL GVI CbP00480 FLDDGTFI EDP GT CQVLPH FLY QSY ATL ISF YGP TFIMV ILN IKI WRE AKRL 234 316 EK SE C KAR KTL GVI Bm3 AKNT------E GK CQVIQN PVY QIY ATI IAF YGP TCIMV ILY AKM WLA AKRH 240 326 EK SE G KAR KTL GIM VI VII CeSER-1 FACFFICWT PFF FIN FLI GFG GEN VQI PDWVA SIF LWL GYVSSTI NPI IYT VFN KRF RQA FVR ILRCQ 398 CbP03892 FVCFFVCWT PFF FIN FVV GFC GHH CTL PSWCG TLF LWL GYVSSTI NPI IYT VFN KRF RQA FVR ILRCQ 445 Bm1 FACFFICWT PFF GGN LVL GFC GKR CAL PPTIA SFF LWL GYFSSTI NPL IYT IFN RQF RRT VLE ILRCH 397 CeSER-4 TGTFVACWT PFF LVS IYR PIC G-- CQI SPVLE QVT LWL GYLNSAL NPI IYT VFS QDF RAA FKR IIKRM 439 CbP03707 TGTFVACWT PFF LVS IYR PIC G-- CQI SPVLE QVT LWL GYLNSAL NPV IYT VFS QDF RAA FKR IIKRI 394 Bm2 TGTFVACWT PFF LVS LYR PIC R-- CEI PILLE SIT NWL GYLNSAL NPI IYT VFS NDF RTA FKR ILAR- 79 CeSER-7 MSVFIICWL PFF ILA IFK SF- G-- MWI PDWLD LLA LWL GYSNSTL NPL IYC KYN KEF RIP FRE MLACR 389 CbP00480 MSVFIICWL PFF ILA IFK SF- G-- MKI PGWLD LTA LWL GYSNSTV NPL IYC KYN KEF RIP FRE MLACR 394 Bm3 MSVFVICWL PFF LLA LLK SQ- G-- FLP PNWLD HLA LWL GYSNSLM NPL IYC KHN REF RIP IRE ML-C- 402
Figure 3: Gene maps and alignments of serotonin receptors A,B,C Gene maps for characterized and predicted serotonin receptors. Exons are boxes and intronic sequence is depicted by solid lines. A gray box within exons depicts sequence coding for transmembrane regions. Dashed lines indicate predicted gene sequence. D, annotated protein sequences aligned and sequence analysis by BOXSHADE. White type on black background indicates identical amino acid residues; black type on grey background indicates similar residues. Predicted TM regions are indicated by a solid line above the alignment.
39 the TM regions (Figure 3A, Table 1). Two alternatively-spliced Bm1 transcripts were amplified from a B. malayi cDNA library, Bm1-a and Bm1-b. Bm1-b has a premature stop codon and encodes a truncated receptor encoding only the first 5 TM regions.
• C. elegans ser-4 shares all 14 intron/exon borders with CbG15112. Partial
Bm2 sequence has been identified by RACE; of the two intron/exon borders predicted in Bm2, both are conserved in ser-4. Only two exons of Bm2, encoding the last two transmembrane regions were amplified by RACE, but the predicted protein sequence has 89% identity to SER-4 within these TM regions (Figure 3B and Table 1).
• C. elegans ser-7 shares all 12 intron/exon borders with CbG02068. Bm3 shares 8 intron/exon borders with ser-7 and the Bm3 predicted protein sequence has
44% identity to SER-7, 64% in the TM region (Figure 3C and Table 1). Four alternatively-spliced Bm3 transcripts were identified by RACE. Bm3 exons 5 and 6 were not predicted by the Softberry algorithm, and the sequence of the amplified transcripts indicated that both exons were alternatively-spliced. Exon 7 is alternatively-spliced at the 5’ end, with two different splice sites leading to two differently-sized exon 7. The presence of exon 5 (no exon 6) and the longer exon 7 leads to a reading frame shift in exon 7 that produces a premature stop codon. This transcript produces a truncated receptor with only the first 5 TM domains, as also noted above for Bm1-b (Figure 3C).
The nine 5-HT receptor protein sequences from all three nematode species were aligned using ClustalW. The eight complete 5-HT receptors have 49 identical amino acids, with the highest identity within the TM regions (Figure 3D and Table 1).
40 Table 1: Comparison of C. elegans biogenic amine receptor gene structure and predicted B. malayi orthologs
Gene 1 Isoforms 5’ gene 2 3’ gene 2 Exons Introns Shared I/E % Identity TM borders 3 (full) 4 region Ce ser-1 2 k08b5.1 F59C12.1 6 5 bm1 2 f11a3.2 C23G10.4 9 8 4 29 64 Ce ser-4 1 y22d7ar.12 Y22D7AR.T1 8 7 5 bm2 ND ND Y55B1BR.4 2 1 ND ND 89 Ce ser-7 3 c09b7.2 T14G11.3 7 6 bm3 3 y45d9a2a T14G11.3 8 7 8 44 64 Ce tyra-2 2 t28b4.2 R173.1 12 11 bm4 1 t28b4.2 T13G4.4 10 9 14 78 83 Ce M03F4.3 3 k05b2.5 M03F4.4 11 10 bm5 1 R03G5.7 F36G3.1 >8 >7 6 37 66 Ce dop-1 4 F15A8.12 F15A8.3 9 8 bm6 1 No similarity E01H11.1 7 6 4 44 68 Ce dop-2 2 K09G1.3 T06E4.11 14 13 bm7 ? Y47A7.2 F49E10.2 16 15 10 27 70
1 C. elegans genes were defined using data from the Wormbase database, B. malayi genes from TIGR contig and RACE information. 2 Genes flanking both the C. elegans most similar B. malayi genes were compared by BLAST analysis. Flanking B. malayi genes are named as the C. elegans gene exhibiting most identity. 3 Intron/exon borders were compared from gene maps constructed in this study (Figures 3, 4 and 5) 4 Percentage identity was calculated from pairwise alignments (Pearson and Lipman method). 5 ND, not determined
41 Identification of tyramine receptor homologs in B. malayi
To examine the conservation of the three TA receptors previously characterized in C. elegans (ser-2, tyra-2 and m03f4.3) with potential homologues in
B. malayi and C. briggsae, gene maps were constructed for each gene, using existing
EST sequence data and RACE sequence data. Gene maps for ser-2, tyra-2, m03f4.3 and C. briggsae homologs were compiled from EST data in the Wormbase database.
B. malayi gene maps were compiled from 5’ and 3’ RACE data and the genomic sequence database.
• C. elegans tyra-2 shares 20 of 22 intron/exon borders with the CbG10952; two of the exons in tyra-2 do not appear to be present in CbG10952 (Figure 4A), but EST data from C. briggsae is more limited than that from C. elegans. tyra-2 has a 5’ exon
(5 nt) that is not observed in CbG10952. Bm4 shares 16 of the 18 intron/exon borders with tyra-2, and the predicted Bm4 protein sequence is 78% identical to TYRA-2, with the highest identity, 83%, in the TM regions (Figure 4B and Table 1).
• C. elegans m03f4.3 shares all 20 intron/exon borders with CbG14571. The near complete Bm5 transcript shares 11 of its anticipated 14 intron/exon borders with m03f4.3 (Figure 4B). Bm5 spans a gap in the genome that has yet to be sequenced, so that an exact identification of intron/exon borders in exon 4 is not possible. Predicted
Bm5 protein sequence is 37% identical to M03F4.3, with the highest identity, 66%, in the TM regions (Figure 4B and Table 1).
All six TA receptor protein sequences from the three nematodes were aligned in using ClustalW in the MegAlign program. All six sequences have 78 identical amino acids with the highest identity within the TM regions (Figure 4C).
42 A Ce tyra-2 49 114 121 188 195 100 101 191 132 122 81 Cb G10952 49 114 121 74 111 210 100 101 191 132 112 81 Scale 100 nt Exon Bm4 1kB Intron 149 117 190 191 97 100 268 120 112 81 TM Predicted sequence B Ce M03F4.3 87 231 187 130 345 93 106 213 115 131 141 Cb G14571 87 231 187 130 345 93 106 213 115 131 141 Bm5 90 115 244 400 224 124 124 35
C III CeTYRA-2 18 QILKGSALFLLVLWTIFANSLVFIVLYKNPRLQTVPNLLVGNLAFSDLALGLIVLPLSSVYAIA CbP02670 18 QILKGSALFLLVIWTIFANSLVFIVLYKNPRLQTVPNLLVGNLAFSDLALGLIVLPLSSVYAIA Bm4 11 NILKGSALTLLVIWTIAANILVFVVLYKNPHLQTVPNLLVANLAFSDSCLGVIVLPLSSIYAIA CeM03F4.3 99 DVFIALFLVMLILLTIFGNILVVLSVVVYKRMRTFTNILLTSLATADLLVGLIVMPMSLLDLLH CbP03587 99 DVFIALFLVMLILLTIFGNILVVLSVVVYKRMRTFTNILLTSLATADLLVGLIVMPMSLLDLLH Bm5 72 QIIIAIVLIILIFFVIVGNTLVILSVIIYKRMRTFTNKLLTSLATADLLVGLFVMPLSLLDLLL III IV CeTYRA-2 G-EWVFPDA LCEVFVSADILCSTASIWNLSIVGLDRYWAITSPVAYMSKRNKRTAGIMI LSV WIS S CbP02670 G-EWVFPDA LCEVFVSADILCSTASIWNLSIVGLDRYWAIT-PVAYMSKRNKRTAGIMI LSV WIS S Bm4 N-EWLFTST LCVVFVSADILCSTASIWNLSIVGLDRYWAITTPRAYMAKRNKRTVAYLI LSV WFS S CeM03F4.3 NHRWPLGRF LCRMWATSDVLLCTASILNLCVISLDRYFAITSPLKYPRTRSRKMAAGLL TAV WAI S CbP03587 NHRWPLGRF LCRMWATSDVLLCTASILNLCVISLDRYFAITSPLKYPRTRSRKMAAGLL TGV WTI S Bm5 NHAWPFDML LCKVWSTSDVLFCTASILNLCVISIDRYLAISKPLKYSRTRNRITAALLL GSV WLI S V CeTYRA-2 ALISLAPLLG--WKQTAQTPNLIYEKNNTVR--QCTFLDLPSYTVYSATGSF FIP TLL MFF VYFKI CbP02670 ALISLAPLLG--WKQTAQTPNLIYEKNNTVR--QCTFLDLPSYTVYSATGSF FIP TLL MFF VYFKI Bm4 ALISLAPFFG--WKQVAERGNMI--KINGTW--QCVFLDLPSYTIYSATGSF FIP LFI MFF VYYKI CeM03F4.3 FVVCSPPWVIPSWNLFIDNNNNTGSSEDFK----CAYSPSVAYRIYSALGSF YLP LLV MLF VYFKI CbP03587 FVVCSPPWVVPSWNLFTDNNNNTGSSEDFK----CAYSPSVAYRIYSALGSF YLP LLV MLF VYFKI Bm5 FIVCTPPWIFHFDDYPITTRANISAPTSCLNMIVCGYPSGIMYRIYSSMASF FIP PLL MCS VYYKI VI CeTYRA-2 YQAFAKH 215 378 AAKERRGVKVLGIILGCFTVCW APF FTM YVL VQF CKD----CSPNAHIEMFI CbP02670 YQAFAKH 214 377 AAKERRGVKVLGIILGCFTVCW APF FTM YVL VQF CKG----CSPNAHIEMFI Bm4 YQTFAKH 207 335 RAKERRGVKVFGIILGCFAICW TPF FIM YVV VQF CSS----CQVDPHIWMFI CeM03F4.3 FRVASER 297 487 YMRERKALKTIGIVVLGFIICW MPF FIM YLV EVF ISDPVAESPIYRITSEFF CbP03587 FRVASER 301 486 YLRERKALKTIGIVVLGFIICW MPF FIM YLV EVF ISDPVAESAVYRITSEFF Bm5 FRIISTR 238 338 YMKERKALKTIAILFFCFSICW LPF FVI YLI EVL VSSP---NDIIYATQEVF VII CeTYRA-2 TWLGYSNSAMNPIIYTVFNRDYQIALKRLFTSE 459 CbP02670 TWLGYSNSAMNPIIYTVFNRDYQIALKRLFTSD 457 Bm4 TWLGYSNSAMNPIIYTIFNHDYQNALKGLFRGN 465 CeM03F4.3 LWLGYSNSVLNPIIYTMYNGDFRRCFRDLLSFG 571 CbP03587 LWLGYSNSVLNPIIYTMYNGDFRRCFRDLLSFG 571 Bm5 LWLGYSNS--NPIIYTMYNHDFRRCFRDLLTLG 426
Figure 4: Gene maps and alignments of tyramine and octopamine receptors A and B, Gene maps for characterized and predicted tyramine and octopamine receptors. Exons are boxes and intronic sequence is depicted by solid lines. A gray box within exons depicts sequence coding for transmembrane regions. Dashed lines indicate predicted gene sequence. C, annotated protein sequences aligned and sequence analysis by BOXSHADE. White type on black background indicates identical amino acid residues; black type on grey background indicates similar residues. Predicted TM regions are indicated by a solid line above the alignment.
43 Identification of dopamine receptor homologs in B. malayi To examine the conservation of DA receptors previously characterized in C. elegans (dop-1 and dop-2) with protein homologues in C. briggsae and B. malayi, gene maps for dop-1 and dop-2 and C. briggsae were complied using EST data from the Wormbase database. Two potential DA receptor genes were identified within the
B. malayi genome. The maps for these two genes were compiled using data generated by 5’ and 3’ RACE (Bm6) and using RACE data (Bm7), (see Materials and Methods).
• C. elegans dop-1 shares all 14 intron/exon borders with CbG14463. The Bm6 transcript shares 8 of its 14 intron/exon borders with dop-1 (Figure 5A). Bm6 exhibits
44% identity to DOP-1, with 68% identity in the TM regions (Figure 5A and Table 1)
• C. elegans dop-2 shares 26 of the 28 intron/exon borders with CbG19317
(Figure 5b), CbG19317 has an extra exon resulting in a longer third intracellular loop.
The predicted Bm7 gene shares only 2 of its 30 intron/exon borders are shared with dop-2. Bm7 has a 22% overall identity to DOP-2, with highest identity (70%) in the
TM regions (Figure 5B and Table 1).
The DA receptor protein sequences from all three nematodes were aligned in using ClustalW algorithm. All six sequences shared 67 aas. DOP-1, CbP03434 and
Bm6 have an additional 86 identical aa not shared with DOP-2 and DOP-2,
CbP04498/9 and Bm7 have 112 identical aa not shared with DOP-1 (Figure 5).
44 A Ce dop-1 174 173 114 214 128 112 146 148 Scale Cb G14463 Exon 180 173 114 227 119 126 146 148 1kB Bm6 Intron 176 172 105 291 137 113 139 68 Transmembrane region
B Ce dop-2 171 123 113 122 108 97 68 115 189 269 258 170 144 171 Cb G19317 167 122 112 115 108 97 68 441 115 189 270 257 170 114 171 Bm7 405 112 122 125 68 100 200 97 101 81 98 138 106 154 211 24
C I II CeDOP-1 5 QWP LLGLFS VLI ILALF GNLLVC AAI LWDRSL RKQ PENLFL VSL AVSDLL VSV LVMLFA AVND I CbP03434 5 QWP LLALFS ILI ILALF GNLLVC AAI LWDRSL RKQ PENLFL VSL AVSDLL VSV LVMVFA AVND I Bm6 5 ASI LLIVYA VII LIALG GNLLVC VAV YCDRIL RRQ QENLFL VSL AVSDLL ISL LVMSFA ASND I CeDOP-2 37 LNY AGLSLI VIP LITLL GNLLVI ISV LRYRAL QS- AINFLI LGL AVADLL VAI IVMPYA VYVY V CbP04499 35 LNY AGLFLI VIP LITLL GNLLVI ISV LRYRAL QS- AINFLI LGL AVADLL VAI IVMPYA VYVY V Bm7 72 LNI SGLFFL IIP TITVL GNSMVI IAV LRFKTL HS- AINFLI FGL AVADLL VGL FVMPYA VYVH V III IV CeDOP-1 LG-YWP FGQ FYC QFWIS FDI TTCTAS ILN LCAISL DRY WHISRP MVY IRYCNR RR-I NYVIVL VWL CbP03434 LG-YWP FGQ FYC QFWIS FDI TTCTAS ILN LCAISL DRY WHISRP MVY IRYCNR RR-I NYVIVL VWL Bm6 LG-YWP FGH LYC QLWIC FDI TCTTAS ILN LSAIAL HRF LHISRP LVY VREGFR RRKV FIVIIF VWL CeDOP-2 TNGDWY LGN LMC DIYMA SDV CCSTAS ILL LAVISF DRY RAVSLP IQY SRQSQN VKRV WTLIAV IWL CbP04499 TNGDWY LGN LMC DVYMA SDV CCSTAS ILL LAVISF DRY RAVSLP IQY SRQSQN VKRV WYVIAA IWL Bm7 QGGYWF LGS MMC DIYSA SDV ACSTAS ILI LTVISF DRY RAVTHP ISY SHNSHD TKRV IFIMAV IWV V CeDOP-1 ISAGI GAA PLGFGF GSK VTINNL TGL P VC EMRLPL PYA IGSSMV SFF LPAMV MVILYT KLY LYAR K CbP03434 ISAGI GAA PLGFDF GSK VTINSL TGL P VC EMRLPL PYA ISSSMV SFF LPAMV MVILYT KLY LYAR K Bm6 ISAII GFT QIILES -AQ RNIANC SSQ P RC ELRLKP LYA LGSSMC SFV IPAAM MILLYT RLY LFAR E CeDOP-2 VSLTL ASP MVFGVN --- --VRPP DAN P YE CRFYNA EFS ILSSMI SFV IPCFL VLFVYI RII AHG- L CbP04499 VSLTL ASP MVFGVN --- --VRPP DAN P FE CRFYNA EFS ILSSFI SFV VPCFL VLFVYI RII AHG- L Bm7 ISLAL ASP MVLGVN --- --IRPS DAD P YE CRFYNP IFS ISSSII SFV IPCFI VLFVYI RLA KETT A VI CeDOP-1 H-- 199 319 ISDQ KAR -LTLGV IMG TFLVCW LPF FTVNIL RAW LPEIFS S------KT IMAVTWL CbP03434 H-- 199 266 SKNV SFF SLTLGV IMG TFLVCW LPF FTVNIL RAW LPEIFS S------KT IMAVTWL Bm6 HFC 213 301 VTDR KAR -LTLGV IMG TFLICW LPF FIVNVI RSF LPALIS D------MQ FKAVTWL CeDOP-2 TMQ 246 741 RKEK RAT -KTLGV VVG VFLVCW VPF VINIL NAV CILLNK DSCQVGYDL FFYCTWI CbP04499 TMQ 226 751 RKEK RAT -KTLGV VVG VFLVCW VPF FVINIL NAV CILLER ESCQVGYDL FFYCTWI Bm7 VSQ 265 597 RMEK RAT -KTLGV VVG IFLACW VPF FSVYIL NAV CIQMDI KSCQVDFYA FFYTTWL VII CeDOP-1 GY ANSS ANPLIY SIF NRDFRR AFK KIIVRV 398 CbP03434 GY ANSS ANPLIY SIF NRDFRR AFK KIIVKV 346 Bm6 GY ANST ANPIIY TIL NRDFRI AFK KILFDD 387 CeDOP-2 GY MNSF MNPIIY TIF NTEFRR AFK SIIF GK 830 CbP04499 GY MNSF MNPIIY TIF NTEFRR AFK SILFGR 836 Bm7 GY INSC INPIIY TIF NVEFRR AFK CILFGK 682
Figure 5: Gene maps and alignments of dopamine receptors A and B, Gene maps for characterized and predicted dopamine receptors. Exons are boxes and intronic sequence is depicted by solid lines. A gray box within exons depicts sequence coding for transmembrane regions. Dashed lines indicate predicted gene sequence. C, annotated protein sequences aligned and sequence analysis by BOXSHADE. White type on black background indicates identical amino acid residues; black type on grey background indicates similar residues. Predicted transmembrane regions are indicated by a solid line above the alignment.
45 Phylogenetic relationships of biogenic amine receptors predict G-protein coupling and ligand binding
A comprehensive search of the Genbank database was conducted to identify and compare all currently characterized BA receptors. An additional 54 G-protein coupled receptors (GPCRs) binding 5-HT, DA, TA or OA were identified using this approach (see Materials and Methods for accession numbers). In addition, the 15 BA
GPCRs from C. elegans, their predicted C. briggsae homologs, and the seven B. malayi BA receptors identified in this study were included in the comparison.
Transmembrane regions for all 94 protein sequences were predicted using TMpred, and abridged to include only the 7 TM regions (see Materials and Methods). A multiple sequence alignment (MSA) was performed using this OTU for all 94 BA receptor proteins from 21 different animal species. A unrooted tree based on this
MSA was generated using maximal parsimony and an unrooted tree compiled using
PAUP (Figure 6). The tree exhibits a radial branching pattern at the center with branch points with low bootstrap values, suggesting that all extant receptors arose from a rapid diversification of an ancestral receptor early in animal evolution. Thus, at the present time, it is not possible to discern from the tree whether one type of extant receptor is more closely related to such an ancestral GPCR progenitor gene.
Further examination of this tree reveals that BA GPCRs do not cluster according to species but, instead, to documented coupling to the three major
G-protein families (Gαs, Gαi/o and Gαq) and to a lesser extent based on their ligand- specificity. For example, G-protein coupling of 54 of the 94 proteins is known or strongly suggested through experimentation, and each of these 54 receptors clusters
46
Figure 6: Unrooted phylogram of aligned biogenic amine G-protein coupled receptors Protein sequences were annotated to include primarily the 7 TM regions; specifically the N- termini were deleted including 19 aa before the first conserved aa in TM I (N1.50), the third intracellular loop was deleted 10 aa after TM V and before TM VI and the C-termini were removed 15 aa after TM VII. Annotated sequences were initially aligned in DNAStar with Clustal W using default parameters and fine-tuned by hand (all alignments available on
47 request). Bootstrapping was undertaken in DNAStar (1000 replicates with random seed) and trees were compiled in PAUP. Caenorhabditus briggsae (Cb) and C. elegans (Ce) protein sequences were downloaded from Wormbase.org all other sequences from NCBI, accession numbers as follows. Serotonin receptors: Aedes aegypti (Aa5HT7: AAG49292), Ascaris suum (As5HT2c: AAC78396), Bombyx mori (Bmo5HT: Q17239), Drosophila melanogaster (Dm5HT2: NP_730859.1, Dm5HT2a: CAA77571, Dm5HT2b: NP_725849, Dm5HT7: AAM49860), Haemonchus contortus (Hc5HT1e: AAO45883), Heliothis virescens (Hv5HT: Q25190), Homo sapiens (Hs5HT1a: CAA40962, Hs5HT1b: P28222, Hs5HT1d: P28221, Hs5HT1e: CAA77558, Hs5HT1f: AAA36605, Hs5HT2a: CAA40963, Hs5HT2b: CAA54513, Hs5HT2c: AAF35842, Hs5HT4: Q13639, Hs5HT5a: CAA57168, Hs5HT6: AAA92622, Hs5HT7: CAH69965). Octopamine/Tyramine receptors: Anopholes gambiae (AgOA: EAA06361), Aplysia californica (AcOA: AAF37686), Aplysia kurdai (AkOA: AAF28802), Apis mellifera (AmOA: NP_001011565), Ascaris suum (AsTA/OA: AAS59268), Bophilus microplus (BmiOA: CAA09335), Bombyx mori (BmoOA: BAD11157), Drosophila melanogaster (DmOA: NP_524669, DmTA: ABE73326), Heliothis virescens (HvOA: CAA64864), Locusta migratoria (LmOA: CAA49269), Lymnaea stagnalis (LsOA1: O77408, LsOA2: O01670), Periplaneta americana (PaOA: AAP93817). Dopamine receptors: Apis mellifera (AmD2), Drosophila melanogaster (DmD2b: NP_524548, DmDOP1: NP_477007, DmDOP2: AAN15957), Homo sapiens (HsD1: CAG46720, HsD2: AAC78779, HsD3: AAH95510, HsD4: AAB59386, HsD5: CAA41360). Mus musculus (MmD1: NP_034206, MmD2: NP_034207, MmD3: NP_031903, MmD4: AAB50730, MmD5: NP_038531). Panulirus interruptus (PiD1: ABB87183, PiD1a: ABB87182), Renilla koellikeri (RkD1: AAL25619), Xenopus laevis (XlD1a: P42289, XlD1b: P42290, XlD1c: P42291). Trace amine receptors: Homo sapiens (HsTAAR: AAI01826), Mus musculus (MmTAAR1: AAK71238) Macaca mulatta (MmuTAAR1: XP_001102243.1), rhesus monkey. C. elegans GAR-1 and C. briggsae CbP01300 sequences were used as outliers for tree analysis. C. elegans sequences are boxed and characterized sequences colour coded depending on G-protein coupling, green: Gαs, purple: Gαi/o and red: Gαq. Bootstrap analysis; **** 100-90 %, *** 89- 80 %, ** 79-70 %, * 69-50 %.
48 based on their known G-protein coupling. Thus, we have designated major clades based on coupling to a similar G-protein subtype and minor subclades based on ligand binding. Clade I includes 38 receptors, 25 of which appear to couple to Gαs. Within clade I three subclades can be discerned; IA, IB and IC represented by CeDOP-1,
CeSER-7 and CeDOP-4 respectively. The IA and IB subclades include receptors from mammals (e.g. human Hs5HT7d, Hs5HT6, HsD1, HsD5 and mouse MmD1A, MmD5 and MmTAAR1), as well as representatives from amphibians (XlD1, XlD1b), insects
(DmOA, Dm5HT7) and nematodes (CeSER-7, Bm6). The distribution of receptors from these two clades in divergent animal genera indicates that these clades evolved early in animal evolution. In contrast, subclade IC does not include any members from higher animals and combined with the closer branching to clade II suggests that subclade IC arose after the evolutionary split from chordates. Within these subclades, the terminal branching is suggestive of stronger functional conservation and more recent divergence. For example, in subclade IA, human HsD1 and Mouse MmD1 are likely to be orthologs, as are the human HsD5/mouse MmD5 and human
HsTAAR1/mouse MmTAAR1 receptors. Similarly, amongst nematodes, subclade IA receptors CeDOPI/CbP03434/Bm6 and subclade IB receptors CeSER-
7/CbP00480/Bm3 are most closely-related to one another and likely to share a closely related function.
Clade II includes 48 receptors, 23 of which appear to couple to Gαi/o. Within this clade two major subclades, IIA and IIB, are represented by the C. elegans receptors CeSER-4, CeGAR1, CeTYRA-2, CeSER-2, and CeDOP-2/CeDOP-3, respectively. Like subclades IA and IB, IIA and IIB contain members from humans to
49 nematodes, again suggesting a divergence of these receptors early in animal evolution.
The fact that B. malayi has two members in subclade IIA (Bm2 and Bm4), while C. elegans and C. briggsae have four (CeSER-4/CbP03703), (CeGAR-1/CbP01300),
(CeTYRA-2/CbP02670), and (CeSER-2/CbP03135), suggests an early gene duplication followed by a second duplication later following the split from chordates.
It is not clear if the latter duplication did not occur in B. malayi or whether B. malayi or its ancestor simply lost the duplicate genes (see Discussion). In subclade IIA, the terminal branches are again indicative of recent duplication events; for example the four human 5HT receptors (Hs5HT1d/Hs5HT1b and Hs5HT1f/Hs5HT1e) and the two
Drosophila 5-HT receptors (Dm5HT2a/Dm5HT2b). The relationship of the uncharacterized C. elegans receptors and related homologs in C. briggsae and B. malayi (CeF14D12.6/CbP18240 and CeM03F4.3/CbP03587/Bm5) to other receptors is less clear. These protein sequences branch near the center of the unrooted tree, where the branching is less reliable and bootstrap values fall below 50%. The other large subclade IIB includes 15 receptors, eight of which are known to couple to Gαi/o.
In this subclade, C. elegans and C. briggsae each have two receptors (CeDOP-
2/CbP04499 and CeDOP-3/CbP17022), while B. malayi has only one (Bm7). The short terminal branches for the characterized mammalian members of this subclade suggest that humans and mice contain three orthologous pairs (HsD3/ MmD3, HsD2/
MmD2, and HsD4/ MmD4).
Clade III includes eight receptors, five of which appear to couple to Gαq
(CeSER-1, As5HT2, Hs5HT2a, Hs5HT2b and Hs5HT2a). This clade branches closest to subclade IIB that includes Gαi/o receptors. The small number of Gαq receptors and
50 its position relative to IIB suggest that clade III diverged from clade II but was not subject to duplication later in evolution, except in humans.
Identification of highly conserved residues amongst diverse GPCR receptors
The structure of the tree described above argues that the GPCR family evolved from a common ancestral gene through duplication to couple to different signaling pathways and bind additional ligands. Overall 10 aas are 100% conserved in all of the
BA receptor sequences: 21 are 99-95% conserved, 8 are 94-90%, 10 are 89-80%, 19 are 79-70%, 16 are 69-60% and 10 are 59-50% (Figure 7). The aa conservation is summarized for each transmembrane domain (TM) and intra/extracellular loop.
Percentages were calculated from MSA and rounded to the nearest whole number
(CeC24A8.1, AsOA/TA and Bm2 receptors were omitted from the calculation since complete sequences were not available; Table 2).
• TM I, N1:50 is 100% conserved. L1:39, T1:46, G1:49, L1:52, V1:56, A1:56,
V1:57 are at least 50% conserved. The initial aa 1:59 in the first intracellular loop is hydrophilic in 73% of the sequences, followed by an R1:60 in 61%. L1:62 is 91% conserved and is followed by a hydrophobic residue in 52% of the receptors.
• TM II, D2:50 is 100% conserved. N2:40, F2:55, S2:45, L2:46, A2:47, A2:49,
L2:51/52, V2:53, M2:58, P2:59 are at least 50% conserved. W2:70 is 100% conserved and G2:68 and G2:73 are over 60% conserved in the first extracellular loop.
• TM III, C3:25, S3:39 and R3:50 are 100% conserved. W3:28, D3:32, V3:33,
C3:36, T3:37, A3:38, I3:40, L3:41, N3:42, L3:43, I3:46, S3:47, D3:49, Y3:51, A3:53,
51 I3:54 are at least 50% conserved. P3:57, Y3:60, T3:65, R3:69 are at least 50% conserved in the second intracellular loop
• TM IV, W4:50 is 100% conserved. I4:46, V4:49, S4:53, I4:56, S4:57,
P4:59/60 are at least 50% conserved. C5:30 is 100% conserved in the second extracellular loop
• TM V, Y5:30, S5:42, S5:43, S5:46, F5:47, Y5:48, P5:50, Y5:58 are at least
50% conserved. I5:61, Y5:62, A5:65, E6:30, K6:61, A6:32, K6:34 are at least 50% conserved in the third intracellular loop.
• TM VI, F6:44 is 100% conserved. T6:36, L6:37, I6:39, I6:40, G6:42, C6:47,
W6:48, P6:50, F6:51, F6:52 are at least 50% conserved. F6:60, C6:61, C7:26 are at least 50% conserved in the third extracellular loop.
• TM VII, S7:46, N7:49, Y7:53 are 100% conserved. W7:40, L7:41, G7:42,
Y7:43, N7:45, P7:50, I7:52, F7:56 are at least 50% conserved. N7:57, F7:60, R7:61,
A7:61, F7:64, Φ7:66, L7:67 are over 50% conserved in the C-terminus (Φ, hydrophobic aa).
Determining amino acids responsible for divergence in the phylogenetic tree
Many of the conserved residues described above have been described previously and are involved in conservation of the GPCR structure, but not specifically in ligand-binding (Livingstone, Strange et al. 1992; Martin and Humphrey
1994; Zuurmond, Hessling et al. 1999; Kroeze, Kristiansen et al. 2002; Chatwin,
Rudling et al. 2003; Xie, Dernovici et al. 2005). The phylogenetic tree was based on an OTU that omitted the more variable third intracellular loop. However, an
52 examination of this loop reveals a number of residues that may be responsible for the specific G-protein coupling of these groups of receptors. Multiple sequence alignments of the second intracellular loop from (W3.50 to W4.50) was performed using Clustal W for the GPCRs with well characterized G-protein coupling (Figure 7).
Y3.59 is present in both Gαi/o- and Gαs-coupled Clade I and II receptors, but is replaced in all of the Gαq-coupled Clade III receptors by H or A. Conversely, K4.45 is present only in characterized Gαq-coupled Clade III receptors and not in the Gαi/o- and Gαs-coupled Clade I and II receptors that have either L/M/T/S/C/A or Y at this position (Figure 7). These two aas also are conserved within the uncharacterized
GPCRs present in the different clades. For example, as predicted, uncharacterized receptors that cluster with either clade I and II also contain Y3.59, but not K4.45. In contrast, those that cluster within clade III do not contain Y3.59, but do contain K4.45.
53 A * CeSE R2 WL LGV TVC QFF TT ADI LLC TSS ILN LCA IAL DRY WAIH-NPINYA---QKRTT-KFVCI*VIV IVW HvOA WV FGI YVC KMW LT CDI MCC TSS ILN LCA IAL DRY WAIT-DPINYA---QKRTL-ERVLLMIG VVW BmoO A WV FGI YVC KMW LT CDI MCC TSS ILN LCA IAL DRY WAIT-DPINYA---QKRTL-ERVLFMIG IVW DmTA WE FGI HLC KLW LT CDV LCC TSS ILN LCA IAL DRY WAIT-DPINYA---QKRTV-GRVLLLIS GVW LmOA WV FGI VVC KMW LT CDV LCC TAS ILN LCA IAL DRY WAIT-DPINYA---QKRTL-RRVLAMIA GVW BmiO A WV FGL HFC ELW LT CDV LCC TAS ILN LCA IAL DRY WAIH-DPINYA---QKRTL-RRVLLSIF LVW LsOA 1 WI FGH VWC QVW LA VDV WLC TAS ILN LCC ISL DRY LAIT-RPIRYP---GLMSA-KRAKTLVA GVW CeTY RA2 WV FPD ALC EVF VS ADI LCS TAS IWN LSI VGL DRY WAIT-SPVAYM---SKRNK-RTAGIMIL SVW CeGA R1 WP LGW VAC QTW LF LDY TLC LVS ILT VLL ITA DRY LSVC-HTAKYL---KWQSP-TKTQLLIV MSW RkD1 WQ LGK HTC QFW IF VDL LCS SAS IVN LSL ISV DRY ISLS-RPLRYL---VLMTT-QRCRIGIF AVW CeSE R4 WN LGV VVC DFW IS VDV LVC TAS ILH LVA IAL DRY WSIT-D-ICYV---QNRTP-KRITLMLA VIW Dm5H T2a WI LGP ELC DIW TS CDV LCC TAS ILH LVA IAV DRY WAVT-N-IDYI---HSRTS-NRVFMMIF CVW Dm5H T2b WI LGP ELC DIW TS CDV LCC TAS ILH LVA IAA DRY WTVT-N-IDYN---NLRTP-RRVFLMIF CVW Hs5H T5a WQ LGR RLC QLW IA CDV LCC TAS IWN VTA IAL DRY WSIT-RHMEYT----LRTRKCVSNVMIA LTW Gαi/o Hs5H T1b WT LGQ VVC DFW LS SDI TCC TAS ILH LCV IAL DRY WAIT-DAVEYS---AKRTP-KRAAVMIA LVW Hs5H T1d WN FGQ ILC DIW LS SDI TCC TAS ILH LCV IAL DRY WAIT-DALEYS---KRRTA-GHAATMIA IVW Hs5H T1e WK LGY FLC EVW LS VDM TCC TCS ILH LCV IAL DRY WAIT-NAIEYA---RKRTA-KRAALMIL TVW Hs5H T1f WI MGQ VVC DIW LS VDI TCC TCS ILH LSA IAL DRY RAIT-DAVEYA---RKRTP-KHAGIMIT IVW HsD3 WN FSR ICC DVF VT LDV MMC TAS ILN LCA ISI DRY TAVV-MPVHYQHGTGQSSC-RRVALMIT AVW MmD3 WN FSR ICC DVF VT LDV MMC TAS ILN LCA ISI DRY TAVV-MPVHYQHGTGQSSC-RRVALMIT AVW HsD2 WK FSR IHC DIF VT LDV MMC TAS ILN LCA ISI DRY TAVA-MPMLYN--TRYSSK-RRVTVMIS IVW MmD2 WK FSR IHC DIF VT LDV MMC TAS ILN LCA ISI DRY TAVA-MPMLYN--TRYSSK-RRVTVMIA IVW HsD4 WL LSP RLC DAL MA MDV MLC TAS IFN LCA ISV DRF VAVA-VPLRYN---RQGGS-RRQLLLIG ATW MmD4 WL LSP RLC DTL MA MDV MLC TAS IFN LCA ISV DRF VAVT-VPLRYN---QQG---QCQLLLIA ATW CeDO P3 WG LGS FFC HVY QA LDV ACS TAS ILN LLA ISL DRY IAIG-HPISYA---QYGARGGRAMISIT IVW CeDO P2 WY LGN LMC DIY MA SDV CCS TAS ILL LAV ISF DRY RAVS-LPIQYS---RQSQNVKRVWTLIA VIW Hs5H T2c WP LPR YLC PVW IS LDV LFS TAS IMH LCA ISL DRY VAVR-SPVEHS---RFNSR-TKAIMKIA IVW Hs5H T2a WP LPS KLC AVW IY LDV LFS TAS IMH LCA ISL DRY VAIQ-NPIHHS---RFNSR-TKAFLKII AVW Hs5H T2b WP LPL VLC PAW LF LDV LFS TAS IMH LCA ISV DRY IAIK-KPIQAN---QYNSR-ATAFIKIT VVW Gαq CeSE R1 WT WSV SMC LLY VY SDV FLC SAS IVH MSV ISL DRY LGIS-QPLR-T---RNRSK-TLIFIKIA IVW As5H T2c WT WSF SVC LLY VY ADV FLC TAS IVH MSM ISL DRF LGIS-RPLK-I---RNRSR-TMTTLKIT FVW DmD2 b WF FGT DWC DIW RS LDV LFS TAS ILN LCV ISL DRY WAIT-DPFSYP---MRMTV-KRAAGLIA AVW PiD1 a WP FGP DFC DVW RS FDV LAS TAS ILN LCV ISL DRY WAIT-DPFSYP---SRMSP-RRACMLIA LVW CeDO P4 WL FGL MMC DVF HA MDI LAS TAS IWN LCV ISL DRY MAGQ-DPIGYR---DKVSK-RRILMAIL SVW LsOA 2 WV FGE FTC TLW LC MDV LYC TAS IWG LCT VAF DRY LATV-YPVWYH---DQRSV-RKAVGCIV FVW PaOA WI FGD VWC SIW LA VDV WMC TAS ILN LCA ISL DRY VAVT-RPVTYP---SIMSS-GRAKLLIA GVW AmOA WI FGD LWC SIW LA VDV WMC TAS ILN LCA ISL DRY LAVT-RPVSYP---QIMSP-RRARLLVA TVW Hs5H T4 WI YGE VFC LVR TS LDV LLT TAS IFH LCC ISL DRY YAICCQPLVYR---NKMTP-LRIALMLG GCW DmOA WN FSP FLC DLW NS LDV YFS TAS ILH LCC ISV DRY YAIV-KPLKYP---ISMTK-RVVGIMLL NTW AcOA WV FGR TMC DIF NA NDV LFS TAS IIH LCC ISM DRY IAIL-HPLQYE---SKMTR-PRALLMLG VTW AkOA WV FGR TMC DIF NA NDV LFS TAS IIH LCC ISM DRY IAIL-HPLQYE---SKMTR-PRAMLMLG VTW MmD5 WP FG- AFC DIW VA FDI MCS TAS ILN LCI ISV DRY WAIS-RPFRYE---RKMTQ-RVALVMVA LAW Gα HsD5 WP FG- AFC DVW VA FDI MCS TAS ILN LCV ISV DRY WAIS-RPFRYK---RKMTQ-RMALVMVG LAW s XLD1 b WP FG- AFC DIW VA FDI MCS TAS ILN LCV ISV DRY WAIS-SPFRYE---RKMTQ-RVALLMIS TAW XlD1 a WP FG- TFC NIW VA FDI MCS TAS ILN LCV ISV DRY WAIS-SPFRYE---RKMTP-KVAFIMIG VAW MmD1 a WP FG- SFC NIW VA FDI MCS TAS ILN LCV ISV DRY WAIS-SPFQYE---RKMTP-KAAFILIS VAW HsD1 WP FG- SFC NIW VA FDI MCS TAS ILN LCV ISV DRY WAIS-SPFRYE---RKMTP-KAAFILIS VAW XLD1 WV FG- DFC DTW VA FDI MCS TAS ILN LCI ISL DRY WAIA-SPFRYE---RKMTQ-RVAFIMIG VAW Hs5H T6 WV LAR GLC LLW TA FDV MCC SAS ILN LCL ISL DRY LLIL-SPLRYK---LRMTP-LRALALVL GAW CeDO P1 WP FGQ FYC QFW IS FDI TTC TAS ILN LCA ISL DRY WHIS-RPMVYI---RYCNR-RRINYVIV LVW DmDO P1 WI FGA QFC DTW VA FDV MCS TAS ILN LCA ISM DRY IHIK-DPLRYG---RWVTR-RVAVITIA AIW PiD1 WP FGS QFC NTW IA CDV MCS TAS IVN LCA ISL DRY IHIK-DPLRYG---RWMTK-RIVTISIA AIW Hs5H T7d WI FGH FFC NVF IA MDV MCC TAS IMT LCV ISI DRY LGIT-RPLTYP---VRQNG-KCMAKMIL SVW CeSE R7 WP LGS TMC SVY TT SDL TLC TAS IVN LCA ISV DRY LVIS-SPLRYS---AKRTT-KRIMMYIA CVW Dm5H T7 WN FGP LLC DIW VS FDV LCC TAS ILN LCA ISV DRY LAIT-KPLEYG---VKRTP-RRMMLCVG IVW B Gαs Gαi/o Gαq
54 Figure 7: Alignment of the second intracellular loop of characterized G-protein coupled biogenic amine receptors.
A, Protein sequences were annotated to include sequences from W3.50 to W4.50, and were aligned DNAStar with Clustal W using default parameters, Boxshade was used to analyze amino acid identity. B, Predicted branch pattern for G-protein coupling divergence.
55
Extracellular loops x x x x x W x x x x x x G x x x x x x x x x x x G C C x x x x x x x x x x x C x x x x x x C x x x x x x x x x x x x x x x x x M P x P x x Y W x x L x W x x P x L V x x x x x x x x x x S S G Y x V D S I x x x x L V x x F F x T x x S x S x S N L D A C x F P x G x x * Y x W x N L A x S A T x W V P N * x x P x C * * x L S x I L N x * x Y x x x I x x F x x V x L x x * G x x N x F x x x x x F A Y x x I I V x x x I S x x R x x x N x R D x x L x Y * x x T I T x R x x Y F x L x A x x K R x I x A x x x F I II III IVx V VIA VII x A L x x x S 100% conserved x x K x P Y x S 99-90% conserved x x x x E x S 89-80% conserved S 79-70% conserved * = Reference residue for Intracellular loops S 69-50% conserved TM indexing < 50% conserved x
Figure 8: GPCR model of conserved amino acids Multiple alignments were examined for conservation and amino acids over 50% were mapped and schematically represented in a GPCR map (see Materials and Methods).
56 Table 2: Location of amino acid residues with greater than 50% conservation amongst 98 BA GPCRs
TM I 1 TM II TM III 2nd iloop TM V TM VI TM VII L1:39 60 N2:40 89 C3:25 100 P3:57 85 Y5:30 66 T6:36 75 W7:40 95 T1:46 60 F2:55 55 W3:28 72 Y3:60 92 S5:42 67 L6:37 77 L7:41 79 G1:49 90 S2:45 90 D3:32 98 T3:65 60 S5:43 51 I6:39 57 G7:42 85 N1:50 100 L2:46 96 V3:33 40 R3:69 54 S5:46 62 I6:40 61 Y7:43 89 L1:52 78 A2:47 97 C3:36 53 F5:47 95 G6:42 66 N7:45 92 V1:56 98 A2:49 67 T3:37 89 TM IV Y5:48 61 F6:44 100 S7:46 100 A1:56 60 N2:50 100 A3:38 83 I4:46 74 P5:50 94 C6:47 94 N7:49 100 V1:57 57 L2:51/5 78 S3:39 100 V4:49 57 Y5:58 93 W6:48 98 P7:50 98 2 V2:53 70 I3:40 98 W4:50 100 P6:50 98 I7:52 76 M2:58 77 L3:41 52 S4:53 76 3rd iloop F6:51 95 Y7:53 100 1st iloop 2 P2:59 91 N3:42 56 I4:56 53 I5:61 75 F6:52 95 F7:56 60 Φ1:60 73 L3:43 87 S4:57 62 Y5:62 77 R1:61 62 1st eloop 2 I3:46 96 P4:59/60 86 A5:65 62 3rd eloop C-term L1:63 91 G2:68 61 S3:47 71 E6:30 92 F6:60 57 N7:57 74 W2:70 100 D3:49 98 2nd eloop K6:61 71 C6:61 71 F7:60 91 G2:73 81 R3:50 100 C5:30 100 A6:32 81 C7:26 66 R7:61 77 Y3:51 94 K6:34 53 A7:61 77 A3:53 63 F7:64 82 I3:54 59 Φ7:66 100 L7:67 72
1 The conservation of amino acid residues amongst GPCRs is summarized for each transmembrane domain (TM) and loop. Percentage conservation was calculated from multiple sequence alignments and rounded to the nearest whole number. (CeC24A8.1, AsTA/OA and Bm2 that lacked complete sequence were omitted from the calculation), 2 iloop -intracellular loop, eloop - extracellular loop
57 Discussion
B. malayi contains fewer BA receptors than C. elegans or C. briggsae
The present study was designed to identify the BA receptors present in the genomes of the free living nematodes C. elegans and C. briggsae and the parasitic nematode, B. malayi. The C. briggsae genome contains 15 genes encoding putative
BA receptors, all of which have apparent homologues in C. elegans. The C. briggsae genome contains three potential 5-HT receptors: CbP03892 (78.1% identity to SER-
1), CbP03707, (91.6 % identity to SER-4), and CbP004800 (87.5% identity SER-7).
Three potential TA receptors [CbP10888 (97.1% identity to SER-2), CbP02670
(97.4% identity to TYRA-2) and CbP03587 (93.2% to TYRA-3)], four potential DA receptors [CbP03434 (89.5% identity DOP-1), CbP04499 (90.8% identity to DOP-2),
CbP17022 (88% identity to DOP-3) and CbP24819 (88% identity to DOP-4)] have been identified. Homologs to five receptors whose ligand binding has yet to be characterized directly were also identified; CbP03135 (90% identity to SER-3),
CbP14966 (84% to F16D3.7), CbP03587 (96.5% identity to F14D12.6), CbP18041
(76% identity to C24A8.1), and CbP05495 (96.4% identity to T02E9.3). The strong conservation between the C. elegans and C. briggsae genes encoding BA receptors suggests that further adaptation since the evolutionary split did not require substantial additional selection in their sensory repertoires.
The B. malayi genome appears to contain only seven genes encoding putative
BA receptors, compared to the 15 found in the genomes of both C. elegans and C. briggsae. However, the seven B. malayi genes have clear homologues in the latter
58 two species and each of the major BA receptor subclasses appear to be represented.
The B. malayi genome contains potential homologs of three C. elegans 5-HT receptors
[ser-1 (Bm1), ser-4 (Bm2) and ser-7 (Bm3)], two TA receptors [tyra-2 (Bm4) and tyra-
3 (Bm5)], and two DA receptors [dop-1 (Bm6) and dop-2 (Bm7)] (Figure 6).
Therefore, although B. malayi appears to have fewer BA receptors than C. elegans,
BA receptors coupling to each of the core G-proteins appear to be present, arguing that the reduced BA receptor number in B. malayi is most likely not the result of incomplete genomic sequence.
Although the reason for the reduced number of BA receptors in B. malayi remains to be determined, our examination of B. malayi BA receptor transcripts suggests that additional receptor diversity in B. malayi is not being generated by alternative-splicing (see below). Instead, we suggest that the reduced number of BA receptors in B. malayi maybe linked to lifestyle differences. B. malayi, in contrast to
C. elegans and C. briggsae, has no free-living stages and resides exclusively in the relatively homeostatic environments of its mosquito and human hosts. Since many of the BA receptors are thought to fine-tune responses to environmental stimuli, the reduced number of BA receptors in B. malayi may be a response to decreased environmental variability. In addition to a reduced number of genes encoding BA receptors in B. malayi, it is not yet clear whether each of the genes actually encode functional proteins. For example, the third intracellular loop of the putative B. malayi
SER-7 homologue (Bm3) contains a large poly N tract that may prevent expression and or effective coupling.
59
Clustering of receptors as a predictor of receptor coupling and ligand-binding
The first published phylogenetic comparison of 5-HT receptors included only
5-HT receptors from, human, mouse, rat, dog, hamster, Drosophila and snail
(Peroutka and Howell 1994). These authors concluded that all BA receptors descended from a single primordial 5-HT receptor; however, no receptors with alternate ligands were used in their analysis. Our study has included 5-HT, DA, OA and TA receptors and indicates a divergence of G-protein coupling prior to the evolution of ligand specificity. For example, we found that all 54 previously characterized BA receptors formed major groups based exclusively on documented or suggested coupling to the three major families of G-protein (Gαs: clade I, Gαi/o: clade
II and Gαq: clade III) and not on ligand specificity. Therefore, our analysis may be useful for predicting the G-protein coupling of the remaining 40 uncharacterized receptors, as well as for receptors from other species identified in the future. For example, As5HT2c, CeSER-1, Hs5HT2a, Hs5HT2b and Hs5HT2c cluster with
Dm5HT2, CbP03892 and Bm1, suggesting that these uncharacterized receptors may couple to Gαq. Similarly, Hs5HT7, Dm5HT7, CeSER-7, PiD1, DmDOP1, CeDOP-1,
Hs5HT6, XlD1, MmD1a, HsD1, XlD1a, XlD1b, HsD5, MmD5, AkOA, AcOA,
DmOA, Hs5HT4, AmOA, PaOA, LsOA1, PiD1a, and DmD2b cluster in clade I. Bm3 and Bm6 also are in clade I suggesting that these receptors couple to Gαs.
The clustering of receptors and the resulting prediction of function may be further supported if synteny can be demonstrated for closely related pairs. The genomes of C. elegans and B. malayi have been compared previously for the presence
60 of specific genes (Streit, Li et al. 1999; Murray, Manoury et al. 2005) and for overall synteny (Blaxter, Aslett et al. 1999; Guiliano, Hall et al. 2002; Lee, O'Dowd et al.
2003; Stein, Bao et al. 2003). Adjacent genes in B. malayi are commonly 2-10 Mb apart in C. elegans (Ghedin, Wang et al. 2004). Similarly, mapping B. malayi BAC ends to C. elegans chromosomes also suggests that the two genomes exhibit little overall synteny (Guiliano, Hall et al. 2002; Whitton, Daub et al. 2004). In contrast, comparison of a 65 kb B. malayi genomic fragment with C. elegans found that 6-7 predicted genes in this region were conserved, suggesting that microsynteny may be present (Blaxter, Aslett et al. 1999). An analysis of synteny between the areas of the B. malayi and C. elegans genomes containing BA GPCRs is hindered by the small size of many of the existing B. malayi contigs, but, in general, in most cases the upstream and downstream genes in C. elegans and B. malayi are different (Table 1). The only area of apparent synteny is on contig 1384551 containing Bm3 and Bm4 and the area on C. elegans chromosome X containing Ceser-7 and Cetrya-2. Bm3 and Bm4 were about 6
Mb apart on contig 1384551, the same distance between Ceser-7 and Cetyra-2 on chromosome X. The flanking 3’ genes of Bm3 and Ceser-7 appear to also be homologues (T14G11.3, see Table 1). In our alignment (Figure 6), Bm3 and CeSER-
7 cluster closely together in subclade IA and Bm4 and CeTYRA-2 in subclade IIA.
Thus, at least in this case, the relationships defined by conservation of gene sequence
(and therefore potential function) are supported by conserved synteny of the genes.
Subclades determining ligand-binding indicate conservation of gene structure. For example, Ceser-1 (clade III) has 6 exons, as do CbP03892, and Bm1 (Figure 3A).
Similarly, gene structure is also conserved in clade IA between Ceser-7, CbP00480
61 and Bm3 (Figure 3C). These observations suggest that gene structures may be conserved within subclades (Figure 3A-C, 4A-B, and 5A-B).
The clustering pattern of five yet uncharacterized C. elegans receptors can be used to predict G-protein coupling, as well as ligand binding. For example, C24A8.1/
T02E9.3 cluster in clade IIB along with DOP-2 and DOP-3 both of which bind DA and couple to Gαi/o, suggesting that these two uncharacterized receptors are Gαi/o - coupled DA receptors. Similarly, F16D3.7 clusters closely with Hs5HT7d within clade IA, suggesting that F16D3.7 is a Gαs-coupled 5-HT receptor. Finally SER-3, initially thought to be a 5-HT receptor, clusters closely with Gαs-coupled OA receptors, suggesting that SER-3 may not bind 5-HT. Indeed recent studies suggest that SER-3 is an OA receptor (Suo, Kimura et al. 2006). The predictions outlined above are strong, based on the clustering within major clades. In contrast, the predictions for the C. elegans F14D12.6 and M03F4.3 receptors are less robust.
Although these receptors both cluster in the Gαi/o group, their branching junction lies close to an area of ambiguity at the center of the tree. For receptors such as these an examination of other less conserved regions of the protein may be more informative in predicting ligand binding and G-protein coupling. The future availability of receptors from more primitive eukaryotes also may help resolve the ambiguity associated with the early diversification of this large receptor family.
In summary, we consider that a careful phylogenetic comparison of receptors and, if possible, support through synteny, permits a strong prediction of some of the pharmacological properties of receptors and will aid in the development of experimental hypotheses regarding receptor function.
62
Identification of conserved amino acid residues in the BA receptor family
By conducting an extensive comparison between nematode GPCRs and those from other species, we have identified aas that have been under strong selection pressure (Figure 6, 7 and Table 2). The functional roles for some of these residues have been illuminated by previous studies that have identified aas in GRCPs (Pollock,
Manelli et al. 1992; Schwinn 1993; Gerhardt, Bakker et al. 1997; Backstrom, Chang et al. 1999; Zuurmond, Hessling et al. 1999; Kroeze, Kristiansen et al. 2002; Chatwin,
Rudling et al. 2003; Xie, Dernovici et al. 2005).
Amongst human 5-HT receptors 33 aa are 100% conserved and 27 are 80% conserved. The 33 aa that are 100% conserved are involved in ligand binding and G- protein coupling, desensitization and receptor trafficking (Kroeze, Kristiansen et al.
2002). In the present study, only 12 aas were conserved amongst all 94 receptors
(Table 2). Two of these aas C.3.25 and C5.30 form a disulfide bond that is responsible for stabilizing receptor conformation (Kroeze, Kristiansen et al. 2002);
R3.50 an integral part of the DRY motif in TM III responsible for isomerization between inactive and active conformations via interactions with E6:30 (in the 3rd iloop) and P6:50 (in TM VI) that are 92% and 98% conserved, respectively (Visiers,
Hassan et al. 2001; Flanagan 2005). N7.49 and Y7.53 lie within TM VII and are part of the NPxxY motif responsible for receptor sequestration and desensitization
(Kroeze, Kristiansen et al. 2002). D2.50, F6.44 and S7.46, also are present in all of the sequences. D2.50A abolishes 5-HT binding in the human 5-HT1a receptor and is thought to interfere with hydrogen-bonding with N7.49, as a double mutation,
63 D2.50A/N7.49D, restores 5-HT binding (Chanda, Minchin et al. 1993; Collins 1993;
Schwinn 1993; Bouvier, Moffett et al. 1995; Sealfon, Chi et al. 1995). No mutational studies have specifically targeted F6.44 or S7.46 and the specific functions of these residues have not been determined. Interestingly, all Gαs- and Gαi/o-coupled BA receptors contain N7.45, but Gαq-coupled receptors have an S in this position, (except
Hs5HT2c that has a C).
Amino acids essential for ligand binding also have been identified previously.
Interestingly, mutations in TM III and TM VII of the Drosophila OA/TA receptor
(S194A and S576A, respectively) reduce coupling to the inhibition of adenyl cyclase without altering ligand affinity (Chatwin, Rudling et al. 2003). In addition, D3.32 and
SS5.42/5.43 are involved in DA binding to rat D2 receptor and that a D3.32, S5.42 and
F6.52 are responsible for 5-HT binding in 5-HT1A, 5-HT2A, 5-HT2B 5-HT4, 5-HT6, 5-
HT1A/B and 5-HT1B, respectively (Livingstone, Strange et al. 1992; Pollock, Manelli et al. 1992; Kroeze, Kristiansen et al. 2002). A K4.45M mutation decreased the agonist efficacy of the rat 5-HT2C by 60% and the C. elegans SER-1 by 40%. In addition,
Y3.60 was conserved in all Gαs-and Gαi/o- coupled receptors (clade I and II) and
K4.45 was conserved in all Gαq-coupled receptors. Xie et al. in a study of 5-HT receptors, noted a conservation of of K4.45 and the absence of Y3.60 in the Gαq- coupled 5-HT receptors (Xie, Dernovici et al. 2005). The results of that study extend and expand these observations to all BA receptors (Figure 7).
Splice variants resulting in protein isoforms of BA receptors have been identified previously. For example, the human D3 receptor has a complex intron/exon arrangement that gives rise to a truncated isoform encoding only the first 5 TM
64 regions, D3nf (Liu, Bergson et al. 1994; Schmauss 1996; Nimchinsky, Hof et al. 1997).
D3nf is unable to bind dopamine, but is able to form dimers with D3 and impair its binding activity and/or trafficking (Fishburn, Belleli et al. 1993; Elmhurst, Xie et al.
2000; Karpa, Lin et al. 2000). The C. elegans homologue of human D3, DOP-3, also has alternatively-spliced isoforms that encode truncated 5 TM receptors. Co- expression of truncated and full length DOP-3 decreased full length expression
(Fishburn, Belleli et al. 1993). The presence of truncated cDNAs from these two B. malayi genes (Bm1 and Bm3) could be an artefact. However these truncated (Bm1 and
Bm3) transcripts for were amplified multiple times from separate cDNA libraries, suggesting both could be real and potentially involved in the regulation of receptor function/localization.
In summary, this study has identified seven genes in the parasitic nematode, B. malayi, that encode putative BA receptors, in contrast to the 15 genes in C. elegans and C. briggsae. However, all of the predicted B. malayi BA receptors have clear homologues in both C. elegans and C. briggsae. Importantly, our bioinformatics approach and phylogenetic analysis accurately predicted both the G-protein coupling and ligand specificity of 48 characterized receptors and, therefore, may also prove useful in predicting the potential G-protein coupling and ligand specificity for uncharacterized BA receptors.
65 CHAPTER III
Are C. elegans receptors useful targets for drug discovery: Pharmacological comparison of tyramine receptor homologues from Caenorhabditis elegans
(TYRA-2) and Brugia malayi (Bm4)
66 Abstract
The biogenic amine, tyramine (TA) modulates a number of key processes in nematodes and a number of TA-specific receptors have been identified. In the present study, we have identified a putative TA receptor (Bm4) in the recently sequenced
Brugia malayi genome and compared its pharmacology to its putative C. elegans orthologue, TYRA-2, under identical expression and assay conditions. TYRA-2 and
Bm4 are the most closely related C. elegans and B. malayi BA receptors and differ by only 14 aa in the TM regions directly involved in ligand binding. Membranes from
HEK-293 cells stably expressing Bm4 exhibited specific, saturable, high-affinity,
3 3 [ H]LSD and [ H]TA binding with Kds of 18 ± 0.9 nM and 15 ± 0.2 nM, respectively.
More importantly, both TYRA-2 and Bm4 TA exhibited similar rank orders of potencies for a number of potential tyraminergic ligands. However, some significant differences were noted. For example, chloropromazine exhibited an order of magnitude higher affinity for Bm4 than TYRA-2 (pKis of 7.6 ± 0.2 and 6.49 ± 0.1, respectively). In contrast, TYRA-2 had significantly higher affinity for phentolamine than Bm4. These results highlight the utility of the nearly completed B. malayi genome and the importance of using receptors from individual parasitic nematodes for drug discovery.
67 Introduction
The biogenic amines, tyramine (TA) and octopamine (OA) modulate a number of key processes in nematodes, including pharyngeal pumping, locomotion and egg- laying, suggesting that TA/OA-mediated signaling may provide useful targets for anthelminthic drug development. In general, TA/OA oppose the action of serotonin
(5-HT) (Alkema, Hunter-Ensor et al. 2005). For example, in the free-living nematode,
Caenorhabditis elegans, exogenous TA or OA cause hyperactivity, inhibit pharyngeal pumping on bacteria or 5-HT and suppress 5-HT stimulated egg-laying (Horvitz,
Chalfie et al. 1982; Segalat, Elkes et al. 1995; Niacaris and Avery 2003). The OA inhibition of pumping appears to involve Gαo, as OA inhibits the firing rate of the pharyngeal muscle in wild-type C. elegans, but has no effect on goa-1 mutants defective for Gαo function (Rogers, Franks et al. 2001).
Recently TA-and OA-specific signaling pathways have been described in both insects and C. elegans (Saudou et al., 1990 and Rex et al., 2002). TA and OA are synthesized from tyrosine. Tyrosine decarboxylase (tdc-1) converts tyrosine to TA and then tyramine β-hydroxylase (tbh-1) hydroxylates TA to form OA (Hare and Loer
2004; Alkema, Hunter-Ensor et al. 2005). In C. elegans, tdc-1 is expressed in RIC and RIM neurons, and the UV1 and gonadal sheath cells, while tbh-1 is expressed only in the RIMs and gondal sheath cells, suggesting that the RICs release only TA and the RIMs release both TA and OA (Alkema, Hunter-Ensor et al. 2005). In C. elegans, a role for TA, independent of OA, has been uncovered by the identification of
G-protein coupled receptors (GPCRs) with much higher affinities for TA than OA and
68 the examination of tdc-1 and tbh-1 null mutants that are unable to synthesize TA/OA and TA, respectively). For example, tdc-1, but not tbh-1, null mutants do not suppress head oscillations in response to anterior touch, suggesting a specific role for TA in this behavior (Alkema et al., 2005). Three distinct TA receptors have been characterized in C. elegans, SER-2, TYRA-2 and TYRA-3 (Rex 2002, Rex et al 2005, Hapiak et al., unpublished). Interestingly, these three TA receptors have different pharmacological profiles with markedly different affinities for TA. For example, SER-2 and TYRA-2 have Kis for TA of 70 nM and 3 nM, respectively. These TA receptors also are expressed in different tissues, suggesting potentially different physiological roles (Rex and Komuniecki, 2002; Rex et al. 2005).
Recently, C. elegans has been used as a model to identify signaling pathways in parasitic nematodes because of its completely sequenced genome, the availability of signaling mutants and its well defined molecular genetics. Although C. elegans is a useful model for the identification and characterization of potential drug targets in core signaling pathways, nematodes exhibit enormous diversity and significant physiological/biochemical/molecular differences between nematode species have been clearly demonstrated. For example, parasitic nematodes appear to have an active nitric oxide synthase and C. elegans does not (Zhu et al. 2004 and Morton et al.,
1999). In contrast, C. elegans proteins are probably not good targets for high throughput screening. Because of the enormous variance among the nematodes, processes in C. elegans may not be exactly duplicated in parasitic nematodes and binding constants and regulation may differ significantly among orthologues nematode proteins. On the positive side, all of anthelminthics currently in use appear
69 to function similarly in both C. elegans and parasitic nematodes (Geary and
Thompson 2001).
Are C. elegans G-protein coupled receptors similar to their homologues in parasitic nematodes? Interestingly, 5-HT1-like receptors from C. elegans (SER-4) and
Haemonchus contortus (5-HT1Hc) were recently characterized by different investigators after heterologous expression (Olde and McCombie 1997; Smith, Borts et al. 2003). Notably, although these two putative 5-HT receptor homologues exhibited markedly different affinities for 5-HT and a number of other ligands, it was not clear whether those differences resulted from true differences in the pharmacologies of the receptors or artefacts of the expression and assay systems. For
3 example, 5HT1Hc was expressed in insect Sf9 cells and assayed using [ H]5-HT and
SER-4 was assayed after expression in mammalian LCER6 cells using [125I]LSD.
Therefore, the present study was designed to compare potentially homologous TA receptors from the free-living nematode, C. elegans (TYRA-2), and the filarial parasite, Brugia malayi (Bm4), under identical expression and assay conditions to examine how these variables might affect ligand binding and to determine how similarly these two receptors might bind potential tyraminergic ligands.
70 Materials and Methods
Materials
Dulbeccos Modified Eagles Medium (DMEM) was purchased from Mediatech
(Herdon, VA) and fetal bovine serum (FBS) from HyCLONE (Logan, UT).
Penicillin/streptomycin, trypsin/EDTA, laminin, geneticin (G418 sulfate) and goat serum were purchased from Sigma (St. Louis, MO). All cell culture plasticware was purchased from Sarstedt (Newton, NC). COS-7 and HEK293t cells were originally ordered from the American Type Culture collection (ATCC) (Rockville, MD).
Restriction enzymes were purchased from New England Biolabs (NEB) (Beverly,
MA). The [3H]LSD was purchased from Perkin Elmer (Wellesley, MA) and [3H]TA was purchased from American Radiolabled Chemicals (St Louis, MO). All other ligands were purchased from Sigma. UnifilterTM plates, top sealerTM and microscint cocktails were from Perkin Elmer (Wellesley, MA). cDNA amplification kits were purchased from BD biosciences (San Diego, CA). B. malayi total RNA was a gift from David Spiro, TIGR (Washington, DC)
Methods
Mining the TIGR B. malayi database for C. elegans TYRA-2 homologues
C. elegans TYRA-2 protein sequence was used to identify genes encoding potential homologous genes in B. malayi. TBLASTN searches of the B. malayi
WGS_database were performed (http://tigrblast.tigr.org/er-
71 blast/index.cgi?project=bma1) using C. elegans protein sequences obtained from
Wormbase (http://www.wormbase.org). TBLASTN searches were performed with
TYRA-2. Contigs with high matches to C. elegans proteins were run through a modified gene prediction program, Softberry
(http://sun1.softberry.com/berry/phtml?topic=fgenesh
&group=program&subgroup=gfind) (Goff, Ricke et al. 2002), specific for B. malayi intron/exon splice patterns. Individual B. malayi genes, from the original contig hit, were translated into protein and motifs characteristic to GPCR identified, e.g., the
DRY motif at the end of TM III and the NPxxY motif within TM VII. Proteins with either of these motifs were then compared with the C. elegans protein database
(www.ncbi.nih.gov/BLAST), to confirm whether the original C. elegans TYRA-2 query had the highest identity to the B. malayi sequence. ExPasy translate
(http://ca.expasy.org/) was used to read through predicted exon splice sites of predicted protein sequences lacking either the conserved DRY or NPxxY motif, to try and find potential exon sequence coding for these motifs within predicted intronic regions.
Screening of 5’ and 3’ RACE libraries from B. malayi
To identify the full length B. malayi TYRA-2 homologue, Bm4, RACE libraries were constructed using total RNA isolated from adult B. malayi (a kind donation from David Spiro, TIGR). Two sets of gene-specific primers were designed: primary and nested (Integrated DNA Technologies, IA) for both 5’ and 3’ RACE of
Bm4, (5’ bm4 5’ race 1º 5’-GTAAATGATTGGATTCATTGCTG-3’, bm4 5’ race
72 nested 5’-CGGTGTCCAACAAATTGC-3’, bm4 3’race 1º 5’-
GAACCATTGCTGCCAATATAC-3’, bm4 3’race nested 5’-
GCCAATCTTGCCTTCAGTG-3’. The specificities of initial PCR products were tested using nested vector and gene-specific primers. Amplified PCR products were cloned into the TOPO 2.1 vector (Invitrogen, Carlsbad, CA) and the DNA sequence determined by MWG (High Point, NC). About 10 to 20 clones were sequenced for each RACE product. Sequence data from both 5’ and 3’ RACE was compiled and the full length cDNA (flcDNA) sequence compiled (Seqman Program, DNASTAR,
Madison, WI) and aligned with the corresponding genomic sequence to confirm gene structure (MegAlign, DNASTAR, Madison WI).
Gene Maps
The C. elegans tyra-2 gene map was created from the EST wormbase database
(www.wormbase.org). The C. briggsae CbG109562 gene map was compiled using genefinder prediction from Wormbase. The B. malayi Bm4 gene map was compiled from 5’ and 3’ RACE data (see Chapter II).
Protein alignments
Protein alignments were carried out using DNAStar MegAlign. Proteins were first annotated to include only the areas of high identity, specifically, 17 aas prior to the first conserved aas in TM I (N1.50), 10 aas after TM V and before TM VI and 15 aas after TM VII, were removed. Annotated protein sequences were uploaded into
MegAlign and initially aligned with Clustal W using default parameters and fine-
73 tuned by hand. Files were saved in a Pileup GCG format and uploaded into
BOXSHADE (http://bioweb.pasteur.fr/seqanal/interfaces/boxshade.html) to identify identical (white on black) and similar (black on grey) aas. BOXSHADE.ps files were downloaded and opened in Canvas X for figure construction.
Phylogenetic tree
TA, OA and trace amine-associated receptor (TAAR) protein sequences were downloaded from NCBI and annotated to include primarily the seven transmembrane regions as for alignments. Annotated sequences were initially aligned using
MegAlign in DNAStar with Clustal W using default parameters and fine-tuned by hand (all alignments available on request). Bootstrapping was undertaken in DNAStar
(1000 replicates with random seed) and trees were compiled in PAUP. C. briggsae
(Cb) and C. elegans (Ce) protein sequences were downloaded from Wormbase
(www.wormbase.org) and all other sequences were from NCBI, accession numbers as follows. Tyramine/Octopamine receptors: Anopheles gambiae (AgOA: EAA06361),
Aplysia californica (AcOA: AAF37686), Anopheles gambiae (AgTA: EAA07468.2),
Aplysia kurdai (AkOA: AAF28802), Apis mellifera (AmOA: NP_001011565, AmTA:
CAB76374.1, AmPTA: XP_394231.2), Ascaris suum (AsTa/OA: AAS59268),
Bophilus microplus (BmiOA: CAA09335), Bombyx mori (BmoOA: BAD11157),
Drosophila melanogaster (DmOA: NP_524669 DmOA2 (OCTB1_DROME)
Q9VCZ3, DmOA3 (OCTB3_DROME) Q4LBB6, DmTA: ABE73326, DmTA2
AAK57748.1), Heliothis virescens (HvOA: CAA64864), Locusta migratoria (LmOA:
CAA49269), Lymnaea stagnalis (LsOA1: O77408, LsOA2: O01670), Manduca sexta
74 (MsPOA: ABI33825.1), Papilio xuthus (PxTA: BAD72869.1), Periplaneta americana (PaOA: AAP93817). TAARs: Homo sapiens (HsTAAR1: AAI01826) and
Mus musculus (MmTAAR1: AAK71238).
Cloning and sequencing Bm4 cDNA.
Full length Bm4 cDNA was amplified from a B. malayi cDNA RACE library constructed using total RNA isolated from male and female specimens (a kind donation from David Spiro, TIGR, Washington, DC) and the BD SMARTTM RACE cDNA Amplification kit (BD Biosciences, CA), using the manufacturer’s protocol.
Gene specific primers were used to amplify full length cDNA via PCR
(5’CCCGGGATGCAAGAGGTGCACATCGAC
3’CCGCGGCTATCGCTTTACATACTCTCTTGC); restriction enzyme sites are in italics and predicted start and stop codons in bold face. Amplified PCR products were run on 1% agarose gels. Bands of correct size were purified and cloned into TOPO
2.1 (Invitrogen) and sequenced by MWG.
Expression of Bm4 in COS-7 and HEK-293 cell lines
Bm4 flcDNA was further cloned into pDisplayTM mammalian expression vector using the restriction enzyme sites 5’ Xma I and 3’ Sac II in frame of an N- terminal HA tag (Invitrogen) and reading frame verified by sequencing MWG. Bm4 constructs were prepared by Eppendorf Maxi prep and transiently transfected into
COS-7, NIH-3T3, CHO and HEK-293t or stably in HEK-293 cells. Bm4 constructs were transiently transfected into COS-7, NIH-3T3 CHO and HEK-293t cells at a
75 range of concentrations (2 ng-8 ng) and at a density of ~5x105 cells per 10 cm dish using Lipofectamine 2000 at 1 µg:3 µl. Temperature shocked cells were moved to a
30 ºC incubator 24 hours post-transfection. Cells were harvested ~48 hours post- transfection. Stable cell lines were created in a similar way, but instead of harvesting at 48 hours, the culture medium was changed to contain 800 µg/ml of antibiotic G418 to select for stable clones. Immun flurescence staining and radioactive binding of membrane fractions approaches were used to select stable colonies.
Immunoflurescence of cell stable expressing Bm4
Cells were grown and fixed on cover slips with HistochoiceTM. Fixed cells were washed three times with phosphate buffered saline (PBS) and non-specific sites were blocked with 10% heat-inactivated goat serum for 30 minutes. Each coverslip was incubated with monoclonal anti-HA Probe (1:2, 000 Santa Cruz Biotechnologies,
Santa Cruz, CA) for 1 hour at RT, washed three times with PBS, and incubated with anti-mouse FITC-conjugated antibody (1:50, Jackman Immunoresearch Laboratories,
Inc. (West Grove, PA) protected from light. Coverslips were washed three times in
PBS, mounted in Vectashield hard set mounting media with DAPI (Vector
Laboratories Inc, Burlingame, CA) and examined for fluorescence using a Zeiss
Axiophot with a FITC filter.
Membrane preparation
Crude membrane fractions were prepared from transfected and stable cell lines. Cells were first washed in ice-cold PBS and incubated in hypotonic solution [15
76 mM Tris-HCl pH 7.4, 1 mM EDTA, 1mM phenylmethylsulfonylfluoride, PMSF)] for five minutes at 4 ºC. Resuspended cells were lysed on ice by sonication (10 pulses of
15 seconds at 45 second intervals) and then centrifuged at 200 x g for 10 minutes.
The supernatant was collected and centrifuged at 98,000 g for 45 minutes at 4 ºC. The pellet containing the membrane fraction was resuspended in TEM buffer (50 mM
Tris-HCl pH 7.4, 0.5 mM EDTA, 10 mM MgCl2, 1 mM PMSF) and stored at -80 ºC.
Radioligand binding assay
Saturation and inhibition assays were conducted in 96 well microtiter plates in a final volume of 100 µl. For saturation binding, 15 µg of membrane protein was incubated with [3H]LSD or [3H]TA at a range of concentrations (1 nM - 40 nM) to obtain the Bmax and Kd. Inhibition assays were performed with various ligands to determine their affinities to displace 10 nM [3H]TA. For both assays, total and non- specific binding were determined in the absence and presence of 1000-fold excess of unlabeled LSD or TA, respectively. Receptor binding experiments were incubated at room temperature for 1 hour in restricted light and terminated by filtration through a
96 well micro G/B filter plates (PerkinElmer), previously soaked in 0.3 % polyethyleneimine. Filters were washed three times in ice-cold TEM buffer, dried and the radioactivity retained was measured by liquid scintillation counting. Binding data was analyzed by curve fitting (DeltaGraph, DeltaPoint Inc, 1993). Each experiment was performed in triplicate at least three times. Student t-tests were performed using http://www.physics.csbsju.edu/stats/t-test.html.
77 Results
Cloning and sequence analysis of Bm4
A gene encoding a putative C. elegans TYRA-2 homolog, Bm4, was identified in the B. malayi genome based on the identity of its predicted ORF with TYRA-2. To isolate the cDNA encoding Bm4, gene-specific primers were designed against the start and stop codons identified from 5’ and 3’RACE. Using a B. malayi cDNA RACE library as a template, a 1.42-kb fragment was isolated. The predicted amino acid sequence of Bm4 contains seven putative TM regions and characteristic GPCR motifs: a DRY motif at the end of TM III and NPxxY motif within TM VII (Moro, Lameh et al. 1993; Barak, Tiberi et al. 1994).
B. malayi gene maps were compiled from the 5’ and 3’ RACE data and the B. malayi genomic sequence database. Bm4 shares 16 of the 18 intron/exon borders with tyra-2, and the predicted Bm4 protein sequence is 78% identical to TYRA-2, with the highest identity, 83%, in the TM regions (Figure 9). Only 14 aas in the TM regions forming the ligand-binding pocket differ between the two receptors: A3.33T and
E3.36V in TM III, V5.40I, T5.51L, L5.52F and L5.52I in TM V, L6.37F, T6.45A,
V6.46I, A6.49T, T6.53I and L6.57V in TM VII and E7.35W and V7.55I in TM VII
(Figure 9).
Identification of Bm4, a putative C. elegans TYRA-2 homologue in B. malayi
Thirty-four TA or OA receptors from 19 different species were identified in an
NCBI search (see Materials and Methods) using TYRA-2. The G-protein coupling of
18 of the 34 receptors is known or strongly suggested through experimentation
78 (Vanden Broeck, Vulsteke et al. 1995; von Nickisch-Rosenegk, Krieger et al. 1996;
Gerhardt, Bakker et al. 1997; Gerhardt, Lodder et al. 1997; Blenau, Balfanz et al.
2000; Chang, Li et al. 2000; Borowsky, Adham et al. 2001; Holt, Subramanian et al.
2002; Bischof and Enan 2004; Ohta, Utsumi et al. 2004; Ono and Yoshikawa 2004;
Balfanz, Strunker et al. 2005; Cazzamali, Klaerke et al. 2005; Maqueira, Chatwin et al. 2005; Dacks, Dacks et al. 2006). A phylogenetic tree of annotated receptors was generated (see Materials and Methods) using maximal parsimony and an unrooted tree compiled using PAUP (Figure 10). Surprisingly, the TA and OA receptors did not cluster according to species, but instead, clustered according to documented G-protein coupling to Gαs and Gαi/o. Two main clades are present within the tree; clade I contains all of the characterized Gαs-coupled OA receptors and clade II all of the Gαi/o receptors. Bm4 clusters in clade II with TYRA-2, CbP02670 (C. briggsae predicted homologue) and Lymnae stagnalis (LsOA2) suggesting that Bm4 is also Gαi/o-coupled
(Figure 9). Interestingly, a distinctive branch, within clade II includes a number of uncharacterized receptors (DmTA2, AmPTA, CeM03F4.3, CbP03587 and Bm5) that based on this alignment are predicted to couple Gαi/o.
Multiple alignments of these TA/OA receptors revealed, that specific aa are conserved in the individual clades (Figure 11). For example, W4.52 and R6.32 are present in all Gαi/o -coupled receptors. In contrast all Gαs-coupled receptors contain L,
V, Y, I or C at 4.52 and an A at 6.32. Similarly, R2.39 and S3.47 are conserved in all
Gαs-coupled receptors, with Gαi/o-coupled receptors containing P/Q at 2.39 and G/A at
3.47. In addition, 65 aas are conserved in both TYRA-2 and Bm4 but not in other
Gαi/o-coupled clade II receptors (Figure 11).
79
A Ce tyra-2 49 114 121 188 195 100 101 191 132 122 81 Scale Bm4 149 117 190 191 97 100 268 120 112 81 100 nt Exon 1kB Intron TM B I II CeTYRA-2 18 QI LKG SAL FLL VL WTI FAN SLV FIV LYK NPR LQT VPN LLV GNL AFS DLA LGL IVL PLS SVYA IAG EW VFP D Bm4 11 NI LKG SAL TLL VI WTI AAN ILV FVV LYK NPH LQT VPN LLV ANL AFS DSC LGV IVL PLS SIYA IAN EW LFT S III IV CeTYRA-2 AL CEV FVS ADI LCS TAS IWN LSI VGL DRY WAI TSP VAY MSK RNK RTA GIMI LSV WIS SAL IS LAP LLG WKQ TA Bm4 TL CVV FVS ADI LCS TAS IWN LSI VGL DRY WAI TTP RAY MAK RNK RTV AYLI LSV WFS SAL IS LAP FFG WKQ VA V CeTYRA-2 QT PNL IYEK NNT VR QC TFL DLP SYT VYS ATGSFF IPT LLM FFV YF KIYQ AFAKH 215 378 A AKE RRG VKV LG Bm4 ER GNM I--K ING TW QC VFL DLP SYT IYS ATGSFF IPL FIM FFV YY KIYQ TFAKH 207 207 R AKE RRG VKV FG VI VII CeTYRA-2 I ILG CFT VCW APF FTM YVL VQF CKDCSP NAH IE MFI TW LGY SNS AMN PII YTV FNR DYQ IAL KRL FTS E 459 Bm4 I ILG CFA ICW TPF FIM YVV VQF CSSCQV DPH IW MFI TW LGY SNS AMN PII YTI FNH DYQ NAL KGL FRG N 465
Figure 9: Gene maps and protein alignment. A, Gene maps were constructed from EST data from Wormbase for Ce tyra-2 and from 5’ and 3’ RACE data for Bm4. Exons are boxes and intronic sequence is depicted by solid lines. A grey box within exons depicts sequence coding for TM regions. B, Annotated protein sequences were aligned by MegAlign using Clustal W and sequence analysis by BOXSHADE. White type on black background indicates identical amino acid residues; black type on grey background indicates similar residues. Predicted TM regions are indicated by a solid line above the alignment.
80 Characterization of Bm4 after heterologous expression in mammalian cells
The Bm4 cDNA was cloned into pDisplayTM and transiently transfected with
Lipofectamine 2000TM into a number of mammalian cell lines (NIH-3T3, COS-7,
HEK-293 or CHO), resulting in the expression of an N-terminal HA tagged receptor.
Unfortunately, expression only reached a maximum of ~7% in each of these lines (as visualized by immunofluorescence to the N-terminal HA tag) and isolated membranes did not exhibit significant saturable [3H]LSD binding. Heat shock 24 hours after transfection, as described for the increased expression of nematode neuropeptide receptors (Kubiak, Larsen et al. 2003), or increasing the amount of transfected DNA
(maximum of 8 µg/10 cm plate) did not increase expression. Therefore, stable HEK-
293 cell lines expressing Bm4 were prepared as described in Methods. Membranes from HEK293 cells stably expressing Bm4 exhibited saturable, high affinity [3H]LSD binding, with a Bmax and Kd of 1.79 ± 0.2 pmol/mg and 18.1 ± 0.93 nM, respectively
(Figure 12). Most importantly, [3H]LSD binding was inhibited with high affinity by
TA (data not shown). As predicted, these isolated membranes also exhibited
3 saturable, high affinity [ H]TA binding with a Bmax and Kd of 1.15 ± 0.05 pmol/mg and 15 ± 0.2 nM, respectively (Figure 12).
To determine the pharmacological profile of the Bm4, potential tyraminergic ligands were tested at varying concentrations for their ability to displace 10 nM [3H]
81 Gαs DmOA
55 DmOA2 36 DmOA3 41 MmTAAR1 * 28 25 HsTAAR1 **** **** 32
43 AkOA 3 **** AcOA 2 LsOA1 63 **** 92 46 63 PaOA 21 CeSER-2 *** 25 MsOA 1 * 23 CbP1888 3 **** 52 **** 42 26 19 *** 21 AsTA/OA 40 **** 18 **** 45 * AgOA 34 * AmTA2 62 I AmOA 46 AgTA2 31 78 30 10 II DmTA **** 36 2 12 ** *** CeSER-3 * 17 34 34 **** 1 34 24 **** CbP03135 BmoOA 19 *** 42 7 2 17 * 5 HvOA 7**** 34 59 III PxTA LmOA 49 BmiOA 47 **** DmTA2 **** 41 88 71 AmPTA
LsOA2 97 **** 55 50 **** 7 CeM03F4.3 CbP03587 30 **** 1 Bm5 CeTYRA-2 **** CbP02670 2 47 50 changes Gαi Bm4 Bootstrap
100-90% **** 89-80% *** 79-70% ** 69-50% *
82 Figure 10: Unrooted phylogenetic tree of TA and OA receptors Protein sequences were annotated to include only the seven TM regions; specifically the N-termini were deleted including 19 aa before the first conserved aa in TM I (N1.50). The third intracellular loop was deleted 10 aa after TM V and before TM VI. C-termini were also removed 15 aa after TM VII. Annotated sequences were initially aligned in DNAStar with Clustal W using default parameters and fine tuned by hand (all alignments available on request). Bootstrapping was undertaken in DNAStar (1000 replicates with random seed) and trees were compiled in PAUP.
83 IIIIII Bm4 11 NILKG SALTLLVIWT IAANILVFVVLYKNPHLQTVPNLLVANLAFSDSC LGVIVLPLSSIYAIAN-EWLF TSTLCVVFVS ADILCSTASIWNLSIVGLDRYW CeTYRA-2 18 QILKG SALFLLVLWT IFANSLVFIVLYKNPRLQTVPNLLVGNLAFSDLA LGLIVLPLSSVYAIAG-EWVF PDALCEVFVS ADILCSTASIWNLSIVGLDRYW CeSER-2 46 LVLGT ITYLVIIAMT VVGNTLVVVAVFSYRPLKKVQNYFLVSLAASDLA VAIFVMPLHVVTFLAGGKWLL GVTVCQFFTT ADILLCTSSILNLCAIALDRYW DmTA 109 ALLTA LVLSVIIVLT IIGNILVILSVFTYKPLRIVQNFFIVSLAVADLT VALLVLPFNVAYSILG-RWEF GIHLCKLWLT CDVLCCTSSILNLCAIALDRYW BmiOA 57 AVGTA LSLSFITVFT VVGNVLVICSVFNHRPLRTVQNVFLVSLALADIA VALLVMPFNVAYSIMG-RWVF GLHFCELWLT CDVLCCTASILNLCAIALDRYW HvOA 52 AICTA IVLTLIIIST IVGNILVILSVFTYKPLRIVQNFFIVSLAVADLT VAILVLPLNVAYSILG-QWVF GIYVCKMWLT CDIMCCTSSILNLCAIALDRYW BmoOA 54 AICTA IILTMIIIST VVGNILVILSVFTYKPLRIVQNFFIVSLAVADLT VAILVLPLNVAYSILG-QWVF GIYVCKMWLT CDIMCCTSSILNLCAIALDRYW AmOA 65 ILVTL IVLAIVNVMV VLGNVLVILAVYHTSKLRNVTNMFIVSLAVADLM VGLAVLPFSATWEVFK-VWIF GDLWCSIWLA VDVWMCTASILNLCAISLDRYL PaOA 25 LIASL VVLLLINVMV IVGNCLVIAAVFMSSKLRSVTNLFIVSLAVADLM VGLAVLPFSATWEVFK-VWIF GDVWCSIWLA VDVWMCTASILNLCAISLDRYV DmOA 153 WVFKA FVMLLIIIAA ICGNLLVIISVMRVRKLRVITNYFVVSLAMADIM VAIMAMTFNFSVQVTG-RWNF SPFLCDLWNS LDVYFSTASILHLCCISVDRYY AcOA 38 LVLRG MAMAAIMVGA IFGNVLVISSVLRFGRLRAITNFFIVSLAFADLL VAILVMPFSASMEISG-KWVF GRTMCDIFNA NDVLFSTASIIHLCCISMDRYI AkOA 38 LVLRG MAMAAIMVGA IFGNVLVISSVLRFERLRAITNFFIVSLAFADLL EAILVMPFSASMEISG-KWVF GRTMCDIFNA NDVLFSTASIIHLCCISMDRYI MmTAAR1 22 QASLY SLMSLIILAT LVGNLIVIISISHFKQLHTPTNWLLHSMAIVDFL LGCLIMPCSMVRTVER-CWYF GEILCKVHTS TDIMLSSASIFHLAFISIDRYC HsTAAR1 23 RASLY SLMVLIILTT LVGNLIVIVSISHFKQLHTPTNWLIHSMATVDFL LGCLVMPYSMVRSAEH-CWYF GEVFCKIHTS TDIMLSSASIFHLSFISIDRYY * * * * * * * * * ** * *** IV V Bm4 AITT PRAYM AKRNKRTVAYLILSVWFSSALISLAPFF-GWKQV AERGNMI------KINGT WQ------CVFLDLPSYTIYSATGSFFIPLFIMFFVY CeTYRA-2 AITS PVAYM SKRNKRTAGIMILSVWISSALISLAPLL-GWKQT AQTPNLIYE------KNNTV RQ------CTFLDLPSYTVYSATGSFFIPTLLMFFVY CeSER-2 AIHN PINYA QKRTTKFVCIVIVIVWILSMLISVPPII-GWNNW QEN------MMED S------CGLSTEKAFVVFSAAGSFFLPLLVMVVVY DmTA AITD PINYA QKRTVGRVLLLISGVWLLSLLISSPPLI-GWNDW PDE------FTSA TP------CELTSQRGYVIYSSLGSFFIPLAIMTIVY BmiOA AIHD PINYA QKRTLRRVLLSIFLVWVISALISVPPLI-GWNDW PEQ------FDET TP------CRLTQETGYVLYSASGSFFIPLLIMSIVY HvOA AITD PINYA QKRTLERVLLMIGVVWVLSLIISSPPLL-GWNDW PDV------FEPD TP------CRLTSQPGFVIFSSSGSFYIPLVIMTVVY BmoOA AITD PINYA QKRTLERVLFMIGIVWILSLVISSPPLL-GWNDW PEV------FEPD TP------CRLTSQPGFVIFSSSGSFYIPLVIMTVVY AmOA AVTR PVSYP QIMSPRRARLLVATVWILSFVICFPPLV-GWKDK RSHPAYNMTF AQNG--PFNTT TIFVPVKPCP WICELTNDAGYVVYSALGSFYIPMLVMLFFY PaOA AVTR PVTYP SIMSSGRAKLLIAGVWVLSFVICFPPLV-GWKDK REDPPSNSSG SLFGSRPLTPP PALQVPAPCP WICELTNDAGYVVYSALGSFYLPMLVMLFFY DmOA AIVK PLKYP ISMTKRVVGIMLLNTWISPALLSFLPIFIGWYTT PQH------QQFVIQNP TQ------CSFVVNKYYAVISSSISFWIPCTIMIFTY AcOA AILH PLQYE SKMTRPRALLMLGVTWVASVLISYIPVYSQLYTT RQN------VQALLTDP DS------CPFIVNKVYAGVSSSVSFWIPCTIMIFVY AkOA AILH PLQYE SKMTRPRAMLMLGVTWVASVLISYIPVYSQLYTT RQN------VQALLTDP DS------CPFIVNKVYAGVSSSVSFWIPCTIMIFVY MmTAAR1 AVCD PLRYK AKINISTILVMILVSWSLPAVYAFGMIFLELNLK GVEELYR------S-QVSDL GG------CSPFFSKVSGVLAFMTSFYIPGSVMLFVY HsTAAR1 AVCD PLRYK AKMNILVICVMIFISWSVPAVFAFGMIFLELNFK GAEEIYY------K-HVHCR GG------CSVFFSKISGVLTFMTSFYIPGSIMLCVY *** * ****** VI VII Bm4 YKIYQTFAKHR 207 386 AKERRGVKVFGIILGCFAICWTPFFIMYVVVQFC -SSCQVDPHIWMFITWLGYSN SAMNPIIYTI FNHDYQNALKGLFRGN 465 CeTYR A-2 FKIYQAFAKHA 216 378 AKERRGVKVLGIILGCFTVCWAPFFTMYVLVQFC -KDCSPNAHIEMFITWLGYSN SAMNPIIYTV FNRDYQIALKRLFTSE 457 CeSER-2 VKIFISARQRV 254 365 AKEKRAAKTIAVIIFVFSFCWLPFFVAYVIRPFC -ETCKLHAKVEQAFTWLGYIN SSLNPFLYGI LNLEFRRAFKKILCPK 448 DmTA IEIFVATRRRL 300 522 SKERRAARTLGIIMGVFVICWLPFFLMYVILPFC -QTCCPTNKFKNFITWLGYIN SGLNPVIYTI FNLDYRRAFKRLLGLN 601 BmiOA LKIFLATRRRL 248 334 SRERRAARVLGIVMGVFVLCWLPFFIMYVTAAFC -DHCVQSDRLVNFITWLGYVN SALNPVIYTV FNTDFRRAFRSLLCSG 413 HvOA FEIYLATKKRL 248 397 TRERRAARTLGIIMGVFVVCWLPFFVIYLVIPFC -ASCCLSNKFINFITWLGYCN SALNPLIYTI FNMDFRRAFKKLLCMK 476 BmoOA FEIYLATKKRL 245 399 TRERRAARTLGIIMGVFVVCWLPFFVIYLVIPFC -VSCCLSNKFINFITWLGYVN SALNPLIYTI FNMDFRRAFKKLLFIK 478 AmOA WRIYNAAVSTF 278 449 RMETKAAKTLG IIVGGFILCW LPFFTMYLVRAFC-RNCIHP-TVFSVLFWLGYCN SAINPCIYAL FSKDFRFAFKSIICKC 528 PaOA WRIYRAAVQTF 239 453 RMETKAAKTLG IIVGGFIVCW LPFFTMYLVRAFC-EDCIHH-LLFSVLFWLGYCN SAINPCIYAL FSKDFRFAFKRIICRC 562 DmOA LAIFREAN-RM 347 403 KREHKAARTLG IIMGTFILCW LPFFLWYTLSMTC-EECQVPDIVVSILFWIGYFN STLNPLIYAY FNRDFREAFRNTLLCL 483 AcOA IRIFLEARKQM 233 269 KREHKAAKTLG IIMGAFILCF LPFFSWYVATTMCRDSCPYPPLLGSALFWVGYFN SCLNPVIYAY FNREFRTAFKKLLRLD 350 AkOA IRIFLEARKQM 233 269 KREHKAAKTLG IIMGAFILCF LPFFSWYVATTMCRDSCPYPPLLGSALFWVGYFN SCLNPVIYAY FNRDFRTAFKKLLRPD 350 MmTAAR1 YRIYFIAKGQQ 219 240 SKETKAAKTLG IMVGVFLVCW CPFFLCTVLDPFL--GYVIPPSLNDALYWFGYLN SALNPMVYAF FYPWFRRALKMVLLGK 319 HsTAAR1 YRIYLIAKEQQ 220 232 SKERKAVKTLG IVMGVFLICW CPFFICTVMDPFL--HYIIPPTLNDVLIWFGYLN STFNPMVYAF FYPWFRKALKMMLFGK 322 * * * * *** * ** ** ** * *
Figure 11: Alignment of characterized TA, OA and TAA receptors Characterized TA, OA and TAA receptor protein sequences were aligned by MegaAlign using ClustalW and sequence analysis by BOXSHADE. White type on black background indicates identical amino acid residues; black type on grey background indicates similar residues. Predicted TM regions are indicated by a solid line above the alignment and identical amino acids are marked with an asterisk *.
84 A
1500
1000
500
0 0 10000 20000 30000 40000 [3H-LSD]pM B 1500
1000
500
0 ² 0 10000 20000 30000 40000 3 [ H-TA]HTA] pM
85
Figure 12: Saturation binding for [3H] LSD and [3H] TA with membranes expressing Bm4. Membranes prepared from stably expressing HEK-293 cells were incubated with [3H]LSD (A) or [3H]TA (B) at concentrations ranging from 1 nM–40 nM in the presence or absence of 1000-fold unlabelled LSD or TA, respectively. Specific binding is represented by closed squares and non-specific binding by triangles. Bm4 3 displays affinity for [ H]LSD with a Kd of 18.1 ± 0.92 nM and Bmax of 1.79 ± 0.2 3 pmol/ mg and an affinity for [ H]TA with a Kd of 15 ± 0.2 nM and Bmax of 1.15 ± 0.05 pmol/ mg.
86 TA in membranes isolated from HEK293 cells stably expressing Bm4. The rank order of potency was as follows; TA > chlorpromazine > cyproheptadine = mianserin = DA
> synephrine > phentolamine > tolazoline = OA, confirming the identification of Bm4 as a TA receptor.
Comparison of the pharmacological profiles of Bm4 and TYRA-2
To determine whether Bm4 and its putative C. elegans orthologue, TYRA-2, bound ligands with similar affinities, the inhibition binding of both receptors was compared under identical expression and assay conditions in membranes isolated from
HEK-293 cells expressing the individual receptors. Interestingly, the pharmacological profiles of Bm4 and TYRA-2 were quite similar (Table 3).
However, Bm4 had a significantly higher affinity for chlorpromazine (pKis = 7.6 ± 0.2 and 6.49 ± 0.1, respectively) and TYRA-2 had a significantly higher affinity for phentolamine (pKis = 5.81 ± 0.1 and 6.4 ± 0.1, respectively). Bm4 also had a significantly greater affinity for DA than OA (pKis = 6.74 ± 0.15 and 5.66 ± 0.12, respectively), while TYRA-2 had identical affinities for these two ligands (pKis = 6.22
± 0.1 and 6.19 ± 0.1, respectively). The rank order of potency for TYRA-2 expressed in HEK-293 cells was as follows: TA > cyproheptadine > chlorpromazine = mianserin
> phentolamine > synephrine > OA = DA > tolazoline, mirroring that of TYRA-2 characterized previously after expression in COS-7 cells (Table 3 and 4 and Rex et al.,
2005).
87 AA 100 TA OA 80 DA
60
40
20
0 1E-101E-91E-81E-71E-61E-51E-4 [Ligand] M
B 100 Chlorpromazine Cyproheptadine Mianserin 80 Phentolamine Synephrine Tolazoline 60
40
20
0 1E-10 1E-91E-8 1E-7 1E-6 1E-5 1E-4 [Ligand] M
Figure 13: Pharmacological profile of Bm4 Membranes of HEK-293 cells expressing Bm4 were incubated with 10 nM [3H]TA in the presence of ligands at a range of concentrations. Data are representative of at least three independent experiments performed in triplicate (see Table 3 for summary of pharmacology).
88
A 100 TA OA 80 DA
60
40
20
0 1E-10-10 1E-9 - 1E-8 - 1E-7 1E-6 - 1E-5 - 1E-4 - [Ligand] M
Figure 14: Pharmacological profile of TYRA-2 Membranes of HEK-293 cells expressing TYRA-2 were incubated with 10 nM [3H]TA in the presence of ligands at a range of concentrations. Data are representative of at least three independent experiments performed in triplicate (see Table 3 for summary of pharmacology).
89
Table 3: Pharmacological profiles for Bm4 and TYRA-2
HEK-293 COS-7 Ligand Bm4 TYRA-2 TYRA-2 Tyramine (TA) 7.9 ± 0.07 7.7 ± 0.15 7.4 ± 0.14 (12.3) (26) (39) Octopamine (OA) 5.66 ± 0.12** 6.19 ± 0.1 5.85 ± 0 .14 (2155) (639) (1412) Dopamine (DA) 6.74 ± 0.15** 6.22 ± 0.1 5.79 ± 0.13 (179.5) (599) (1621) Chlorpromazine 7.6 ± 0.2 * 6.49 ± 0.1 6.6 ± 0.2 (21) (322) (229) Cyproheptadine 6.8 ± 0.1 6.96 ± 0.1 6.59 ± 0.1 (146) (109) (112) Mianserin 6.78 ± 0.13 6.56 ± 0.2 6.62 ± 0.14 (165) (271) (239) Synephrine 6.04 ± 0.2 6.11 ± 0.1 6.07 ± 0.03 (893) (775) (851) Phentolamine 5.81 ± 0.1 * 6.4 ± 0. 1 6.1 ± 0.1 (1545) (483) (794) Tolazoline 5.66 ± 0.14 5.28 ± 0.13 5.53 ± 0.3 (2150) (5150) (2951) Membranes of HEK-293 cells expressing Bm4 or TYRA-2 were incubated with 10 3 nM [ H] TA in the presence of ligands at a range of concentrations. pKis are expressed ± SEM and are an average of at least three independent experiments performed in triplicate (Kis in nM in parenthesis). TYRA-2 in COS-7 reproduced from Rex et al. (2005). * indicates significance difference from TYRA-2 in HEK-293 cells using Student’s paired t-test P * < 0.001, ** < 0.005 (95% confidence interval).
Table 4: Comparison of pharmacological profiles of Bm4 and TYRA-2
Receptor Pharmacological profile
Bm4 TA > Chlo > Cyp = Mia = DA > Syn > Phe > Tol = OA
TYRA-2 (COS-7) TA > Cyp > Chlo = Mia > Phe > Syn > OA = DA > Tol
TYRA-2 (HEK-293) TA > Cyp > Chlo = Mia > Phe > Syn > OA = DA > Tol
Chlo, Chlorpromazine; Cyp, Cyproheptadine; Mia, Mianserin; Syn, Synephrine; Phe, Phentolamine and Tol, Tolazoline.
90 Discussion
Tyramine and octopamine play important neuromodulatory roles in nematodes.
For example, in C. elegans, exogenous TA and OA cause hyperactivity and inhibit 5-
HT dependent increases in pharyngeal pumping and egg-laying (Horvitz, Chalfie et al.
1982; Niacaris and Avery 2003). In general, the effects of TA and OA appear to oppose the action of 5-HT (Arakawa, Gocayne et al. 1990). Unfortunately, given the relative impermeability of the nematode cuticle, the effects of TA and OA on intact nematodes have been measured at very high, unphysiological concentrations and given our recent identification of TA-specific receptors in C. elegans, it is unclear whether the effects reported previously for OA are not, in fact, mediated by TA receptors. Clearly, TA and OA signaling is complex and is mediated by at least 5 different receptors expressed at different levels throughout the nervous system (Rex and Komuniecki 2002; Rex, Molitor et al. 2004; Alkema, Hunter-Ensor et al. 2005;
Rex, Hapiak et al. 2005; Suo, Kimura et al. 2006). Based on studies in insects and C. elegans, TA receptors appear to couple to an inhibition of adenyl cyclase activity through Gαo and OA receptors to a stimulation of adenyl cyclase activity, presumably through Gαs, although few studies demonstrating direct G-protein coupling are available (Nathanson 1985; Gerhardt, Lodder et al. 1997; Alkema, Hunter-Ensor et al.
2005; Balfanz, Strunker et al. 2005). Most importantly, OA receptors have been useful targets for pesticide development and presumably TA and OA receptors might serve a similar role in parasitic nematodes (Hiripi, Nagy et al. 1999; McClung and
Hirsh 1999). For example, the formamidine pesticides are high affinity OA receptor agonists and activate adenyl cyclase (Nathanson 1985; Hiripi, Nagy et al. 1999).
91 These compounds induce behavioral changes, such as hyperactivity, identical to the behavioral changes seen in OA-treated insects (Evans and Gee 1980; Wierenga and
Hollingworth 1990). Similarly, cocaine, a naturally occurring insecticide, functions by blocking OA re-uptake and potentiates octopaminergic transmission (Nathanson,
Hunnicutt et al. 1993; McClung and Hirsh 1999).
In general, parasitic nematodes are difficult to culture and refractory to modern molecular genetics, so that the free-living nematode, C. elegans recently has been used as a model to potentially define key signaling pathways. Importantly, all of the commercially available anthelminthics also appear to be effective against C. elegans
(Geary and Thompson 2001; Brown, Jones et al. 2006). However, nematodes exhibit enormous diversity and it is not clear whether pathways or receptors identified in C. elegans will faithfully translate to parasitic species. Indeed, the recent characterization of putative 5-HT1-like receptor homologues from C. elegans and H. contortus suggests that ligand-binding to receptors from the two species might differ significantly (Olde and McCombie 1997; Smith, Borts et al. 2003). However, since these receptors were characterized under dramatically different expression and assay conditions it is difficult to make any definitive comparisons. In fact, species differences in the pharmacologies of homologous receptors are not uncommon. For example, the human trace amine associated receptor 1 (HsTAAR1) and its rat homologue (RnTAAR1) have markedly different agonist potencies after expression in
AV12-664 cells (Wainscott, Little et al. 2007). Therefore, the present study was designed to characterize the two most identical of the C. elegans/B. malayi BA receptor homologues, TYRA-2 and Bm4, under identical expression and assay
92 conditions to gain insight into how useful C. elegans BA receptors might be as surrogates for drug discovery.
The genome of the filarial parasite is nearly complete. Surprisingly, in contrast to the 15 genes encoding BA receptors in C. elegans and its close relative
Caenorhabditis brigssae, the B. malayi genome appears to contain only seven predicted BA receptors, although all of the predicted B. malayi receptors appear to have clear homologues in C. elegans. TA and OA receptors cluster according to predicted G-protein coupling, suggesting that the tree constructed in the present study may be useful for predicting the G-protein coupling of the as yet uncharacterized
TA/OA receptors. For example, all Gαs-coupled receptors cluster in clade I and we predict that MsOA, AgOA and CbP03135 are also Gαs-coupled OA receptors. As predicted, HsTAAR1 that has high affinity to TA and couples to Gαs, is found in clade
I, supporting clustering based on coupling and not ligand specificity (Borowsky,
Adham et al. 2001). In contrast, all Gαi/o characterized receptors fall into clade II and we predict that AsTA/OA, CbP1888, AgTA2, PxTA, BmiOA, LsOA2, Bm4, Bm5,
CbP02670, CeM03F4.3, AmPTA, DmTA2 and CbP03587 are Gαi/o-coupled TA receptors. Based on an analysis of the sequence alignments, 41 aa are conserved in the TA/OA GPCR family; this number is significantly higher than the 33 aas that are conserved in the 12 human 5-HT receptors reported previously (Kroeze, Kristiansen et al. 2002). Interestingly, 65 aas that are conserved in both TYRA-2 and Bm4 are different in other Gαi/o clustered receptors, supporting the pharmacological differences between TYRA-2 and SER-2 reported previously (Rex and Komuniecki 2002; Rex,
Hapiak et al. 2005).
93 TYRA-2 and Bm4 differ by only 14 amino acids in the transmembrane regions forming the ligand-binding pocket (TM III, V, IV and VII) and both receptors also share the S1xxxxS5 motif in TM V thought to be responsible for TA binding (Ohta,
Utsumi et al. 2003). The pharmacological profile of TYRA-2 expressed in HEK-292 cells mimicked that of TRYA-2 expressed in COS-7 cells published previously (Rex,
Hapiak et al. 2005). This result is perhaps surprising as pharmacologies of other
GPCRS are often affected by cell line and expression level. In contrast, although
Bm4 and TYRA-2 exhibited nearly identical affinities for predicted agonists, such as
TA and mianserin, they exhibited significantly different affinities for a number of antagonists, most notably, chlorpromazine and phentolamine (Table 3).
Chlorpromazine is a relatively non-specific antagonist and binds to DA (D2), 5-HT1 and 5-HT2, histamine (H1) and muscarinic (M1/M2) receptors with high affinity.
Chlorpromazine interacts with aas in TM II of the γ subunit of the nicotinic actetylcholine receptor (CTL*SIS*xxL* with *indicating aa bound to chlorpromazine), but no studies on the specificity of chloropromazine binding to GPCRs have been reported (Revah, Galzi et al. 1990). The aas binding chlorpromazine in the nicotinic receptor are spaced about 10.7Å apart in the TMII α−helix, a distance compatible with the structure of chlorpromazine. Phentolamine is a non-selective alpha-adrenergic antagonist whose structure is similar to chlorpromazine (Figure 15). Both chlorpromazine and phentolamine have differential affinities for the two receptors, suggesting that the 10 aa differences between the two receptors in the TM regions could account for the differences in binding between these two structurally similar ligands (Figure 9 and 15).
94
N
N N H
N Cl- N OH
S chlorpromazine phentolamine
Figure 15: Chemical structure of chlorpromazine and phentolamine
The physiological role of TYRA-2 in C. elegans has yet to be determined.
Although, GFP fluorescence from tyra-2::gfp translational fusions is observed in a number of neurons in the pharynx, nerve ring and tail (Rex, Hapiak et al. 2005), TA dependent phenotypes have yet to be expressed in tyra-2-null worms. The results of the present study demonstrate that even two closely related nematode BA receptors, differing by only 14 aas in the ligand binding TM regions, still exhibit significant pharmacological differences. These results highlight the importance of using receptors from the parasitic nematode species of interest for drug discovery. These results highlight the utility of a bioinformatics approach for predicting ligand specificity and coupling of receptors from parasitic nematodes and the importance of using receptors from specific parasitic nematodes for drug discovery.
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