The FLP-side of nematodes Paul McVeigh 1 , Timothy G. Geary 2 , Nikki J. Marks 1 and Aaron G. Maule 1 1 Parasitology, School of Biological Sciences, Queen’s University Belfast, 97 Lisburn Road, Belfast BT9 7BL, UK 2 Institute of Parasitology, McGill University, 21111 Lakeshore Road, Ste. Anne de Bellevue, Quebec, H9X 3V9, Canada The central role of FMRFamide-like peptides (FLPs) in nematode motor and sensory capabilities makes FLP signalling an appealing target for new parasiticides. Accumulating evidence has revealed an astounding level of FLP sequence conservation and diversity in the phylum Nematoda, and preliminary work has begun to identify the nematode FLP receptor complement in Caenorhabditis elegans, with a view to investigating their basic biology and therapeutic potential. However, much work is needed to clarify the functional aspects of FLP signalling and how these peptides exert their effects at the organismal level. Here, we summarize our current knowledge of nematode FLP signalling. A relatively short history The most recent reviews in this journal concerning parasitic nematode FMRFamide-like peptides (FLPs, named from the sequence Phe-Met-Arg-Phe-NH 2 ) date back to 1996 [1,2], published during the first wave of research into this large family of neuropeptides. At that early stage, data had accumulated on the structure, immunochemical localization and physiological effects of FLPs in nematode tissue assays, complemented by results from a small number of gene knockout studies in Caenorhabditis elegans. Ten years on, these basic data still form the foundation of our understanding of the nematode FLP system. However, significant strides in mapping FLP expression and FLP receptor biology have been made in the past 5–6 years, due almost entirely to the exploitation of C. elegans [3,4]. By contrast, nothing has been published on FLP receptors in parasitic nematodes, and only recently have data begun to accumulate on the flp gene complement of these organisms [5]. With only one nematode parasite gen- ome-sequencing project under way (Brugia malayi [6]), it is unlikely that the level of molecular knowledge of any nematode parasite will rival that in C. elegans in the immediate future. However, the more than 340 000 expressed sequence tags (ESTs) currently available from 44 species of parasitic nematode [5] represent a resource which, if exploited in the context of known C. elegans neurobiology data, can be used to improve our understanding of the parasite FLP system. FLPs: the basics FLPs (also known as FMRFamide-related peptides or FaRPs) are the largest and most diverse family of neuropeptides known [7]. As their name suggests, they show similarity to FMRFamide, a cardioactive tetrapep- tide isolated from the clam Macrocallista nimbosa [8]. Although RFamide peptides have been identified through- out the invertebrates and to some extent in vertebrates [9], the FLP system of nematodes is unusually complex – at least 32 flp genes are known in the phylum Nematoda [5] (Table 1). Each flp gene encodes distinct and characteristic variations on the FLP C-terminal tetrapep- tide motif x-x o -Arg-Phe-NH 2 , (where x is any amino acid except cysteine and x o is any hydrophobic amino acid except cysteine; cysteine has not been reported in a FLP; Table 1). This structural diversity is reflected in the range of FLP-induced physiological responses, which comprise a variety of different effects on muscle, motorneurons, behaviour and sensory abilities [10–15] (Table 2). Mechanisms involved in processing of FLP propeptides are detailed in Box 1. The FLP tetrapeptide motif means that some peptides not previously considered as ‘true FLPs’ are included in the grouping, such as many vertebrate neuropeptides ending in Arg-Phe-NH 2 (–RFamide peptides). This more inclusive view of FLPs might represent a more realistic vision of the evolutionary relations of –RFamide peptides, especially in view of current evidence showing a conserved role for these peptides and their receptors in the control of feeding behaviour [9]. It is clear that nematode FLPs have a broad role in their nervous systems, whereas vertebrate FLPs appear to have somewhat restricted distributions and a more focussed remit of roles. Currently, the expanding knowledge of vertebrate FLPs does not serve to weaken the potential of parasitic helminth FLP signalling as a target for parasite control. The nematode FLP system undoubtedly poses some interesting biological questions, and understanding this signalling system is a valuable goal in itself. Nevertheless, the drive towards discovery of novel therapeutic compounds to target the system in parasites has also provided a powerful impetus for FLP research. FLPs are inextricably linked to parasite motor and sensory function [7] and are conserved in a range of invertebrate pest species [16], so the parasite FLP system has obvious potential as a target for new anthelmintics (helminth- specific drugs) or even endectocides (drugs that act on both endo- and ectoparasites). All nematodes have a similar FLP complement Caenorhabditis elegans is currently known to have at least 29 flp genes, encoding at least 68 distinct FLPs [3,5,17]. Although data on the FLP complement of other Corresponding author: McVeigh, P. ([email protected]). Review TRENDS in Parasitology Vol.22 No.8 August 2006 www.sciencedirect.com 1471-4922/$ - see front matter Q 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.pt.2006.06.010
Transcript
The FLP-side of nematodesPaul McVeigh1, Timothy G. Geary2, Nikki J. Marks1 and Aaron G. Maule1
1Parasitology, School of Biological Sciences, Queen’s University Belfast, 97 Lisburn Road, Belfast BT9 7BL, UK2Institute of Parasitology, McGill University, 21111 Lakeshore Road, Ste. Anne de Bellevue, Quebec, H9X 3V9, Canada
The central role of FMRFamide-like peptides (FLPs) in
nematode motor and sensory capabilities makes FLP
signalling an appealing target for new parasiticides.
Accumulating evidence has revealed an astounding
level of FLP sequence conservation and diversity in the
phylum Nematoda, and preliminary work has begun to
identify the nematode FLP receptor complement in
Caenorhabditis elegans, with a view to investigating
their basic biology and therapeutic potential. However,
much work is needed to clarify the functional aspects of
FLP signalling and how these peptides exert their effects
at the organismal level. Here, we summarize our current
knowledge of nematode FLP signalling.
A relatively short history
The most recent reviews in this journal concerningparasitic nematode FMRFamide-like peptides (FLPs,named from the sequence Phe-Met-Arg-Phe-NH2) dateback to 1996 [1,2], published during the first wave ofresearch into this large family of neuropeptides. At thatearly stage, data had accumulated on the structure,immunochemical localization and physiological effects ofFLPs in nematode tissue assays, complemented byresults from a small number of gene knockout studiesin Caenorhabditis elegans. Ten years on, these basic datastill form the foundation of our understanding of thenematode FLP system. However, significant strides inmapping FLP expression and FLP receptor biology havebeen made in the past 5–6 years, due almost entirely tothe exploitation of C. elegans [3,4]. By contrast, nothinghas been published on FLP receptors in parasiticnematodes, and only recently have data begun toaccumulate on the flp gene complement of theseorganisms [5]. With only one nematode parasite gen-ome-sequencing project under way (Brugia malayi [6]), itis unlikely that the level of molecular knowledge of anynematode parasite will rival that in C. elegans in theimmediate future. However, the more than 340 000expressed sequence tags (ESTs) currently availablefrom 44 species of parasitic nematode [5] represent aresource which, if exploited in the context of knownC. elegans neurobiology data, can be used to improve ourunderstanding of the parasite FLP system.
FLPs: the basics
FLPs (also known as FMRFamide-related peptides orFaRPs) are the largest and most diverse family of
www.sciencedirect.com 1471-4922/$ - see front matter Q 2006 Elsevier Ltd. All rights reserved
neuropeptides known [7]. As their name suggests, theyshow similarity to FMRFamide, a cardioactive tetrapep-tide isolated from the clam Macrocallista nimbosa [8].Although RFamide peptides have been identified through-out the invertebrates and to some extent in vertebrates[9], the FLP system of nematodes is unusually complex –at least 32 flp genes are known in the phylum Nematoda[5] (Table 1). Each flp gene encodes distinct andcharacteristic variations on the FLP C-terminal tetrapep-tide motif x-xo-Arg-Phe-NH2, (where x is any amino acidexcept cysteine and xo is any hydrophobic amino acidexcept cysteine; cysteine has not been reported in a FLP;Table 1). This structural diversity is reflected in the rangeof FLP-induced physiological responses, which comprise avariety of different effects on muscle, motorneurons,behaviour and sensory abilities [10–15] (Table 2).Mechanisms involved in processing of FLP propeptidesare detailed in Box 1. The FLP tetrapeptide motifmeans that some peptides not previously considered as‘true FLPs’ are included in the grouping, such asmany vertebrate neuropeptides ending in Arg-Phe-NH2
(–RFamide peptides). This more inclusive view of FLPsmight represent a more realistic vision of the evolutionaryrelations of –RFamide peptides, especially in view ofcurrent evidence showing a conserved role for thesepeptides and their receptors in the control of feedingbehaviour [9]. It is clear that nematode FLPs have a broadrole in their nervous systems, whereas vertebrate FLPsappear to have somewhat restricted distributions and amore focussed remit of roles. Currently, the expandingknowledge of vertebrate FLPs does not serve to weakenthe potential of parasitic helminth FLP signalling as atarget for parasite control.
The nematode FLP system undoubtedly poses someinteresting biological questions, and understanding thissignalling system is a valuable goal in itself. Nevertheless,the drive towards discovery of novel therapeuticcompounds to target the system in parasites has alsoprovided a powerful impetus for FLP research. FLPs areinextricably linked to parasite motor and sensory function[7] and are conserved in a range of invertebrate pestspecies [16], so the parasite FLP system has obviouspotential as a target for new anthelmintics (helminth-specific drugs) or even endectocides (drugs that act on bothendo- and ectoparasites).
All nematodes have a similar FLP complement
Caenorhabditis elegans is currently known to have atleast 29 flp genes, encoding at least 68 distinct FLPs[3,5,17]. Although data on the FLP complement of other
Review TRENDS in Parasitology Vol.22 No.8 August 2006
Review TRENDS in Parasitology Vol.22 No.8 August 2006 387
www.sciencedirect.com
nematode species have been limited, a recent bioinfor-matic analysis of FLP-encoding ESTs reported the inter-species structural conservation of nematode FLPs [5](Table 1). Some of the FLPs predicted by this and previousstudies have also been characterized using mass spec-trometry [17]. Available evidence suggests, therefore, thatflp gene complements are largely comparable acrossthe phylum.
In light of this intra-phylum conservation of FLPs, ithas been proposed that nematode flp genes be namedaccording to their C. elegans sequelog and appended witha species-specific two-letter designator [5]. (The term‘sequelog’ implies sequence similarity but does not suggestany evolutionary or functional link between sequences.Terms such as ortholog or homolog are inappropriate inthe case of FLPs, because we have no hard evidence forany such relationships.) For example, the Onchocercavolvulus gene with similarity to flp-24 would be namedOv-flp-24. Crucially, this system recognizes the stronginter-species conservation of FLPs and/or FLP signaturesand facilitates easy recognition of the type of FLP inquestion, regardless of species.
FLPs are not the only peptide transmitters used in thenematode nervous system – C. elegans also has w42 nlpgenes encoding w124 neuropeptide-like proteins, and 38genes encoding R76 insulin-like peptides (such as ins-1–ins-37 and daf-28) [3]. These neuropeptides are utilized inaddition to a range of classical transmitters [18,19]. Thechallenge of deciphering how such a bewilderinglycomplex system is integrated in a simple nervous systemis daunting. One initial approach must be to delineate theexpression of individual FLPs and FLP receptors as ameans of clarifying their potential for functional inter-relationships.
Individual FLPs have distinct, restricted expression
patterns
Immunocytochemical localization of neuropeptidesrevealed FLP immunoreactivity in all of the mainnematode neural structures [1], but most of the antibodiesused could not reliably distinguish between the highlyconserved FLP C-termini. Use of N-terminally directedantibodies has been proposed [7] to take advantage of FLPN-terminal variation but, to date, no such studies innematodes have been published. Immunocytochemistry isnevertheless a useful technique for investigating grosspatterns of FLP distribution, but for information onexpression patterns of individual FLPs there is a need toapply more stringent techniques that enable analysis offlpgene expression.
The first data on nematode flp gene expression weregleaned from C. elegans using reporter-construct studies.The majority of C. elegans flp genes have been localized inthis way [3,20], showing that they have distinct butoverlapping expression patterns. This implies that someC. elegans neurons use a repertoire of FLPs; to take onecell as an example, the ASE anterior sensory neuronsexpress flp-4, -5, -6, -13, and -20, producing a total of 13distinct FLPs, as well as the six neuropeptide-likeproteins (NLPs) encoded by nlp-3 and -7 [3]. Althoughwe cannot yet assign functions to individual peptides, it
flp-29 ILMRFa AF16 As PI: negligible NE As PP DE2– EPSPY; DI–
FDRDFMHFa AF17 As PI: ma bwRT3 DE2YRinYEPSPY EPSPaY;
DIYRinYaIn peptide sequences, a indicates amide.bPeptide titles indicate the species from which peptides have been biochemically isolated: AF, A. suum FLP; PF, P. redivivus FLP.c‘As PI’ denotes analysis of worm behaviour following pseudocoelomic injection into adult A. suum; [, increased movement; Y, decreased movement; ma, movement
abolished; negligible, negligible effect. Only gross descriptions of effects, based on qualitative descriptions of Ref. [15], are given; for more detailed descriptions of
behavioural effects, see Refs [13,14].dbwRT denotes A. suum dorsal body wall muscle response types 1–4: 1, slow inhibitory; 2, fast inhibitory; 3, excitatory; 4, biphasic;eovRT denotes A. suum ovijector response types 1–5: 1, inhibitory; 2, excitatory; 3, transient contraction; 4, transient contraction followed by spastic paralysis; 5, relaxation
followed by increased activity.f‘As PP’ denotes effects on A. suum 5-HT stimulated pharyngeal pumping; Ce APF denotes effects on C. elegans pharyngeal action potential frequency; NE, no effect.gDE2 denotes a hyperpolarising (Y), depolarising ([) or negligible (–) effect on A. suum DE2 motorneurons; Rin details an increase ([), decrease (Y) or biphasic effect ([Y) on
neuronal input resistance; EPSP denotes an increase ([) or decrease (Y) on excitatory postsynaptic potential frequency; EPSPa denotes changes in excitatory postsynaptic
potential amplitude; DI denotes a hyperpolarizing (Y), depolarising ([), biphasic ([Y) or negligible (-) effect on A. suum DI motorneurons;hMcVeigh, P., Marks, N.J., Maule, A.G., unpublished.
Review TRENDS in Parasitology Vol.22 No.8 August 2006 389
seems logical to infer that this range of neuropeptideexpression would confer these cells with multifunctionalstatus. Similar neurochemical diversity is shown byother neurons. This could be one method of counteringthe functional restrictions imposed by a nervous systemconsisting of just 302 cells (C. elegans hermaphrodite),as this architecturally simple structure can never-theless orchestrate the sophisticated behaviours shownby nematodes.
Analysis of flp gene expression in parasitic nematodeshas generally been performed using in situ hybridization(ISH). ISH has been used in our laboratory to localize flpgene expression in Globodera pallida [21], Haemonchuscontortus and Trichostrongylus colubriformis (S. Leech,PhD Thesis, Queen’s University Belfast, 2003), Meloido-gyne incognita (M. Johnston, PhD Thesis, Queen’sUniversity Belfast, 2006), Panagrellus redivivus (C.L.Moffett, PhD Thesis, Queen’s University Belfast, 2001)and Teladorsagia circumcincta (I.R. Miskelly, PhD Thesis,Queen’s University Belfast, 2006); we have observedhighly restricted expression patterns for individualflp genes.
A comparative analysis of the localization of corre-sponding flp genes between C. elegans and G. pallidashows notable differences. For example, flp-1 is quitewidely expressed in C. elegans compared with G. pallida,in which Gp-flp-1 is expressed only in the retrovesicularganglion. Similarly, Gp-flp-6 expression was observed in
www.sciencedirect.com
the circumpharyngeal nerve ring and in cells of theposterior lumbar ganglia, whereas flp-6 in C. elegans wasreported solely in the ASE head neurons. Furtherdifferences are seen with other flp genes (see Ref. [21]for a comparative discussion). Although these differencesmight represent a species-specific difference in flpexpression, more confidence could be placed in compara-tive studies that used identical techniques. There areinherent caveats with localization studies using greenfluorescent protein (GFP) fusions, as the transcriptionalreporters used in these studies might not contain all ofthe cis-regulatory sequences associated with the gene andso might not give a complete representation of a gene’sexpression pattern.
Yet another approach to FLP localization is thatperformed by Yew et al. [22], who dissected individualnerve ganglia from Ascaris suum and subjected these tomass spectrometric analysis, producing a peptidomicmap of the individual anterior ganglionic groupings.This study seems to show a much less restrictedexpression of FLPs than is apparent from the gene-expression studies in either C. elegans or G. pallida,with the majority of FLPs being identified fromnumerous ganglia in the head. These data could providefurther evidence for FLP expression differences betweennematode species, but caution is warranted as theauthors of this work also identify limitations of thesepeptidomic studies (see Ref. [22] for a discussion). Given
down products identified in nematodes are shown for each enzyme in
Figure Ie.
TRENDS in Parasitology
…GSVKRKNEFIRFGKRKNEFIRFGKRFTA…
SPC
KNEFIRFGKR KNEFIRFGKR
KNEFIRFG KNEFIRFG
EP
AP DA
EP
AP DA
CP CP
SPC SPC
PHM and PAL PHM and PAL
Intracellular
Synaptic cleft
KNEFIRF.NH2 KNEFIRF.NH2
AP: K + NEFIRF.NH2
EP: KNE + FIRF.NH2
DA: KNEFIRF + NH2
(a)
(b)
(c)
(d)
(e)
Figure I.
Review TRENDS in Parasitology Vol.22 No.8 August 2006390
that both the basic nervous architecture and FLPcomplement are largely conserved throughout thephylum Nematoda, these putative differences in flpgene expression pattern seem surprising. This evidencecould point to the role of individual FLPs (or theirparent neurons) varying between species, although weshould bear in mind that the dissimilarities couldalternatively be attributed to evolutionary, develop-mental or experimental differences. More detailed andtechnique-matched interspecies comparisons arerequired to address these issues.
www.sciencedirect.com
FLPs signal mostly through G-protein coupled receptors
The past two years have seen a shift in research focus fromFLP ligands towards the biology of their receptors, withseveral studies reporting ‘deorphanization’ (the process ofmatching receptors with their cognate ligands) of the firstnematode FLP receptors [4]. C. elegans has been at theforefront of this research, which was initially propelled bypharmaceutical investment in FLP receptors as targetsfor the next generation of novel anthelmintics [23].Indeed, C. elegans is presently the only nematode speciesin which such work has been described. Eleven C. elegans
Review TRENDS in Parasitology Vol.22 No.8 August 2006392
www.sciencedirect.com
FLP receptors have been identified, all of which are seven-pass G-protein coupled receptors (GPCRs; Table 3). Atypical approach to receptor deorphanization uses hetero-logously-expressed receptors (usually in Chinese hamsterovary (CHO), human embryonic kidney (HEK) or Xenopusoocyte cells), which are screened with a range of putativeligands. Cellular responses are measured [e.g. using Ca2C
fluorescence-based assays, or binding assays using thenon-hydrolyzable analogue of GTP (GTPgS)] to gauge thepotency of the ligands, and ligand–receptor matches canthen be made on the basis of the most potent ligand(s) foreach receptor.
These studies have used high-throughput assays toscreen a maximum of 200 [24], but more typically 30–70[25–28], neuropeptides against each of the heterologouslyexpressed GPCRs. This is by no means a full represen-tation of the C. elegans neuropeptide complement, whichis currently known to comprise at least 250 distinctpeptides [3,5,17]. Therefore, until each receptor ischallenged with the full neuropeptide complement, theidentification of endogenous ligands is equivocal. Eventhen, further criteria should perhaps be addressed beforea receptor can properly be dubbed ‘deorphanized’, such asappropriate in vivo localization of ligand and receptor andsimilar phenotypes after knockout or silencing of receptorand ligand genes. Such functional studies should ulti-mately aim to match individual ligand–receptor pairings,their downstream signalling and their associatedbehavioural phenotypes.
For example, the relationships of one FLP receptor todownstream behavioural activities in C. elegans arebeginning to be unravelled. NPR-1, the first-discoveredC. elegans neuropeptide GPCR, occurs in two forms thatdiffer at position 215 by a single amino acid (phenyl-alanine or valine). Worms expressing the version with avaline, NPR-1.215V, mainly show a ‘solitary feeding’(wild-type) phenotype; worms expressing the phenyl-alanine version, NPR-1.215F, have a propensity toaggregate during feeding (a ‘social feeding’ phenotype)[29]. Heterologous expression studies enabled identifi-cation of the activating ligands as the AF9 peptide(GLGPRPLRFamide) encoded by the flp-21 gene and the-PGVLRFamide peptides encoded by flp-18; the workrevealed different selectivity for these ligands by the tworeceptor isoforms [24,30]. The NPR-1.215V isoform, whenexpressed in Xenopus oocytes, differs from NPR1.215F intwo ways: first, NPR-1.215V is at least ten-fold moresensitive to the ligand AF9/FLP-21 than NPR-1.215F;second, NPR-1.215V is additionally activated by FLP-18peptides, but this activity could depend on cellularcontext, as a separate study that expressed both NPR-1isoforms in Chinese hamster ovary cells found that AF9/FLP-21 was the only activating ligand [24]. Thisapparent receptor promiscuity is interesting from apharmacological standpoint, as, if this is a generalproperty of nematode FLP receptors, it could providesome explanation as to the source of FLP diversity: ifFLP receptors are promiscuous, there could be littleselective pressure acting to restrict FLPsequence diversification.
Review TRENDS in Parasitology Vol.22 No.8 August 2006394
The neurons that express NPR-1 are believed to have acomplex role in the integration of chemical stimuli thatregulate feeding behaviour and other sensory functions,including ethanol tolerance [4,29–31]. Despite this level offunctional knowledge, NPR-1 is probably an unsuitabledrug target candidate in parasites; FLP receptors associ-ated with phenotypes that are incompatible with aparasitic lifestyle (e.g. those directly involved in loco-motion) would offer more obvious appeal as drug targetcandidates. Unfortunately, data on the biology (orbiological relevance) of other nematode FLP receptorsare not currently available to direct selection ofdrug targets.
Even in the absence of any further deorphanizationdata beyond the 11 reported receptors (Table 3), it is clearthat most nematode FLPs exert their effects throughunidentified GPCRs. Indeed, C. elegans genome datareveal that a total of w60 GPCRs might act as peptidereceptors [4,32,33]. Gene silencing techniques, such asRNA interference (RNAi) are valuable in functionalstudies of GPCRs. One such study screened 60 C. elegansGPCRs for locomotory and reproductive phenotypes [33];six of the GPCRs analyzed had a role in reproduction, andanother seven were involved in locomotion. Interestingly,four of these receptors have been matched with FLPligands (Table 3).
It is possible to monitor GPCR activation indirectly,through elements of downstream signalling, suchas G-protein activation. For example, evidence thatthe receptor for the peptide encoded by flp-14, AF2(KHEYLRFamide), is a GPCR was derived from studieson AF2/FLP-14-induced binding of radioactively labelledGTPgS to Ascaris membrane preparations in vitro;the results imply that AF2/FLP-14 triggers the processof GDP–GTP exchange on the Ga subunit via a GPCR [34].Identification of second messenger pathways can alsoimplicate GPCRs in neuropeptide action (see Figure 1and later).
FLPs are active in bioassays for neuromuscularfunction in several types of muscle, including somatic,ovijector and pharyngeal muscle, as well as when injectedinto whole, live Ascaris (Table 2). We can thereforepostulate that some FLP receptors reside on muscle;however, denervation of somatic muscle strips can alter orabolish the activity of many FLPs [7,10], suggesting thatin addition to their muscle-based effects, some peptides actthrough modulation of neuronal conductance. Electro-physiology shows that FLPs can indeed influence motor-neuron activity, with the majority of peptides tested
Figure 1. Nematode FLP signalling. (a) Data from Ascaris suum and Ascaridia galli. Eight
suum. The majority of these are associated with G-protein coupled receptors (GPCR sub
FLP triggering of downstream second messengers allied to an identifiable physiological
to adenylate cyclase, with X1 and X2 producing upregulation of adenylate cyclase and
AVPGVLRFamide) and GPCRX3 is also reported in Ascaridia galli) [14,39,70]. Note that al
there is no evidence to suggest that these effects are mediated by a single receptor, only
mediated by GPCRs, but a single FLP, PF4 (FLP-1, KPNFIRFamide) is thought to trigger a
muscle [35–37]. This channel has been shown, in electrophysiology experiments, to be lo
locomotory muscle and in the absence of evidence to the contrary, we presume that my
linking the inhibitory effect of PF1 to nitric oxide (NO), a gaseous transmitter produced by
[43,44]. The close association between the hypodermis and body wall muscle would allow
Data obtained from Caenorhabditis elegans. Although the identity of the PF1 receptor in
signals through a G protein subunit [15], although the location of this receptor in C. ele
www.sciencedirect.com
producing changes in activity of both excitatory andinhibitory A. suum motorneurons. One study delineatedfive major neuronal response types, theoretically corre-sponding to at least five FLP receptor subtypes (whichmight be attributed to different receptors, second messen-ger pathways or combinations of both) on Ascaris nerves[13]. Similar studies on muscle physiology have shown atleast four FLP responses on Ascaris somatic muscle andfive on the Ascaris ovijector, as well as varied effects onworm behaviour. These have been described elsewhere[11] and are summarized in Table 2. In the absence ofexpression data, it is difficult to place these response typesin an in vivo context, but such studies help provide crudeestimates of FLP receptor diversity in these tissues.
The GPCR-mediated effects of FLPs on muscles andnerves are consistent with the more traditional neuro-modulatory effects attributed to neuropeptides, because ofthe relatively slow time-course of signal transductionthrough a GPCR and associated second messengerpathways. However, some FLPs act like faster classicalneurotransmitters, exerting their effects by directlygating ion channels (Figure 1). There is evidence that atleast one FLP, PF4 (KPNFIRFamide) encoded by flp-1,acts in this way [35–37] by directly triggering influx of ClK
into Ascarismuscle cells. This ClK influx produces a rapidhyperpolarization [comparable in time-course to theactivation of g-aminobutyric acid (GABA)-gated ClK
channel] and relaxation of Ascaris somatic muscle, andis not affected by a G-protein inhibitor [36]. Although theevidence for a FLP-gated ClK channel is strong, absoluteconfirmation will require functional expression ofchannels so that indirect activation by other mechanisms,such as G-protein subunits, can be discounted.
Travelling downstream: intracellular signalling by FLPs
Despite the considerable amount of data on FLP responsetypes in neuronal and neuromuscular bioassays, we stillknow relatively little about the downstream signallingprocesses through which these effects are exerted. This isof interest as the signalling molecules involved in thesepathways, if pharmacologically different from those invertebrate hosts, could also be targets through which FLPsignalling could be disrupted. Much of what is known hasbeen learned from A. suum, but this knowledge amountsto only partial reconstructions of a small number of FLPpathways, most of which appear to signal throughadenylate cyclase (Figure 1). One example is thattriggered by AF2/FLP-14, which, as well as causing anincrease in cAMP of up to 100 fold over basal levels
partial signalling pathways associated with eleven FLPs have been delineated in A.
types X1-X5), and have been inferred indirectly from biochemical evidence showing
effect on A. suum body wall muscle. FLP-triggered GPCR pathways X1-X4 are linked
X3 and X4 downregulating adenylate cyclase (the pathway through AF3 (FLP-18,
though the GPCRX4 (blue) pathway is associated with six distinct FLPs [AF11 (FLP-1,
QRSMVRFamide); FLP-9 (KPSFVRFamide); and FLP-13 (APEASPFIRFamide)] [14],
that these FLPs have similar downstream effects. Most FLP signalling seems to be
ligand-gated ClK channel, producing ClK influx and relaxation of A. suum body wall
cated on A. suum somatic muscle membranes [36,37]. On the basis of their effects on
oactive GPCRs are situated on body wall muscle cell membranes. However, studies
nitric oxide synthase (NOS), have localized NOS activity to the A. suum hypodermis
hypodermally produced NO to exert its effect by diffusion into the muscle cells. (b)
Ascaris has not been reported, there is evidence from C. elegans that this receptor
Review TRENDS in Parasitology Vol.22 No.8 August 2006 395
(thought to be linked to the inhibitory phase of thebiphasic muscle response of this peptide), is also impli-cated in the control of glycolysis and might be involved inpotentiation of the nicotinic acetylcholine response[14,38–42]. These wide-ranging effects seem to be con-gruent with bioinformatic and biochemical evidence,which indicate that AF2/FLP-14 is one of the mostabundant nematode FLPs [5]. This peptide clearly hasan important role in nematode neurobiology and, presum-ing that its receptor is as conserved as AF2/FLP-14 itself,could provide a high-value target for pharmacologicalinterference. Other known components of FLP-triggeredsecond messenger pathways are shown in Figure 1.
Apart from adenylate cyclase and nitric oxide [14,38–44], no other second messengers have been reported inFLP actions on Ascaris somatic muscle, but it should benoted that only one study has investigated messengersother than adenylate cyclase – no evidence was foundfor stimulation of Ins(1,4,5)P3 levels by AF1/FLP-8,KNEFIRFamide, or AF2/FLP-14 [39]. By contrast, pre-liminary studies on selected FLP activities in the Ascarisovijector have found no evidence for involvement of theadenylate cyclase pathway, but instead implicate thephosphatidylinositol pathway, comprising both inositoltriphosphate (Ins(1,4,5)P3) and protein kinase C (PKC) (P.McVeigh, PhD thesis, Queen’s University Belfast, 2004).
Concluding remarks
Recent years have witnessed a shift in research focus fromFLPs per se towards their receptors and signallingmechanisms, as nematode neurobiologists have harnessedtechniques pioneered in other spheres of biologicalresearch. This is not to suggest that we have a completeportfolio of knowledge on the ‘upstream’ aspects of FLPs –far from it. Despite recent insights into the breadth of FLPconservation and diversity in nematodes, we do not yetknow the full FLP complement for any parasitic nematodeand we still know little about the specific functions ofindividual peptides. The inherent complexity thatabounds in this signalling system demands a hugeresearch effort, particularly if we are to decipher its rolein nematode biology.
It is clear that nematode FLPsmodulate and coordinatesophisticated behavioural activities. The potential of FLPreceptors as novel drug targets is obvious, and the recentbreakthroughs in receptor expression anddeorphanizationhave invigorated this area of research from both a drug-screening and a biological perspective. Although recentbreakthroughs have been significant, we remain largelyignorant of FLP–receptor interplay and the associatedbehavioural consequences of FLP action. Expanding ourknowledge of the FLP ligands, their receptors, theirsignalling pathways and their impact on worm biology iscrucial if we are to make rational judgements on targetselection and screening paradigms.
Acknowledgements
Work in the laboratory of A.G.M. is supported by The Department forAgriculture and Rural Development (DARD) for Northern Ireland and theNational Institutes of Health (R01-AI49162).
www.sciencedirect.com
References
1 Brownlee, D.J.A. et al. (1996) Nematode neuropeptides: localization,isolation and functions. Parasitol. Today 12, 343–351
2 Maule, A.G. et al. (1996) The pharmacology of nematode FMRFamide-related peptides. Parasitol. Today 12, 351–357
3 Li, C. (2005) The ever expanding neuropeptide gene families in thenematode Caenorhabditis elegans. Parasitology 131, S109–S127
4 Geary, T.G. and Kubiak, T.M. (2005) Neuropeptide G-protein-coupledreceptors, their cognate ligands and behaviour in Caenorhabditiselegans. Trends Pharmacol. Sci. 26, 56–58
5 McVeigh, P. et al. (2005) Analysis of FMRFamide-like peptide(FLP) diversity in phylum Nematoda. Int. J. Parasitol. 35,1043–1060
6 Ghedin, E. et al. (2004) First sequenced genome of a parasiticnematode. Trends Parasitol. 20, 151–153
7 Maule, A.G. et al. (2002) Neuropeptide signalling systems – potentialdrug targets for parasite and pest control. Curr. Top. Med. Chem. 2,733–758
8 Price, D.A. and Greenberg, M.J. (1977) Structure of a molluscancardioexcitatory neuropeptide. Science 197, 670–671
9 Dockray, G.J. (2004) The expanding family of –RFamide peptides andtheir effects on feeding behaviour. Exp. Physiol. 89, 229–235
10 Maule, A.G. et al. (1995) Inhibitory effects of nematode FMRFamide-related peptides (FaRPs) on muscle strips from Ascaris suum. Invert.Neurosci. 1, 255–265
11 Moffett, C.L. et al. (2003) The ovijector of Ascaris suum: multipleresponse types revealed by Caenorhabditis elegans FMRFamide-related peptides. Int. J. Parasitol. 33, 859–876
12 Papaioannou, S. et al. (2005) Role of a FMRFamide-like family ofneuropeptides in the pharyngeal nervous system of Caenorhabditiselegans. J. Neurobiol. 65, 304–319
13 Davis, R.E. and Stretton, A.O.W. (2001) Structure-activity relation-ships of 18 endogenous neuropeptides on the motornervous system ofthe nematode Ascaris suum. Peptides 22, 7–23
14 Reinitz, C.A. et al. (2000) Changes in locomotory behaviour and cAMPproduced in Ascaris suum by neuropeptides from Ascaris suum orCaenorhabditis elegans. Mol. Biochem. Parasitol. 111, 185–197
15 Nelson, L.S. et al. (1998) Disruption of a neuropeptide gene, flp-1,causes multiple behavioural defects in Caenorhabditis elegans.Science 281, 1686–1690
16 Mousley, A. et al. (2004) Neuropeptide signalling: a repository oftargets for novel endectocides? Trends Parasitol. 20, 482–487
17 Husson, S.J. et al. (2005) Discovering neuropeptides in Caenorhabdi-tis elegans by two dimensional liquid chromatography and massspectrometry. Biochem. Biophys. Res. Commun. 335, 76–86
18 Brownlee, D. et al. (2000) The range and biological activity ofFMRFamide-related peptides and classical transmitters in nema-todes. Adv. Parasitol. 45, 109–180
19 Rex, E. et al. (2005) TYRA-2 (F01E11.5): a Caenorhabditis eleganstyramine receptor expressed in theMC andNSM pharyngeal neurons.J. Neurochem. 94, 181–191
20 Kim, K. and Li, C. (2004) Expression and regulation of anFMRFamide-related neuropeptide gene family in Caenorhabditiselegans. J. Comp. Neurol. 475, 540–550
21 Kimber, M.J. et al. (2002) Localisation of Globodera pallidaFMRFamide-related peptide encoding genes using in situ hybrid-isation. Int. J. Parasitol. 32, 1095–1105
22 Yew, J.Y. et al. (2005) Mass spectrometric map of neuropeptideexpression in Ascaris suum. J. Comp. Neurol. 488, 396–413
23 Lowery, D.E. et al. (2003) Pharmacia & Upjohn Company, UnitedStates. G protein-coupled receptor-like receptors and modulatorsthereof. Patent 6,632,621
24 Kubiak, T.M. et al. (2003) Differential activation of “social” and“solitary” variants of the Caenorhabditis elegans G protein-coupledreceptor NPR-1 by its cognate ligand AF9. J. Biol. Chem. 278,33724–33729
25 Mertens, I. et al. (2005) Characterisation of an RFamide-relatedpeptide orphan GPCR in C. elegans. Ann. N. Y. Acad. Sci. 1040,410–412
26 Mertens, I. et al. (2004) Functional characterisation of the putativeorphan neuropeptide G-protein coupled receptor C26F1.6 in Caenor-habditis elegans. FEBS Lett. 573, 55–60
Review TRENDS in Parasitology Vol.22 No.8 August 2006396
27 Mertens, I. et al. (2005) Molecular characterisation of two G protein-coupled receptor splice variants as FLP2 receptors in Caenorhabditiselegans. Biochem. Biophys. Res. Commun. 330, 967–974
28 Mertens, I. et al. FMRFamide related peptide ligands activate theCaenorhabditis elegans orphan GPCR Y59H11AL.1. Peptides (inpress)
29 de Bono, M. and Bargmann, C.I. (1998) Natural variation in aneuropeptide Y receptor homolog modifies social behaviour and foodresponse in C. elegans. Cell 94, 679–689
30 Rogers, C. et al. (2003) Inhibition of Caenorhabditis elegans socialfeeding by FMRFamide-related peptide activation of NPR-1. Nat.Neurosci. 6, 1178–1185
31 Davies, A.G. et al. (2004) Natural variation in the npr-1 gene modifiesethanol responses of wild strains of C. elegans. Neuron 42, 731–743
32 Bargmann, C.I. (1998) Neurobiology of the Caenorhabditis elegansgenome. Science 282, 2028–2033
33 Keating, C.D. et al. (2003) Whole-genome analysis of 60 G protein-coupled receptors in Caenorhabditis elegans by gene knockout withRNAi. Curr. Biol. 13, 1715–1720
34 Kubiak, T.M. et al. (2003) AF2 interaction with Ascaris suum bodywall muscle membranes involves G-protein activation. Biochem.Biophys. Res. Commun. 301, 456–459
35 Maule, A.G. et al. (1995) Isolation and preliminary biologicalcharacterization of KPNFIRFamide, a novel FMRFamide-relatedpeptide from the free-living nematode, Panagrellus redivivus.Peptides 16, 87–93
36 Purcell, J. et al. (2002) PF4, a FMRFamide-related peptide, gates lowconductance ClK channels in Ascaris suum. Eur. J. Pharmacol. 456,11–17
37 Holden-Dye, L. et al. (1997) The effects of the peptide KPNFIRFamide(PF4) on the somatic muscle cells of the parasitic nematode Ascarissuum. Br. J. Pharmacol. 120, 379–386
38 Geary, T.G. et al. (1999) Pharmacology of FMRFamide-relatedpeptides in helminths. Ann. N. Y. Acad. Sci. 897, 212–227
39 Thompson, D.P. et al. (2003) Effects of KHEYLRFamide andKNEFIRFamide on cyclic adenosine monophosphate levels in Ascarissuum somatic muscle. Int. J. Parasitol. 33, 199–208
40 Trailovic, S.M. et al. (2005) Brief application of AF2 produces longlasting potentiation of nAChR responses in Ascaris suum. Mol.Biochem. Parasitol. 139, 51–64
41 Rex, E. et al. (2004) Regulation of carbohydrate metabolism in Ascarissuum body wall muscle: a role for the FMRFamide AF2, not serotonin.Mol. Biochem. Parasitol. 133, 311–313
42 Pang, F.Y. et al. (1995) The effects of the nematode peptide,KHEYLRFamide (AF2) on the somatic musculature of the parasiticnematode Ascaris suum. Parasitology 110, 353–362
43 Bowman, J.W. et al. (1995) Nitric oxide mediates the inhibitory effectsof SDPNFLRFamide, a nematode FMRFamide-related neuropeptide,in Ascaris suum. J. Neurophysiol. 74, 1880–1888
44 Bascal, Z.A. et al. (2001) Characterisation of a putative nitric oxidesynthase in the neuromuscular system of the parasitic nematode,Ascaris suum. Parasitology 122, 219–231
45 Waggoner, L.E. et al. (2000) Effect of a neuropeptide gene onbehavioural states in Caenorhabditis elegans egg-laying. Genetics154, 1181–1192
46 Cowden, C. et al. (1989) AF1, a sequenced bioactive neuropeptideisolated from the nematode Ascaris suum. Neuron 2, 1465–1473
47 Cowden, C. and Stretton, A.O. (1993) AF2, an Ascaris neuropeptide:isolation, sequence, and bioactivity. Peptides 14, 423–430
48 Cowden, C. and Stretton, A.O. (1995) Eight novel FMRFamide-likeneuropeptides isolated from the nematode Ascaris suum. Peptides 16,491–500
49 Marks, N.J. et al. (1999) Isolation, pharmacology and gene organiz-ation of KPSFVRFamide: a neuropeptide from Caenorhabditiselegans. Biochem. Biophys. Res. Commun. 254, 222–230
www.sciencedirect.com
50 Marks, N.J. et al. (2001) Isolation and preliminary biologicalassessment of AADGAPLIRFamide and SVPGVLRFamide fromCaenorhabditis elegans. Biochem. Biophys. Res. Commun. 286,1170–1176
51 Fellowes, R.A. et al. (1998) Modulation of the motility of the vaginavera of Ascaris suum in vitro by FMRFamide-related peptides.Parasitology 116, 277–287
52 Fellowes, R.A. et al. (2000) Nematode neuropeptide modulation of thevagina vera ofAscaris suum: in vitro effects of PF1, PF2, PF4, AF3 andAF4. Parasitology 120, 79–89
53 Brownlee, D.J. et al. (1995) The action of serotonin and the nematodeneuropeptide KSAYMRFamide on the pharyngeal muscle of theparasitic nematode, Ascaris suum. Parasitology 111, 379–384
54 Brownlee, D.J. and Walker, R.J. (1999) Actions of nematode FMRF-amide-related peptides on the pharyngeal muscle of theparasitic nematode, Ascaris suum. Ann. N. Y. Acad. Sci. 897,228–238
55 Rogers, C.M. et al. (2001) Regulation of the pharynx ofCaenorhabditiselegans by 5-HT, octopamine, and FMRFamide-like neuropeptides.J. Neurobiol. 49, 235–244
56 Davis, R.E. and Stretton, A.O. (1996) The motornervous system ofAscaris: electrophysiology and anatomy of the neurons and theircontrol by neuromodulators. Parasitology 113, S97–S117
57 Kubiak, T.M. et al. (2003) Functional annotation of the putativeorphan Caenorhabditis elegans G-protein-coupled receptor C10C6.2as a FLP15 peptide receptor. J. Biol. Chem. 278, 42115–42120
58 Canaff, L. et al. (1999) Peptide hormone precursor processing: gettingsorted? Mol. Cell. Endocrinol. 156, 1–6
59 Steiner, D.F. (1998) The proprotein convertases. Curr. Opin. Chem.
Biol. 2, 31–3960 Gomez-Saladin, E. et al. (1994) Isolation and in situ localisation of a
cDNA encoding a Kex2-like prohormone convertase in the nematodeCaenorhabditis elegans. Cell. Mol. Neurobiol. 14, 9–25
61 Kass, J. et al. (2001) The EGL-3 proprotein convertase regulatesmechanosensory responses of Caenorhabditis elegans. J. Neurosci. 21,9265–9272
62 Kovaleva, E.S. et al. (2002) Human proprotein convertase 2homologue from a plant nematode: cloning, characterisation, andcomparison with other species. FASEB J. 16, 1099–1101
63 Jacob, T.C. and Kaplan, J.M. (2003) The EGL-21 carboxypeptidase Efacilitates acetylcholine release at Caenorhabditis elegans neuromus-cular junctions. J. Neurosci. 23, 2122–2130
64 Han, M. et al. (2004) Drosophila uses two distinct neuropeptideamidating enzymes, dPAL1 and dPAL2. J. Neurochem. 90,129–141
65 Avery, L. et al. (1993) The Caenorhabditis elegans unc-31 geneaffects multiple nervous system-controlled functions. Genetics 134,455–464
66 Charlie, N.K. et al. (2006) Presynaptic UNC-31 (CAPS) is required toactivate the G alpha (s) pathway of the Caenorhabditis elegans
synaptic signaling network. Genetics 172, 943–96167 Isaac, R.E. et al. (2000) Conserved roles for peptidases in the
processing of invertebrate neuropeptides. Biochem. Soc. Trans. 28,460–464
68 Sajid, M. et al. (1996) Metabolism of AF1 (KNEFIRF-NH2) in thenematode, Ascaris suum, by aminopeptidase, endopeptidase anddeamidase enzymes. Mol. Biochem. Parasitol. 75, 159–168
69 Sajid, M. et al. (1997) Purification and properties of a membraneaminopeptidase from Ascaris suum muscle that degrades neuro-peptides AF1 and AF2. Mol. Biochem. Parasitol. 89, 225–234
70 Trim, N. et al. (1998) The role of cAMP in the actions of the peptideAF3 in the parasitic nematodes Ascaris suum and Ascaridia galli.Mol. Biochem. Parasitol. 93, 263–271
