Martha J. Larsen

Differential Activation of “Social” and “Solitary” Variants of the Caenorhabditis elegans G Protein-coupled Receptor NPR-1 by Its Cognate Ligand AF9

Natural variations of wild Caenorhabditis elegans isolates having either Phe-215 or Val-215 in NPR-1, a putative orphan neuropeptide Y-like G protein-coupled receptor, result in either “social” or “solitary” feeding behaviors (de Bono, M., and Bargmann, C. I. (1998) Cell 94, 679–689). We identified a nematode peptide, GLGPRPLRF-NH2 (AF9), as a ligand activating the cloned NPR-1 receptor heterologously expressed in mammalian cells. Shifting cell culture temperatures from 37 to 28 °C, implemented 24 h after transfections, was essential for detectable functional expression of NPR-1. AF9 treatments linked both cloned receptor variants to activation of Gi/Go proteins and cAMP inhibition, thus allowing for classification of NPR-1 as an inhibitory G protein-coupled receptor. The Val-215 receptor isoform displayed higher binding and functional activity than its Phe-215 counterpart. This finding parallels the in vivo observation of a more potent repression of social feeding by the npr-1 gene encoding the Val-215 form of the receptor, resulting in dispersing (solitary) animals. Since neuropeptide Y shows no sequence homology to AF9 and was functionally inactive at the cloned NPR-1, we propose to rename NPR-1 and refer to it as an AF9 receptor, AF9-R1.

Pharmacology of FMRFamide-related Peptides in Helminths

Abstract: Nervous systems of helminths are highly peptidergic. Species in the phylum Nematoda (roundworms) possess at least 50 FMRFamide‐related peptides (FaRPs), with more yet to be identified. To date, few non‐FaRP neuropeptides have been identified in these organisms, though evidence suggests that other families are present. FaRPergic systems have important functions in nematode neuromuscular control. In contrast, species in the phylum Platyhelminthes (flatworms) apparently utilize fewer FaRPs than do nematodes; those species examined possess one or two FaRPs. Other neuropeptides, such as neuropeptide F (NPF), play key roles in flatworm physiology. Although progress has been made in the characterization of FaRP pharmacology in helminths, much remains to be learned. Most studies on nematodes have been done with Ascaris suum because of its large size. However, thanks to the Caenorhabditis elegans genome project, we know most about the FaRP complement of this free‐living animal. That essentially all C. elegans FaRPs are active on at least one A. suum neuromuscular system argues for conservation of ligand‐receptor recognition features among the Nematoda. Structure‐activity studies on nematode FaRPs have revealed that structure‐activity relationship (SAR) “rules” differ considerably among the FaRPs. Second messenger studies, along with experiments on ionic dependence and anatomical requirements for activity, reveal that FaRPs act through many different mechanisms. Platyhelminth FaRPs are myoexcitatory, and no evidence exists of multiple FaRP receptors in flatworms. Interestingly, there are examples of cross‐phylum activity, with some nematode FaRPs being active on flatworm muscle. The extent to which other invertebrate FaRPs show cross‐phylum activity remains to be determined. How FaRPergic nerves contribute to the control of behavior in helminths, and are integrated with non‐neuropeptidergic systems, also remains to be elucidated.

FMRFamide‐like peptides encoded on the flp‐18 precursor gene activate two isoforms of the orphan Caenorhabditis elegans G‐protein‐coupled receptor Y58G8A.4 heterologously expressed in mammalian cells

Two alternatively spliced variants of an orphan Caenorhabditis elegans G‐protein‐coupled receptors (GPCRs; Y58G8A.4a and Y58G8A.4b) were cloned and functionally expressed in Chinese hamster ovary (CHO) cells. The Y58G8A.4a and Y58G8A.4b proteins (397 and 433 amino acid residues, respectively) differ both in amino acid sequence and length of the C‐terminal tail of the receptor. A calcium mobilization assay was used as a read‐out for receptor function. Both receptors were activated, with nanomolar potencies, by putative peptides encoded by the flp‐18 precursor gene, leading to their designation as FLP‐18R1a (Y58G8A.4a) and FLP‐18R1b (Y58G8A.4b). Three Ascaris suum neuropeptides AF3, AF4, and AF20 all sharing the same FLP‐18 C‐terminal signature, ‐PGVLRF‐NH2, were also potent agonists. In contrast to other previously reported C. elegans GPCRs expressed in mammalian cells, both FLP‐18R1 variants were fully functional at 37°C. However, a 37 to 28°C temperature shift improved their activity, an effect that was more pronounced for FLP‐18R1a. Despite differences in the C‐terminus, the region implicated in distinct G‐protein recognition for many other GPCRs, the same signaling pathways were observed for both Y58G8A.4 isoforms expressed in CHO cells. Gq protein coupling seems to be the main but not the exclusive signaling pathway, because pretreatment of cells with U‐73122, a phospholipase inhibitor, attenuated but did not completely abolish the Ca2+ signal. A weak Gs‐mediated receptor activation was also detected as reflected in an agonist‐triggered concentration‐dependent cAMP increase. The matching of the FLP‐18 peptides with their receptor(s) allows for the evaluation of the pharmacology of this system in the worm in vivo.

Effects of KHEYLRFamide and KNEFIRFamide on cyclic adenosine monophosphate levels in Ascaris suum somatic muscle.

KHEYLRF-NH(2) (AF2) is a FMRFamide-related peptide (FaRP) present in parasitic and free-living nematodes. At concentrations as low as 10 pM, AF2 induces a biphasic tension response, consisting of a transient relaxation followed by profound excitation, in neuromuscular strips prepared from Ascaris suum. In the present study, the effects of AF2 on cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP) and inositol-1,4,5-triphosphate (IP(3)) levels were measured following muscle tension recordings from 2 cm neuromuscular strips prepared from adult A. suum. AF2 induced a concentration- and time-dependent increase in cAMP, beginning at 1 nM; cAMP levels increased by 84-fold following 1 h exposure to 1 microM AF2. cGMP and IP(3) levels were unaffected by AF2 at concentrations </=1 microM. AF2-induced stimulation of cAMP was unaffected by removal of the dorsal or ventral nerve cord, even though this form of denervation abolished the excitatory phase of the tension response. The effects of 0.1 and 1 microM AF2 on cAMP were also unaffected by 10 microM SDPNFLRF-NH(2) (PF1, an inhibitory FaRP) and 10 microM PF1022A (an inhibitory cyclodepsipeptide), even though each of these peptides abolished the excitatory phase of the tension response induced by AF2. Within an alanine-scan series of AF2 analogues, only KHAYLRF-NH(2) stimulated cAMP production with equipotency to AF2; the effects of this peptide on muscle tension also mimicked AF2. Another excitatory FaRP present in nematodes, KNEFIRF-NH(2) (AF1), also stimulated cAMP production, but was 100-fold less potent than AF2. The stimulatory effects of AF1 on tension and cAMP levels were blocked by an alanine-substituted analogue of this peptide (Ala(6)-AF1, KNEFIAF-NH(2)), while the stimulatory effects of AF2 on tension and cAMP were not affected by this analogue. AF2 and AF1 increase A. suum somatic muscle cAMP by targeting different receptors. Increases in cAMP stimulated by AF2 can be decoupled from the excitatory response caused by this peptide, and it is not possible to establish a causal linkage between the contractile response elicited by this peptide and its effects on cAMP accumulation.