Molecular characterization of two G protein-coupled receptor splice variants as FLP2 receptors in Caenorhabditis elegans.

Two alternatively spliced Caenorhabditis elegans G protein-coupled receptors, T19F4.1a and T19F4.1b, were cloned and functionally characterized. The T19F4.1b receptor protein is 30 amino acids longer than T19F4.1a, and the difference in amino acid constitution is exclusively conferred to the intracellular C-terminal region, suggesting a potential difference in G protein-coupling specificity. Following cloning of the receptor cDNAs into the pcDNA3 vector and stable or transient transfection into Chinese hamster ovary cells, the aequorin bioluminescence/Ca2+ assay was used to investigate receptor activation. This is the first report of the construction of a cell line stably expressing a C. elegans neuropeptide receptor. Our experiments identified both receptors as being cognate receptors for two FMRFamide-related peptides encoded by the flp-2 precursor: SPREPIRFamide (FLP2-A) and LRGEPIRFamide (FLP2-B). Pharmacological profiling using truncated forms of FLP2-A and -B revealed that the active core of both peptides is EPIRFamide. Screening of peptides encoded by other flps did not result in a significant activation of the receptor. In contrast to other C. elegans receptors tested in heterologous expression systems, the functional activation of both T19F4.1a and T19F4.1b was not temperature-dependent. Screening in cells lacking the promiscuous Galpha16 suggests that T19F4.1a and b are both linked to the Gq pathway.

Postgenomic characterization of G-protein-coupled receptors.

G-protein-coupled receptors (GPCRs) constitute one of the largest families of membrane-spanning proteins. Their importance in drug development has been proven over and over again. Therefore, they remain one of the most significant groups of molecules to be characterized. In the postgenomic era, the methods used for the characterization of GPCRs have dramatically changed: the predicted orphan receptors are now often used to ascertain the ligands (reverse pharmacology), whereas, in the past, the bioactive ligand was used to identify the receptor (classic approach). In this review, we will give an overview of the recent postgenomic functional assays that are frequently used to link the orphan GPCR of both vertebrate and invertebrate organisms with their ligands.

Functional characterization of the putative orphan neuropeptide G-protein coupled receptor C26F1.6 in Caenorhabditis elegans.

In this study, we describe the cloning and the characterization of the third FMRFamide-related peptide (FaRP) receptor in Caenorhabditis elegans, the VRFa receptor 1. Numerous structurally different FaRPs were synthesized and used to screen the orphan C26F1.6 receptor for activation. Two peptides ending in M(orL)VRFamide elicited a calcium response in receptor expressing mammalian cells. The response is dose-dependent and appeared to be very specific, since very closely related FaRPs were less active, even the other peptides ending in M(orL)VRFamide. Pharmacological profiling of the most active peptide suggests that SMVRFa is the most active binding core. N-terminal extension decreases peptide activity.

G protein-coupled receptors in invertebrates: a state of the art.

G protein-coupled receptors (GPCRs) constitute one of the largest and most ancient superfamilies of membrane-spanning proteins. We focus on neuropeptide GPCRs, in particular on those of invertebrates. In general, such receptors mediate the responses of signaling molecules that constitute the highest hierarchical position in the regulation of physiological processes. Until recently, only a few of these receptors were identified in invertebrates. However, the availability of a plethora of genomic information has boosted the discovery of novel members in several invertebrate species, such as Drosophila, in which 18 neuropeptide GPCRs have been characterized. The finalization of genomic projects in other invertebrates will lead to a similar expansion of GPCR understanding. Many new insights regarding neuropeptide regulation have followed from the discovery of their cognate receptors. Furthermore, information on GPCR signaling is still fragmentary and the elucidation of these pathways in model insects such as Drosophila will lead to further insights in other species, including mammals. In this review we present the current status of what is known about invertebrate GPCRs, discuss some novel perceptions that follow from the identified members, and, finally, present some future prospects.

Identification in Drosophila melanogaster of the invertebrate G protein-coupled FMRFamide receptor.

We here describe the cloning and characterization of the functionally active Drosophila melanogaster (Drm) FMRFamide receptor, which we designated as DrmFMRFa-R. The full-length ORF of a D. melanogaster orphan receptor, CG 2114 (Berkeley Drosophila Genome Project), was cloned from genomic DNA. This receptor is distantly related to mammalian thyroid-stimulating hormone-releasing hormone receptors and to a set of Caenorhabditis elegans orphan receptors. An extract of 5,000 central nervous systems from the related but bigger flesh fly, Neobellieria bullata (Neb), was used to screen cells expressing the orphan receptor. Successive purification steps, followed by MS, revealed the sequence of two previously uncharacterized endogenous peptides, APPQPSDNFIRFamide (Neb-FIRFamide) and pQPSQDFMRFamide (Neb-FMRFamide). These are reminiscent of other insect FMRFamide peptides, having neurohormonal as well as neurotransmitter functions. Nanomolar concentrations of the Drm FMRFamides (DPKQDFMRFamide, TPAEDFMRFamide, SDNFMRFamide, SPKQDFMRFamide, and PDNFMRFamide) activated the cognate receptor in a dose-dependent manner. To our knowledge, the cloned DrmFMRFa-R is the first functionally active FMRFamide G protein-coupled receptor described in invertebrates to date.

Characterization of the short neuropeptide F receptor from Drosophila melanogaster.

A seven transmembrane G-protein coupled receptor has been cloned from Drosophila melanogaster. This receptor shows structural similarities to vertebrate Neuropeptide Y(2) receptors and is activated by endogenous Drosophila peptides, recently designated as short neuropeptide Fs (sNPFs). sNPFs have so far been found in neuroendocrine tissues of four other insect species and of the horseshoe crab. In locusts, they accelerate ovarian maturation, and in mosquitoes, they inhibit host-seeking behavior. Expression analysis by RT-PCR shows that the sNPF receptor (Drm-sNPF-R) is present in several tissues (brain, gut, Malpighian tubules and fat body) from Drosophila larvae as well as in ovaries of adult females. All 4 Drosophila sNPFs clearly elicited a calcium response in receptor expressing mammalian Chinese hamster ovary cells. The response is dose-dependent and appeared to be very specific. The short NPF receptor was not activated by any of the other tested arthropod peptides, not even by FMRFamide-related peptides (also ending in RFamide), indicating that the Arg residue at position 4 from the amidated C-terminus appears to be crucial for the response elicited by the sNPFs.