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United States Department of Agriculture

Agricultural Research Service

Title: Molecular Characterization of Two G Protein-Coupled Receptor Splice Variants As Flp2 Receptors in Caenorhabditis Elegans

Authors
item Mertens, Inge - CATHOLIC UNIV/BELGIUM
item Meeusen, Tom - CATHOLIC UNIV/BELGIUM
item Janssen, Tom - CATHOLIC UNIV/BELGIUM
item Nachman, Ronald
item Schoofs, Liliane - CATHOLIC UNIV/BELGIUM

Submitted to: Biochemical and Biophysical Research Communications
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 19, 2005
Publication Date: May 13, 2005
Citation: Mertens, I., Meeusen, T., Janssen, T., Nachman, R.J., Schoofs, L. 2005. Molecular characterization of two G protein-coupled receptor splice variants as FLP2 receptors in Caenorhabditis elegans. Biochemical and Biophysical Research Communications. 330:967-974.

Interpretive Summary: Because of problems with the development of resistance to conventional pesticides, there is a critical need for new concepts and alternative approaches in controlling invertebrate pests such as insects and nematode worms. The basic premise of this research is that neuropeptides (short chains of amino acids) serve as potent messengers in invertebrates such as nematode worms and insects to regulate vital functions. Nevertheless, neuropeptides in and of themselves hold little promise as pest control agents because of susceptibility to being degraded in the target pest. We must design neuropeptide mimics that resist degradation by enzymes in the digestive tract and blood of pest invertebrates and interact with the active site within the agricultural or medical pest in such a sway as to either over-activate or block critical, neuropeptide-regulated life functions. We report on the isolation, identification and characterization of active sites for specific members of the FMRFamide class of invertebrate neuropeptides (known as FLP2-A and FLP2-B) from the nematode labeled ‘C. elegans’, a prime model for parasitic nematode worms that plague man, livestock and crops. These also represent the first active sites of neuropeptides in nematode worms to be isolated in a manner that allows for further investigation of the molecular characteristics of their interaction with these neuropeptides. This discovery will aid in the design of neuropeptide-like compounds capable of disrupting critical life functions in nematode worms. This work represents a spin-off of our primary focus on insect pests and brings us one step closer to the development of practical neuropeptide-like substances that will be effective in controlling invertebrate pests, including nematode worms and insects, in an environmentally friendly fashion.

Technical Abstract: Two alternatively spliced Caenorhabtidiis 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 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.

Last Modified: 9/21/2014
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