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.
Mertens
Inge
I
Laboratory of Developmental Physiology, Genomics and Proteomics, Katholieke Universiteit Leuven, Naamsestraat 59, B-3000 Leuven, Belgium. Inge.Mertens@bio.kuleuven.ac.be
Meeusen
Tom
T
Janssen
Tom
T
Nachman
Ronald
R
Schoofs
Liliane
L
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D017173:Caenorhabditis elegans / Q000235:genetics* / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D003001:Cloning, Molecular; D006224:Cricetinae; D008969:Molecular Sequence Data; D019151:Peptide Library; D012326:RNA Splicing / Q000235:genetics*; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D018010:Receptors, Invertebrate Peptide / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016415:Sequence Alignment; D015398:Signal Transduction
C055209:FMRFamide receptor; D019151:Peptide Library; D043562:Receptors, G-Protein-Coupled; D018010:Receptors, Invertebrate Peptide; D002118:Calcium