Cloning, mammalian expression and knockout of a C elegans serotonin receptor Bjorn Olde, Richard McCombie+, Verena Gobble* and John Flaming* Neurogenetics Section, NINDS, NIH; +Cold Spring Harbor Research Laboratories; *MGH Cancer Center, Dept. of Pediatrics We have cloned and expressed in murine Ltk- cells a C elegans serotonin receptor. The cDNA (SHT-Ce) is 1528 bp long and codes for a peptide of 445 amino acids. The protein displays strong homology to the seven transmembrane family of G- protein coupled receptors especially to the Iymnae, Drosophilo and mammalian 5HT- 1 receptors. The sequence contains the Aspl49, that is part of the DRY-motif conserved in atl aminergic receptors; also conserved is the Ser217 that is fount in att 5HT receptors except for the three Drosophila setotonin receptors. It contains a short carboxy terminal and the presence of a positively enlarged amino acid residue in a conserved position (Arg434) which are structural athnbutes that have been associatod with a negative coupling to adenylate cyclaoe. The sequence of this receptor was compared to all availabte seven transmembrane receptor sequences in the database to determine phytogenetic relationships using a new form of maximum panimony anatysis ( with the help of FranK Kolakowki who has set up a 7-transmembrane receptor database). It was found that this receptor is much closer to the mammatian 5HT-1 receptors than to 5HT-2 or 5HT-5. Within the 5HT-1 cluster it is closest to the Drosophila 5HT-2a and SHT- 2b receptors and to tho Lymnae serotonin receptors. Considering the multiplicity of serotonin receptots in vertebrate as wolt as invertebrate species, it is likely that other serotonin receptor surpes
exi6t in C elegans. When expressod in murine Ltk- celts the 5HT-Ce receptor displays high affinity for the serotonergic radiotigand [125I]-LSD (Kd=0.33nM)) Ergot alkaloids particulary ergoline derivatives (i.e. LSD and tisuride) display high affinity for the receFcor as did the nonspecific sercrconergic antagonist methhthepin. Indolealkylamines which normatty bind to mammalian 5HT-I roceptors with affinitia raoging from 2-400nM all displayed affinities in the micromolecular range from the C elegans receptor. The high affinty for ergot derivatives and the Iow affinity for indoleattcylamines both appear to be common characteristics of invertebrate receptors. Application of serostonin to the colonial cell-line attenuates forskolin stisnulatect cAMP production. The serotonin geno was isotated from a lambda genornic library and sent to the Sanger Centre for fingerprinting where no match with previosly placed DNA was found The gene
i6 6.6 kb in size and is highly interrupted in structure (not intron-less as has been the case with some of the seven transmembrane receptors cloned in other species). The clone did not hybridize to the YAC grid but did weekly hybridize to Yl
l9D3 on the supplementary grit which itself has a somewhat dubious locatiosn left of mgP30 on the left arm of III (around eat- 8). We have screened muator stain MT3126 for tcl insertions in the vicioity csf the receptor by PCR and have identified 4 indepentent lines after sib-selection that require further selection to get down to a single worm. This work was greatly facilitated by the help of Ed Maryon Joel Rothman and Susan Mango. We are currently trying to localize the expression pattern of the receptor using the GFP vectors as a first step towards a screen looking for mutants that effect the placement of this receptor.