Worm Breeder's Gazette 13(4): 69 (October 1, 1994)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Cloning, Mammalian Expression and Knockout of a C. elegans Serotonin Receptor.

Bjorn Olde1, Richard McCombie2, Verena Gobel3, John Fleming3

1 Neurogenetics Section, NINDS, NIH
2 Cold Spring Harbor Research Laboratories
3 MGH Cancer Center, Dept. of Pediatrics

Figure 1

  We have cloned and expressed in murine Ltk- cells a C. elegans serotonin
receptor. The cDNA (5HT-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 lymnae, Drosophila and mammalian
5HT-1 receptors. The sequence contains the A sp149 ,that is part of the DRY-motif
conserved in all aminergic receptors; also conserved is the S er217 that is found in all
5HT receptors except for the three Drosophila serotonin receptors. It contains a short
carboxy terminal and the presence of a positively charged amino acid residue in a
conserved position (A rg434 )which are structural attributes that have been associated
with a negative coupling to adenylate cyclase. The sequence of this receptor was
compared to all available seven transmembrane receptor sequences in the database to
determine phylogenetic relationships using a new form of maximum parsimony
analysis ( with the help of Frank Kolakowki who has set up a 7-transmembrane
receptor database). (see figure )
  It was found that this receptor is much closer to the mammalian 5HT-1 receptors
than to 5HT-2 or 5HT-5. Within the 5HT-1 cluster it is closest to the Drosophila
5HT-2a and 5HT-2b receptors and to the Lymnae serotonin receptor. Considering the
multiplicity of serotonin receptors in vertebrate as well as invertebrate species, it is
likely that other serotonin receptor subtypes exist in C. elegans.
  When expressed in murine Ltk- cells the 5HT-Ce receptor displays high affinity
for the serotonergic radioligand [[125I]-LSD (K[d]= 0.33nM) Ergot alkaloids particularly
ergoline derivatives (i.e., LSD and lisuride), display high affinity for the receptor, as did
the nonspecific serotonergic antagonist methiothepin. Indolealkylamines, which
normally bind to mammalian 5HT-1 receptors with affinities ranging from 2-400mM,
all displayed affinities in the micromolar range for the C elegans receptor. The high
affinity for ergot derivatives and the low affinity for indolealkylamines, both appear to
be common characteristics of invertebrate receptors. Application of serotonin to the
clonal cell-line attenuates forskolin stimulated cAMP production.
  The serotonin gene was isolated from a lambda genomic library and sent to the
Sanger Centre for fingerprinting where no match with previously placed DNA was
found The gene is 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
Y119D3 on the supplementary grid which itself has a somewhat dubious location left of
mgP30 on the left arm of III (around eat-8 ).We have screened mutator strain MT3126
for tc1 insertions in the vicinity of the receptor by PCR and have identified 4
independent 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

Figure 1