Worm Breeder's Gazette 13(4): 68 (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 the Inositol 1,4,5 Triphosphate Receptor.

Howard Baylis, Mike Squire, David Sattelle

The Babraham Institute Laboratory of Molecular Signalling, Department of Zoology, University of Cambridge, Downing Street, Cambridge UK.

  The intracellular second messenger inositol 1,4,5 trisphosphate (InsP[3]) binds to
a specific receptor (InsP[3]R) that releases Ca[2+] from intracellular storage sites,
thereby modulating various Ca[2+]-associated proteins in cells (Mikoshiba et al 1993
Ann. NY Acad Sci 707, 178-197). As part of a project to study inositol 1,4,5
trisphosphate (InsP[3]) signalling using C. elegans we have been attempting to clone
the nematode InsP[3] receptor. Using degenerate primers to two sequences conserved
amongst InsP[3] receptors in mammals, Xenopus and Drosophila we were able to
amplify a 240bp (IP240) fragment of the coding sequence, either from genomic DNA or
from random primed 1st strand cDNA. In addition, a search of the NCBI EST
database with the Drosophila sequence revealed that one of Yuji Kohara's (WBG 13
20-21) cDNAs ( yk33 g8 )encoded 445 bp covering the end of the gene and part or all of
the 3' untranslated region. This clone was clearly different from cDNA clones
encoding the known ryanodine receptor (eg yk4 h3 ).The amino acid sequences encoded
by the two sequences are clearly much closer to InsP[3] receptors than to the ryanodine
receptors. Thus the evidence to date suggests that this is the InsP[3] receptor. An
alignment of one part of the C. elegans sequence and InsP[3] receptor sequences from
other organisms (figure 1) shows that some of the residues which are identical in all
the other sequences are not conserved in C. elegans. Thus the sequence of this
molecule may provide insights into structure and function of the protein. We have
recently obtained a PCR product covering the 3kb between the two fragments and are
currently working towards obtaining a full length cDNA (expected to be in the region of
8-9kb) and a genomic clone. From here we plan to study the function of the receptor
using C. elegans genetics and in vitro expression and electrophysiology. We are
indebted to Drs Katsuhiko Mikoshiba, Teiichi Furuichi, Yuji Kohara and John
Fleming for their assistance during this study.
  IP240 2056 (Drosophila amino acid numbering)
  RAT 1
  RAT 2
  RAT 3
  Figure 1. An alignment of the amino acid sequence encoded by IP240 to InsP[3]
receptors from other species. Positions which are identical tothe consensus are shown
in upper case. The consensus is where 7 (of the 8) sequences are identical.