Expression of the Calcium-independent Receptor of -Latrotoxin (Cirl) in developing Drosophila melanogaster. Steve M Saylor, Mark FA VanBerkum. Biological Sciences, Wayne State University, Detroit, MI.

   Latrophilins are a family of G-protein coupled receptors (GPCRs) that interact with -Latrotoxin, a component of black widow spider venom, to stimulate exocytosis. They have been implicated in axon guidance, neural connectivity, and synaptogenesis. Here we characterize the gene structure and expression of the Drosophila homolog, cirl (CG8649), as a first step in understanding its function. There are eight splice variants of the cirl gene encoding two distinct proteins that share a common extracellular N-terminus, but having either one transmembrane domain (short isoform) or the seven commonly associated with GPCRs (long isoform). RT-PCR confirmed the expression of at least five cirl isoforms in embryonic tissue including long and short isoforms. Unexpectedly, none of these isoforms are predicted to have a canonical signal sequence, a feature also seen in the cirl gene of eight Drosophila species. Flybase identifies a full-length clone, RE25258, but it actually contains a fortuitous, in frame, signal sequence from an unrelated gene (CG42238). We have subcloned the native cirl cDNA and RE25258 into an expression vector and are verifying the membrane localization and topology of Cirl in S2 cells. Using in situ hybridization, the expression of both the long and short variants were determined in embryos and third instar larvae CNS and imaginal discs. The long isoform exhibits dynamic expression especially in tissues actively undergoing invagination and/or morphogenesis. This includes expression in the embryonic foregut, midgut, and hindgut, then spreading to the ventral nerve cord, and dorsal pouch, as well as in larval CNS, wing, leg, and eye imaginal discs. The short isoform joins the long form in a subset of these tissues; the embryonic foregut, hindgut, dorsal pouch, and larval leg/eye discs. It is notably absent from the nervous system. To begin assessing cirl function we are examining embryos of a P-element insertion line, KG03335, and mobilizing this P-element to create additional alleles. UAS constructs are being developed for overexpression studies. Funded by the NICHHD.