Characterization of GABA B receptor-like proteins in Dictyostelium discoideum

GPCRs are a superfamily of diverse integral membrane proteins that allow detection and transduction of a large variety of extracellular signals. In Dictyostelium only seven members of one family, the crl (cAMP receptor like) family had been identified and studied in detail. The analysis of the genome sequence uncovered 48 additional putative GPCRs grouped into the secretin (family 2), metabotropic glutamate/GABAB (family 3) and the frizzled / smoothened (family 5) families of receptors. The presence of family 2, 3 and 5 receptors in Dictyostelium was indeed surprising because they had been thought to be animal-specific. GABA (gamma amino butyric acid), the principal inhibitory neurotransmitter in mammalian brain, signals through ionotropic (GABA(A)/GABA(C)) and metabotropic GABA(B) receptor systems. The functional GABAB receptor is a heterodimer of receptor 1 and receptor 2 subtypes. The Dictyostelium genome harbours 17 different genes encoding GABAB receptor like proteins each having slightly closer resemblance to one of each subtype. Yeast two-hybrid studies led us speculate that the C termini of these receptors alone may not be sufficient for their interactions to form homo or heterodimers. The detailed analysis of one of each type of receptors namely - GrlA and GrlJ was undertaken. Both these receptors are expressed throughout the development of Dictyostelium with a marked elevation in the later developmental stages. Mutant analysis revealed both GrlA and GrlJ to be involved in post aggregation morphogenesis in Dictyostelium. Strains lacking GrlA displayed a delay in development consistent with its expression pattern and completed development 3-4 hours later than the wild type cells. Furthermore, GrlA was found to be involved in controlling sporulation events as grlA- cells were defective in the generation of the signal required for spore differentiation, SDF-2. This may be due to a decrease in the levels of TagC, a protease present on the surface of prestalk cells that cleaves AcbA (formed in prespore cells and released) to form SDF-2 and the SDF-2 receptor, DhkA. Transcriptional profiling of the grlA- (16 h) displayed downregulation in the serum response factor (srfA) induced genes, which further strengthened the role of GrlA as an important component in sporulation and spore maturation. Our data suggest that GrlJ controls events during the transition from aggregation to post aggregation. grlJ - develops precociously after aggregation stages forming thin fingers that break several times on their way until culmination and form smaller fruiting bodies with malformed spores which are less viable. However, grlJ - does not exhibit any defect in the production or response, neither to the peptides controlling sporulation such as SDF-1 or SDF-2 nor towards GABA or Glutamate like the wild type cells