This amended proposal from Dr. Joan Hooper addresses the mechanisms of Hedgehog signalling in Drosophila segment patterning. Dr. Hooper's research during the previous grant period (supported by a FIRST award) concerned intercellular signalling mechanisms involved in the functioning of segment polarity genes. This study, and those of numerous other labs, have described various gene interactions in two signalling pathways required for segment specification, those of Wingless and Hedgehog. Wingless, secreted from cells along the posterior edge of each parasegment, and Hedgehog, secreted from cells at the anterior edge, produce discrete effects depending upon whether responding cells are contacted by one or the other protein or by both together. These effects become distinguished further by virtue of the production in these cells of the transcription factor engrailed (en; anterior edge of each parasegment) or sloppy paired (slp; posterior compartment of each parasegment. While work on these pathways has been extraordinarily active in recent years, there are large gaps in understanding the molecular mechanisms by which these pathways specify pattern. The precise point of action of many of the involved gene products is unclear, and it may be that not all members of the pathways are known. Dr. Hooper is particularly interested in identifying the receptor of the Hedgehog protein and in understanding the intracellular responses to ligand-receptor interactions. Dr. Hooper has cloned and characterized smoothened (smo), a gene whose mutant phenotype is very similar to that of hh mutants. SMO is required for proper hh function, apparently acting downstream of hh and upstream of cubitus interruptus (ci), a transcription factor which appears to mediate Hh signalling. The sequence of smo suggests that the protein is a seven - transmembrane receptor protein related to Frizzled, but expressed in a quite distinct pattern. Dr. Hooper hypothesized that smo encodes the hh receptor, and she proposes experiments to test this hypothesis. Dr. Hooper has also been investigating ci. The null phenotype of ci is like that of hh, and distinct in some features from wg. In the presence of a null ci allele, wg, gsb, and ptc are not expressed in the ectoderm in late stage 10 and early stage 11 embryos, an effect which mimics mutant hh. A dominant gain-of -function ci allele, ciD, obviates the need for Hh signaling. The Ci protein is a sequence specific DNA binding protein of the zinc finger class. Dr. Hooper's group has established that Ci can activate the Hh-reponsive wg promoter in Schneider-2 cells after transient transfection and has shown that Ci binding sites are required for the activation. When Ci and Hh are co-expressed in Schneider cells, the activity on Ci in wg promoter-luciferase expression is enhanced, and co-expression of Hh with Patched (a known repressor of Hedgehog signalling) also represses Ci activation of the wg promoter. It turns out that smo is expressed at levels comparable to embryonic expression in this cell line, so if it is indeed a Hh receptor, it is present in adequate amounts for this function. ci, ptc, and hh are expressed at much lower levels. Finally, Dr. Hooper identified in genetic screens for modifiers of a dominant ptc phenotype, two new alleles of smo which show allele specific interactions with ptc , which suggests a close functional and, possibly a physical interaction between these two gene products. The current proposal focuses on smo and its functions.
The specific aims are: (1) To determine whether there is a physical interaction between Smo and Hh, using binding assays in cultured cells and co-immunoprecipitation and cross-linking experiments in cultured cells and in vivo. (2) To investigate the possibility that heterotrimeric G proteins are involved in Hh signalling. This hypothesis is based on the fact that many of the structural features of the conceptually translated smo gene product are common to heterotrimeric G protein- linked receptor proteins. A series of pharmacological tests used in G protein studies will be used, taking advantage of the cell culture system. (3) The relationship between ptc and smo will be examined. It will be determined whether Ptc acts downstream of Smo, using constitutively activated Ga to determine whether Ptc effects can be modulated. It will also be determined whether Ptc can modulate the number or affinity of Hh binding sites, an effect which would suggest that Ptc acted directly on Smo. (4) It will be determined whether Smo is regulated by cAMP-dependent protein kinase (PKA). PKA is known to be a negative regulator of Hh signaling and to act upstream of or in parallel to Ptc. Since Ptc has no putative PKA phosphorylation sites, while Smo has six consensus sites, Dr. Hooper hypothesizes that the receptor might be regulated by PKA-dependent phosphorylation. These studies will use the GaON mutants to place the site of PKA dependence, as well as determination of PKA effects on the number and affinity of Hh binding site. PKA binding sites on Smo will be mapped also.
