DESCRIPTION: Structural analogies strongly suggest that DLAR acts as a receptor for an unknown navigational cue. This proposal aims to elucidate the structural requirements for DLAR to function correctly in guidance decisions, to characterize interactions between DLAR and components of better characterized signaling pathways, and to characterize a new mutation that phenocopies mutations in DLAR. In the first specific aim a number of engineered alterations in DLAR will be tested for their ability to rescue DLAR function in a DLAR mutant background. Constructs will be engineered into multiple lines by P-element insertion and then crossed into a DLAR mutant line that expresses GAL4 in postmitotic neurons. GAL4 activates a driver that causes the expression of the construct so that it can be tested for rescuing activity. Rescue has already been achieved with intact DLAR. Constructs to be tested include: extracellular domain deleted, cytoplasmic domain deleted, deletion of each of the two phosphatase domains individually, and inactivating point mutations within each of the phosphatase domains. So long as the engineered constructs are appropriately expressed, these experiments will likely determine whether DLAR acts as a receptor and whether its phosphatase activity is required for its guidance function. At the suggestion of a previous reviewer, DLAR will be tested directly for phosphatase activity by an appropriate assay. The only difficulty here is that it is hard to know what the appropriate substrate is for DLAR to act upon. There are two separate parts to specific aim #2. The first depends upon the observation that the DLAR phenotype is synergized by a weak mutation in Rac and that a human relative of DLAR affects Rho and Rac activity through a guanine nucleotide exchange factor named Trio. It is therefore proposed to further study the effects of dominant negative and constitutively active (or overexpressed) Rho, Rac, and CD42 on motor neuron advance, guidance, and the DLAR phenotype. A GAL4-UAS system would be used to drive expression of the constructs appropriately. The second set of proposed experiments are motivated by the finding that the DLAR phenotype is suppressed by a mutation in the abl tyrosine kinase. It is possible that DLAR's phosphatase activity is somehow in competition with kinase activity in the able signaling pathway. This possible interaction will be further examined by the construction of double mutants with DLAR and other known members of the abl pathway. Kinase-active and kinase-inactive forms of abl will also be compared for their ability to suppress the DLAR phenotype. These experiments offer the potential of better characterizing the signaling pathway involved in DLAR function. Specific aim #3 involves the further analysis of a new mutation, circumfirential, that phenocopies DLAR. Several important issues will be addressed: the mutation will be mapped, the specificity of the navigation errors it evokes will be better characterized, new alleles will be collected, and a mosaic analysis will be performed to determine if circumfirential acts cell autonomously. This last experiment is very important. If circumfirential is required in the motor neuron, then it could be a member of the DLAR signaling pathway. If it is required outside the motor neuron, it could be the still elusive ligand for DLAR. All of these studies will use techniques that are standard in the field and are feasible for this investigator.