The long range goal of this project is to understand the cellular mechanisms underlying axon guidance. These mechanisms are likely to apply to a number of important biological processes including cell migration and cancer metastasis. Specifically the research will focus on the identification of a glycoprotein on the surface of chick dorsal root ganglion (DRG) neurons recognized by the B-oligomer of pertussis toxin (bPTX) and the intracellular signals elicited by the binding of the B-oligomer to this molecule. The intracellular signals elicited by binding pertussis toxin (PTX) to this glycoprotein interfere with guidance cues provided by three distinct guidance molecules; laminin,thrombin and a chick brain collapsing factor. Thus, identification of the glycoprotein bound by the B-oligomer and the intracellular signals elicited by this binding may provide insight into the molecular mechanisms of axon guidance. The five specific aims of the project are l) to identify additional reagents that mimic or block the effects of the bPTX, 2) to use protein cross- linking to identify candidates for the PTX receptor 3) tone rate antibodies that mimic or block the effects of the bPTX., 4) to purify the receptor(s) through which bPTX acts and 5) to test the involvement of a known signaling system in the action of bPTX. Several lectins, anti-carbohydrate antibodies and a monoclonal antibody that blocks growth cone collapse will be screened as potential candidates to block or mimic the bPTX. PTX conjugated to a 125I-labeled crosslinker will be used to label DRG neurons or membranes to identify the molecular weights of molecules bound by PTX. Mice will be immunized with glycoproteins isolated from chick DRG or brain to generate antibodies that mimic or block the effects of the B-oligomer. The B-oligomer """"""""receptor"""""""" will be purified from chick DRG or brain through a combination of lectin PTX and antibody affinity columns as well as conventional chromatography. The C3 exoenzyme of Clostridium botulinum inactivates the GTP-binding protein rho and will be used to test the involvement of rho in axon guidance.
