- Determination of the function of lipid rafts in vivo
- GPI-anchored proteins reside in membrane microdomains known as lipid rafts which function as specialized signaling platforms. Maximal Ret signal transduction in vitro requires its translocation into lipid rafts by GFRas. We have recently discovered that Ret is rapidly ubiquitinated and degraded after activation with GFLs. Interestingly, lipid rafts sequester Ret away from the degradation machinery located in non-raft membrane domains, thereby sustaining Ret signaling. Although the composition and function of lipid rafts has been analyzed in great detail with regard to signal transduction, controversy continues as to whether lipid rafts are membrane structures that exist under normal physiologic conditions. We have produced a knockin mouse that blocks the translocation of the Ret signaling complex into rafts in vivo. The analysis of these mice is allowing us to test the hypothesis that the recruitment of Ret to lipid rafts by GPI-anchored GFRa1 is required for GDNF functions such as development of the kidneys and enteric nervous system.
- Determination of whether GFLs function as long-distance growth factor
- Neurotrophic factors are often produced by the targets of neuronal innervation and activate receptors on the nerve terminals. The distance between the axon terminal and the cell body, where the neurotrophic signal must ultimately travel in order to promote the survival and growth of the neuron, is often a huge distance. The distance between the motor endplate and the cell body of a motor neuron that resides in the spinal cord, for example, can be a meter long. How neurotrophic factors promote signals retrogradely over long distances is an area of intense interest in neurobiology. Whether GFLs act over long distances as target-derived neurotrophic factors is unresolved. Using compartmentalized cultures of sympathetic and sensory neurons, a unique culture system that separates biochemically the cell bodies from the distal axons, my laboratory is examining whether GFLs can signal from axon terminals, and by what mechanism this is accomplished.
• Determination of the postnatal functions of GDNF and Ret
GDNF deletion is lethal perinatally. Ret deletion phenocopies the knockout of GDNF in mice in regards to development of the kidneys and enteric nervous system. Although GDNF is expressed in the nervous system and periphery in adulthood, the functions of GDNF are largely unresolved due to the early death of the GDNF and Ret knockout mice. To avoid this limitation, we are taking two complementary approaches. First, we have generated function-blocking GDNF antibodies that were cleaved to produce Fab fragments. Injection of these anti-GDNF Fab fragments into rodents inhibits GDNF without eliciting a non-specific immune response. Second, we are taking a genetic approach by crossing a Ret conditional knockout mouse with several neuronal subtype-specific and tamoxifen-inducible Cre lines. These approaches are providing us with exquisite control over when and where GDNF and Ret are blocked. These methods are allowing, for the first time, the examination of GDNF and Ret in the postnatal development and maintenance of the neuromuscular junction, nociceptive sensory neurons, craniofacial sensory and motor neurons, and taste buds.