For the past several years, this laboratory has been studying the relationship between the ordered environment of the synaptic membrane and G protein mediated signal transduction systems. Of particular interest has been the capability of elements of the cytoskeleton to alter the coupling among receptors and G proteins involved in the stimulation or inhibition of adenylyl cyclase. Given the increasing number of processes attributed to G proteins, some mechanism which channels individual receptors, G proteins and effectors is likely to exist. This might be particularly true in the nervous system, where rapid and discrete response is a hallmark of synaptic transmission. Most recently, it has been observed that a subunits of G proteins may form complexes with synaptic membrane tubulin and undergo a directed transfer of nucleotide from the latter. This appears to be a highly specific process, as tubulin has been shown to bind, with high affinity, to only two G proteins, as and ail. Even though several other G proteins (alphai2, alphai3, alpha o and transducin (alpha r) are quite closely related to alpha il, their affinity for tubulin is much lower. The initial objective of research in this proposal is designed to determine the binding sites on tubulin for ail or as and the binding sites on these G proteins for tubulin. Studies will be done whereby a variety of proteolytic digestion (both tubulin and G proteins) products are subjected to binding of labelled G protein or labelled tubulin. These studies will be done with native and unfolded proteins and pep- tides, and the interaction will be quantified. Studies with chimeric G proteins, which contain portions of high- and low-tubulin affinity G alphas,will also be performed in the hope of identifying accessible tubulin binding regions. Since different regions on the tubulin molecule appear to be involved in G protein binding and nucleotide transfer, attempts to identify both regions are planned. Reconstitution studies with purified components will be employed to see whether other constituents of the synaptic membrane, which are known to interact with tubulin, might influence the complex formation, or the transfer of nucleotide, between tubulin and G proteins. Finally, peptides corresponding to the tubulin or G protein """"""""binding sites' and """"""""nucleotide transfer domains' will be synthesized. After a thorough characterization of these peptides in reconstituted systems, those which disrupt tubulin-G protein interaction will be tested for their effects on G protein-mediated processes in neural membranes and cells. It is hoped that these studies will provide insight as to how the cytoarchitecture of the neuron contributes to neuroreceptor response and responsiveness. Increase in the understanding of synaptic function must lead to increased understanding of the inner working of the brain.
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