| Activated Gi/o-type G-protein coupled receptors (GPCRs) for neurotransmitters such as noradrenaline, 5-HT, and dopamine are capable of mediating the inhibition of secretory vesicle cargo release in neurons, adrenochromaffin cells and beta cells in the islets of Langerhans. The mechanisms for secretory vesicle exocytosis and the component|
proteins involved are well-conserved between different populations of secretory-granule containing cells. One mechanism for which this phenomenon can occur is via the direct interaction between G protein beta gamma subunits (Gβγ) and the SNARE (soluble NSF attachment protein receptor) proteins of the cell’s vesicle fusion machinery to block vesicle fusion and subsequent cargo release. We have previously shown that Gβγ subunits directly bind to the t-SNARE proteins SNAP-25 and syntaxin1A as well as the v-SNARE protein synaptobrevin. We have established that Gβγ regulates vesicle fusion by competing with synaptotagmin I in a Ca2+ -sensitive manner for binding sites on SNARE proteins. To identify regions on SNARE proteins required for interaction with Gβγ as well as regions on Gβγ required for interaction with SNARE, we have utilized a peptide screening technique. From this, we have identified a series of residues upon SNAP-25 that reduce binding of Gβγ in in vitro protein binding studies. We have also generated a series of residues on Stx1A and Gβγ that we hypothesize are required for the Gβγ-Stx1A interaction. We intend to create full-length Gβγ and Stx1A proteins in which these residues have been mutagenized to Ala. We will then perform binding studies with these full-length Gβγ and Stx1A proteins, much as we did with SNAP-25. Furthermore, we will inject or transfect these proteins into neurons and neuroendocrine cell lines and look for disruption of Gi/o-coupled GPCR-mediated inhibition of exocytosis. From this, we will gain a great deal of structural insights on the nature of the interaction between Gβγ and SNARE. In addition, if introduction of a SNARE protein that specifically cannot bind Gβγ into a cell disrupts the ability of a given Gi/o-coupled GPCR to inhibit exocytosis, our hypothesis of Gβγ-SNARE interactions being vital to Gi/o-coupled GPCR-mediated inhibition of exocytosis will further be validated. Finally, this work will greatly facilitate the identification of additional cell types for which Gβγ -SNARE interactions play a significant role in inhibition of exocytosis, as we will be able to introduce our mutant SNAREs and Gβγ subunits into previously unexplored secretory cell types and look for significant changes in the ability of a given Gi/o-coupled GPCR to inhibit exocytosis.