This past year we have continued our research efforts to develop appropriate in vitro techniques for the examination of G-protein-receptor interactions at the molecular level. This includes adapting our baculoviral expression vectors for simplified isolation of recombinant expressed 5HT2c receptors; modifying our chaotropic salt and G-protein reconstitution procedures for bombesin (GRP) receptors expressed in BalbC fibroblast; and improving the immobilization chemistries for coupling G- protein subunits and rhodopsin for surface plasmon resonance studies of protein-protein interaction. In addition, we have completed a detailed characterization of the molecular bases for """"""""inverse"""""""" agonism of antagonist of the 5HT2c receptor expressed in insect Sf9 cells using the baculovirus system (Hartman & Northup, J. Biol. Chem. In press) and an initial characterization of the GRP receptor expressed in BalbC fibroblasts (Hellmich, Battey & Northup, submitted). We have as well continued to exploit visual transduction models for a detailed examination of the molecular interactions of G-protein subunits, receptor and guanine nucleotides. These studies have led us to propose that the catalytic function of receptors in G-protein activation involves an ordered interaction in which Gbeta-gamma binding to receptor proceeds Galpha-GDP and the product of the catalysis is a dissociated G`[empty] (Hoon, Clark, Ryba & Northup, manuscript in preparation). Prior models of G-protein activation have not ordered subunit interaction and have hypothesized that receptors stabilize the Galpha[empty] requiring GTP binding to dissociate the activated Galpha-GTP. Further, we have devised strategies of nucleotide competition which allow us to examine the receptor-catalysis function in some detail. While these experiments were designed to establish optimal kinetic protocols for testing models of G- protein-receptor interaction, they have provided as a by-product an enormously improved procedure for assay of selective G-protein-receptor interaction which promises to allow refined examination of these processes at the level of cellular membrane factions (Clark, Gutierrez & Northup, manuscript in preparation). Further, these data confirm by independent methods the conclusions we have reached using immobilized rhodopsins and measuring G-protein interactions by surface plasmon resonance (Clark, Mitchell, Chen & Northup, manuscript in preparation).
