In the past year we have characterized and extended our new in situ receptor-G-protein assay systems to increase their utility for a diverse family of receptor structures. The retinal activation of apo-opsin has been examined as a molecular model for ligand-regulated G-protein coupled receptors; and the relationships between G-protein alpha and betagamma subunit and retinal inreactions and rhodopsin conformations tested. these experiments show that as seen for bona fide ligand-regulated receptors retinal activation of apo-opsin displays two affinity states. However the high affinity interaction of opsin with retinal appears to be controlled betagamma rather than a subunit interaction. We have exploited the nucleotide regulation of these interactions to devise a highly specific sensitive biochemical assay for receptor-activation of G-proteins and confirmed its utility for bovine and squid rhodpsins and 5HT2c receptors. We have extended the expression of G-protein-coupled receptors using baculorvirus to the bombesin receptor family and initiated examination of the properties of the uncoupled and G-protein- coupled GRP receptor using our in situ reconstitution procedures. Lastly, as an initial test of the structural basis for Gbetagamma functional differences we have constructed mutations of the Ggamma2 gene to encode for differing isoprenoid modification. These studies show that the betagamma interaction with alpha or receptor is dependent upon isoprenylation, but that the specific isoprenoid modification of the gamma2 makes no difference to the alpha interaction and only modestly alters receptor interaction. These data support the hypothesis that the diverse betagamma protein structures encode receptor specific contacts.
