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 beta-gamma subunit and retinal interactions 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 by beta- gamma rather than alpha subunit interaction. As an extension of the rhodopsin model for G protein coupling we have utilized surface plasmon resonance (SPR) measurements for kinetic analysis of G- protein-rhodopsin interaction monitored in real time. Functionally active rhodopsin was immobilized on an SPR surface via binding to immobilized concanavalin A. Rhodopsin immobilized in this manner retained full biochemical specific activity for catalysis of nucleotide exchange on the retinal G-protein alpha subunit. The binding interactions of retinal alpha and beta-gamma subunits with rhodopsin were profoundly synergistic. Binding of beta-gamma dimers with distinct gamma subunits to rhodopsin, independent of alpha subunit, was readily observable by SPR. Further, these dimers displayed dramatically different binding affinities and kinetics. The physiologically appropriate retinal dimer displayed rapid association and dissociation kinetics, while the other beta-gamma dimers dissociated at more than 100-fold slower rates. These data suggest that the duration of a G-protein-coupled receptor signaling event is an intrinsic property of the G protein coupling partners, in particular, the beta-gamma dimer. These findings are being followed up by analysis of the binding interactions of beta-gamma dimers and rhodopsin with chimeric gamma subunit chains. We initiated a molecular investigation of the G protein signaling of the human cannabinoid receptors. Two genes have been identified encoding receptors for cannabinoid ligands, CB1 and CB2. The CB1 and CB2 cannabinoid receptors mediate antinociception, antiemisis and immunosupression through Gi and Go signaling, however due to their wide range of effects and psychoactive properties the therapeutic utility of currently available agonists is limited. We constructed baculoviruses encoding the human receptors and examined their ligand regulation and G protein selectivity by in situ reconstitution. While the CB1 and CB2 receptors both activate Gi and Go, the efficacy of CB1 activation of Gi or Go is agonist-dependent. Our data thus demonstrate agonist-selective G-protein signaling by the CB1 receptor and suggest that therapeutic agents may be designed to regulate individual G-protein signaling pathways selectively. Our in situ assay procedures may provide a tool for the rational design of non-intoxicating cannabinoid agents. We have continued our investigations of the molecular properties of the bombesin receptor family by mutational analysis of the Gastrin Releasing Peptide Receptor (GRP-R). Site-directed GRP-R mutants were expressed in fibroblasts and assayed for their ability to bind ligands and to catalyze exchange of guanine nucleotides. Two amino acid positions were predicted to form an intramolecular salt-bridge (D98 and R309) and a similar potential ion pair is found in most G protein coupled receptors. Alanine substitution at these positions reduced agonist binding and G-protein activation compared to wild type GRP-R. Single swapGRP-Rmutations (D98R, R309D) wereinactive. In contrast, the double swap mutation (D98R/R309D) had high affinity agonist binding, and had both a higher intrinsic basal activity and overall catalytic exchange activity compared to wild type.Thesefindings are consistent with a saltbridge interaction between these two polar and oppositely charged amino acids that maintains the proper receptor conformation necessary to interact with G proteins. Of the ten residues predicted as potential ligand binding contacts, two mutations showed selective loss for agonist (Y285A) or antagonist (F313A) binding to the GRP-R. In addition, we identified two amino acid residues, F270 and N281, in the sixth transmembrane segment which are essential for the receptor conformational change to a high affinity, catalytically active state. In a competition binding assay using an antagonist radioligand, bombesin showed a 20-100 fold decreased affinity for the N281A and F270A mutant GRP-R as compared to the wild-type receptor. The saturation binding isotherms are best fit by a two-site model, indicating that the receptors are in either a low affinity (KDL) or a high affinity (KDH) state. The ratio of the two affinities (KDL/KDH) was significantly increased in both mutants, while the fraction of mutant receptors in the high affinity state (RH/RTOTAL) was decreased. We conclude that these two residues are important for receptor conformational change to the high affinity state required for efficient catalysis of nucleotide exchange on Gq.
Showing the most recent 10 out of 19 publications
