Independent and synergistic interaction of retinal G-protein subunits with bovine rhodopsin measured by surface plasmon resonance

Abstract

We have used surface plasmon resonance (SPR) measurements for the kinetic analysis of G-protein-receptor interaction monitored in real time. Functionally active rhodopsin was immobilized on an SPR surface, with full retention of biochemical specific activity for catalysis of nucleotide exchange on the retinal G-protein α subunit, via binding to immobilized concanavalin A. The binding interactions of bovine retinal αt and β1γ1 subunits with rhodopsin measured by SPR were profoundly synergistic. Synergistic binding of the retinal G-protein subunits to rhodopsin was not observed for guanosine 5′-[γ-thio]triphosphate-bound Gαt, nor was binding observed with squid retinal Gαq, which is not activated by bovine rhodopsin. The binding affinity (336±171nM; mean value±S.D.) of retinal βγ for rhodopsin in the presence of retinal α subunit measured by SPR confirmed the apparent affinity of 254nM determined previously by nucleotide exchange assays. Binding of β1γ1, β1γ2, and β1γ8-olf dimers to rhodopsin, independently of the α subunit, was readily observable by SPR. Further, these dimers, differing only in their γ subunit compositions, displayed markedly distinct binding affinities and kinetics. The β1γ2 dimer bound with a kinetically determined Kd of 13±3nM, a value nearly identical with the biochemically determined K1/2 of 10nM. The physiologically appropriate β1γ1 displayed rapid association and dissociation kinetics, whereas the other β1γ dimers dissociated at a rate less than 1/100 as fast. Thus rhodopsin interaction with its native signalling partners is both rapid and transient, whereas the interaction of rhodopsin with heterologous Gβγ dimers is markedly prolonged. These results suggest that the duration of a G-protein-coupled receptor signalling event is an intrinsic property of the G-protein coupling partners; in particular, the βγ dimer.

Overview of Biacore Systems and Their Applications

“…Other examples of specificity assays include identification of binding sites (Jokiranta et al, ), monitoring steps involved in complex formation (Schuster et al, ; Clark et al, ; Thai and Ogata, ), and assessing cofactor requirements for an interaction to occur (e.g., Ca 2+ ; Schlattner et al, ).…”

Structural Insights into M1 Muscarinic Acetylcholine Receptor Signaling Bias between Gαq and β-Arrestin through BRET Assays and Molecular Docking

“…Though the calcium immobilization assay has been commonly used to assess the coupling of Gαq to the receptor, it does not measure the direct binding to the receptor and may be affected by the complex regulatory mechanisms of calcium influx, such as Gαi activation [ 14 ]. Recently, novel techniques, such as surface plasmon resonance and flow cytometry, have been developed to measure real-time interactions between GPCRs and G proteins [ 15 , 16 ]. However, these methods are performed outside the cell and cannot determine the effects of other factors inside the cell in real time.…”