title: 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 alpha subunit, via binding to immobilized concanavalin A. The binding interactions of bovine retinal alpha(t) and beta(1)gamma(1) 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'-[gamma-thio]triphosphate-bound Galpha(t), nor was binding observed with squid retinal Galpha(q), which is not activated by bovine rhodopsin. The binding affinity (336+/-171 nM; mean value+/-S.D.) of retinal betagamma for rhodopsin in the presence of retinal alpha subunit measured by SPR confirmed the apparent affinity of 254 nM determined previously by nucleotide exchange assays. Binding of beta(1)gamma(1), beta(1)gamma(2), and beta(1)gamma(8-olf) dimers to rhodopsin, independently of the alpha subunit, was readily observable by SPR. Further, these dimers, differing only in their gamma subunit compositions, displayed markedly distinct binding affinities and kinetics. The beta(1)gamma(2) dimer bound with a kinetically determined K(d) of 13+/-3 nM, a value nearly identical with the biochemically determined K(1/2) of 10 nM. The physiologically appropriate beta(1)gamma(1) displayed rapid association and dissociation kinetics, whereas the other beta(1)gamma 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 Gbetagamma 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 betagamma dimer.
authors.affiliation: Laboratory of Cellular Biology, National Institute on Deafness and Other Communication Disorders, Room 2A-11, 5 Research Court, Rockville, MD 20850, USA.
authors.forename: W A
authors.initials: WA
authors.lastname: Clark
authors.forename: X
authors.initials: X
authors.lastname: Jian
authors.forename: L
authors.initials: L
authors.lastname: Chen
authors.forename: J K
authors.initials: JK
authors.lastname: Northup
mesh_terms: D000818:Animals; D002417:Cattle; D003208:Concanavalin A / Q000378:metabolism; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism*; D007700:Kinetics; D011485:Protein Binding; D021122:Protein Subunits; D012160:Retina / Q000378:metabolism*; D012243:Rhodopsin / Q000378:metabolism*; D020349:Surface Plasmon Resonance
chemical_list: C118981:G-protein Beta gamma; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D021122:Protein Subunits; D003208:Concanavalin A; D012243:Rhodopsin; D020962:Heterotrimeric GTP-Binding Proteins