Sequence of interactions in receptor-G protein coupling.
Guanine nucleotide exchange in heterotrimeric G proteins catalyzed by G protein-coupled receptors (GPCRs) is a key event in many physiological processes. The crystal structures of the GPCR rhodopsin and two G proteins as well as binding sites on both catalytically interacting proteins are known, but the temporal sequence of events leading to nucleotide exchange remains to be elucidated. We employed time-resolved near infrared light scattering to study the order in which the Galpha and Ggamma C-terminal binding sites on the holo-G protein interact with the active state of the GPCR rhodopsin (R*) in native membranes. We investigated these key binding sites within mass-tagged peptides and G proteins and found that their binding to R* is mutually exclusive. The interaction of the holo-G protein with R* requires at least one of the lipid modifications of the G protein (i.e. myristoylation of the Galpha N terminus and/or farnesylation of the Ggamma C terminus). A holo-G protein with a high affinity Galpha C terminus shows a specific change of the reaction rate in the GDP release and GTP uptake steps of catalysis. We interpret the data by a sequential fit model where (i) the initial encounter between R* and the G protein occurs with the Gbetagamma subunit, and (ii) the Galpha C-terminal tail then interacts with R* to release bound GDP, thereby decreasing the affinity of R* for the Gbetagamma subunit. The mechanism limits the time in which both C-terminal binding sites of the G protein interact simultaneously with R* to a short lived transitory state.
Herrmann
Rolf
R
Institut für Medizinische Physik und Biophysik (Campus Mitte), Charité-Universitätsmedizin Berlin, Schumannstrasse 20-21, 10098 Berlin, Germany.
Heck
Martin
M
Henklein
Petra
P
Henklein
Peter
P
Kleuss
Christiane
C
Hofmann
Klaus Peter
KP
Ernst
Oliver P
OP
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002417:Cattle; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000737:chemistry; D019204:GTP-Binding Proteins / Q000378:metabolism*; D007313:Insecta; D007700:Kinetics; D008027:Light; D008055:Lipids / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017368:Protein Prenylation; D017434:Protein Structure, Tertiary; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D017948:Retinal Rod Photoreceptor Cells; D012243:Rhodopsin / Q000737:chemistry; D012542:Scattering, Radiation; D013997:Time Factors; D015293:Transducin / Q000737:chemistry
D018076:DNA, Complementary; D008055:Lipids; D010455:Peptides; D043562:Receptors, G-Protein-Coupled; D012243:Rhodopsin; D019204:GTP-Binding Proteins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D015293:Transducin