Rapid-mix flow cytometry measurements of subsecond regulation of G protein-coupled receptor ternary complex dynamics by guanine nucleotides.
We have used rapid-mix flow cytometry to analyze the early subsecond dynamics of the disassembly of ternary complexes of G protein-coupled receptors (GPCRs) immobilized on beads to examine individual steps associated with guanine nucleotide activation. Our earlier studies suggested that the slow dissociation of Galpha and Gbetagamma subunits was unlikely to be an essential component of cell activation. However, these studies did not have adequate time resolution to define precisely the disassembly kinetics. Ternary complexes were assembled using three formyl peptide receptor constructs (wild type, formyl peptide receptor-Galpha(i2) fusion, and formyl peptide receptor-green fluorescent protein fusion) and two isotypes of the alpha subunit (alpha(i2) and alpha(i3)) and betagamma dimer (beta(1)gamma(2) and beta(4)gamma(2)). At saturating nucleotide levels, the disassembly of a significant fraction of ternary complexes occurred on a subsecond time frame for alpha(i2) complexes and tau(1/2)< or =4s for alpha(i3) complexes, time scales that are compatible with cell activation. beta(1)gamma(2) isotype complexes were generally more stable than beta(4)gamma(2)-associated complexes. The comparison of the three constructs, however, proved that the fast step was associated with the separation of receptor and G protein and that the dissociation of the ligand or of the alpha and betagamma subunits was slower. These results are compatible with a cell activation model involving G protein conformational changes rather than disassembly of Galphabetagamma heterotrimer.
Wu
Yang
Y
Department of Pathology and Cancer Research Facility, University of New Mexico Health Sciences Center, University of New Mexico, Albuquerque, NM 87131, USA.
Buranda
Tione
T
Simons
Peter C
PC
Lopez
Gabriel P
GP
McIntire
William E
WE
Garrison
James C
JC
Prossnitz
Eric R
ER
Sklar
Larry A
LA
D003001:Cloning, Molecular; D003902:Detergents / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods*; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006150:Guanine Nucleotides / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008863:Microspheres; D011485:Protein Binding; D044042:Receptors, Formyl Peptide / Q000235:genetics / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012995:Solubility; D013050:Spectrometry, Fluorescence; D020298:U937 Cells
D003902:Detergents; D006150:Guanine Nucleotides; D008024:Ligands; D044042:Receptors, Formyl Peptide; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go