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 Gα and Gβγ 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-Gαi2 fusion, and formyl peptide receptor-green fluorescent protein fusion) and two isotypes of the α subunit (αi2 and αi3) and βγ dimer (β1γ2 and β4γ2). At saturating nucleotide levels, the disassembly of a significant fraction of ternary complexes occurred on a subsecond time frame for αi2 complexes and τ1/2 ≤ 4 s for αi3 complexes, time scales that are compatible with cell activation. β1γ2 isotype complexes were generally more stable than β4γ2-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 α and βγ subunits was slower. These results are compatible with a cell activation model involving G protein conformational changes rather than disassembly of Gαβγ heterotrimer. © 2007 Elsevier Inc. All rights reserved.

Full Text

Duke Authors

Cited Authors

  • Wu, Y; Buranda, T; Simons, PC; Lopez, GP; McIntire, WE; Garrison, JC; Prossnitz, ER; Sklar, LA

Published Date

  • 2007

Published In

Volume / Issue

  • 371 / 1

Start / End Page

  • 10 - 20

International Standard Serial Number (ISSN)

  • 0003-2697

Digital Object Identifier (DOI)

  • 10.1016/j.ab.2007.08.011