Real-time analysis of the assembly of ligand, receptor, and G protein by quantitative fluorescence flow cytometry

Shawn P. Fay, Richard G Posner, William N. Swann, Larry A. Sklar

Research output: Contribution to journalArticle

80 Scopus citations

Abstract

We describe a general approach for the quantitative analysis of the interaction among fluorescent peptide ligands (L), receptors (R), and G proteins (G) using fluorescence flow cytometry. The scheme depends upon the use of commercially available fluorescent microbeads as standards to calibrate the concentration of fluorescent peptides in solution and the receptor number on cells in suspension. We have characterized a family of fluoresceinated formyl peptides and analyzed both steady-state and dynamic aspects of ligand formyl peptide-receptor interactions in digitonin-permeabilized human neutrophils. Detailed receptor-binding studies were performed with the pentapeptide N-formyl-Met-Leu-Phe-Phe-Lys-fluorescein. Equilibrium studies showed that GTP[S] caused a loss of binding affinity of approximately two orders of magnitude, from ∼0.04 nM (LRG) to ∼3 nM (LR), respectively. Kinetic studies revealed that this change in affinity was principally due to an increase in the dissociation rate constants from ∼1 × 10-3 s-1 (LRG) to ∼1 × 10-1 s-1 (LR). In contrast, the association rate constants in the presence and absence of guanine nucleotide (∼3 × 107 s-1 M-1) were statistically indistinguishable and close to the diffusion limit. In the presence of guanine nucleotide (LR), the kinetic data were adequately fit by a single-step reversible-binding model. In the absence of guanine nucleotides, not all receptors have rapid access to G to form the LRG ternary complex. Mathematically, those R that have rapid access to G are either precoupled to R or the association of G with R is fast compared to the association of L with R. The physiological consequences of coupling heterogeneity are discussed.

Original languageEnglish (US)
Pages (from-to)5066-5075
Number of pages10
JournalBiochemistry
Volume30
Issue number20
StatePublished - 1991
Externally publishedYes

    Fingerprint

ASJC Scopus subject areas

  • Biochemistry

Cite this