Real-time analysis of the assembly of ligand, receptor, and G protein by quantitative fluorescence flow cytometry.
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 approximately 0.04 nM (LRG) to approximately 3 nM (LR), respectively. Kinetic studies revealed that this change in affinity was principally due to an increase in the dissociation rate constants from approximately 1 x 10(-3) s-1 (LRG) to approximately 1 x 10(-1) s-1 (LR). In contrast, the association rate constants in the presence and absence of guanine nucleotide (approximately 3 x 10(7) 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.
Fay
S P
SP
Cytometry, University of New Mexico, School of Medicine, Albuquerque 87131.
Posner
R G
RG
Swann
W N
WN
Sklar
L A
LA
D000595:Amino Acid Sequence; D002463:Cell Membrane Permeability; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000378:metabolism*; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence; D013861:Thiocyanates
D005452:Fluoresceins; D005456:Fluorescent Dyes; D008024:Ligands; D009842:Oligopeptides; D011956:Receptors, Cell Surface; D013861:Thiocyanates; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019204:GTP-Binding Proteins; D016650:Fluorescein-5-isothiocyanate