RT Journal Article
SR Electronic
T1 Subsecond modulation of formyl peptide-linked guanine nucleotide-binding proteins by guanosine 5'-O-(3-thio)triphosphate in permeabilized neutrophils.
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 734
OP 740
VO 43
IS 5
A1 R R Neubig
A1 L A Sklar
YR 1993
UL http://molpharm.aspetjournals.org/content/43/5/734.abstract
AB Rapid activation of guanine nucleotide-binding protein (G protein)-mediated signal transduction mechanisms occurs in many tissues. The human neutrophil provides a useful model for studying the mechanisms of these fast processes. Fluorescent chemotactic tetrapeptide and pentapeptide exhibit 30-50% quenching of fluorescence upon binding to the neutrophil formyl peptide receptor, and their binding affinity is strongly regulated by the G protein Gi. We used rapid kinetic spectrofluorometric methods to study the assembly and disassembly of the ternary complex of ligand, receptor, and G protein in digitonin-permeabilized human neutrophils. Binding was studied up to 20 nM ligand, where the half-time for association was 1.2 sec. The rate constant of association was near that for diffusion-limited reactions of ligands and proteins, 2 x 10(7) M-1 sec-1. The rate of uncoupling of formyl peptide receptor from G protein in the presence of high concentrations of guanine nucleotide was > or = 5 sec-1 (i.e., t1/2 of 0.14 sec). Thus, disassembly of the formyl peptide receptor-G protein complex occurs in the millisecond time domain and may be faster than the next step in the signal transduction process.