Subsecond modulation of formyl peptide-linked guanine nucleotide-binding proteins by guanosine 5'-O-(3-thio)triphosphate in permeabilized neutrophils.
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.
Neubig
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RR
Department of Pharmacology, University of Michigan Ann Arbor 48109-0626.
Sklar
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LA
D019204:GTP-Binding Proteins / Q000187:drug effects / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology*; D006801:Humans; D009010:Monte Carlo Method; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D011487:Protein Conformation; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000187:drug effects / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019204:GTP-Binding Proteins