FIXATION TRAPS FORMYL PEPTIDE RECEPTORS IN HIGH AND LOW-AFFINITY FORMS THAT CAN BE REGULATED BY GTP[S] IN THE ABSENCE OF LIGAND

The formyl peptide receptor on human neutrophils recognizes bacterial, N-formylated peptides and initiates a cascade of intracellular signal s via a pertussis toxin sensitive G(i) protein. We used fluorescence t echniques to investigate the interactions of ligand (L), receptor (R), and G proteins (G), the ternary complex, in both live and fixed human neutrophils. By lightly fixing permeabilized neutrophils with a proce dure that retained ligand binding, we were able to ''capture'' R and G in different configurations in the absence of ligand, Fixed receptors were trapped in a high affinity form (attributed to LRG) that could n ot be rapidly converted to low affinity by the addition of GTP[S]. Add ing saturating nucleotide prior to fixation trapped receptors in a low affinity form (attributed to LR). The low affinity receptors retained the sensitivity of the native receptors to the presence of Na+. The d istribution between high and low affinity receptors was modulated by G TP[S] in a dose dependent manner. The ability to redistribute low and high affinity receptor forms prior to fixation was unique to GTP[S], a s compared to other non-activating nucleotides, suggesting that GTP[S] can regulate the distribution between R and RG. We suggest that preco upled receptors that give rise to high affinity ligand binding are lik ely to exist in native membranes in human neutrophils.