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 signals via a pertussis toxin sensitive Gi protein. We used fluorescence techniques 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 procedure 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 not be rapidly converted to low affinity by the addition of GTP[S]. Adding 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 distribution between high and low affinity receptors was modulated by GTP[S] in a dose dependent manner. The ability to redistribute low and high affinity receptor forms prior to fixation was unique to GTP[S], as compared to other non-activating nucleotides, suggesting that GTP[S] can regulate the distribution between R and RG. We suggest that precoupled receptors that give rise to high affinity ligand binding are likely to exist in native membranes in human neutrophils.
Domalewski
M D
MD
Division of Cytometry, University of New Mexico School of Medicine, Albuquerque 87131, USA.
Guyer
D A
DA
Freer
R J
RJ
Muthukumaraswamy
N
N
Sklar
L A
LA
D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology*; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000378:metabolism*; D009504:Neutrophils / Q000166:cytology / Q000378:metabolism*; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D018000:Receptors, Peptide / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013873:Thionucleotides / Q000378:metabolism
D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D018000:Receptors, Peptide; D013873:Thionucleotides; D006153:Guanosine Diphosphate; D006165:Guanylyl Imidodiphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D009240:N-Formylmethionine Leucyl-Phenylalanine; C023427:guanosine 5'-O-(2-thiodiphosphate); D006160:Guanosine Triphosphate; D012964:Sodium