Desensitization uncouples the formyl peptide receptor-guanine nucleotide-binding protein interaction in HL60 cells.
Formyl peptide receptors on differentiated HL-60 cells were desensitized to formyl-methionyl-leucyl-phenylalanine (FMLP)-stimulated superoxide production in a concentration-dependent manner, similar to that previously described for neutrophils. Membranes isolated from desensitized (DM) and normal (NM) HL-60 cells were used to compare receptor numbers and affinities between NM and DM and compare the ability of receptors on DM and NM to interact normally with their guanine nucleotide regulatory proteins (G proteins). Exposure of differentiated HL-60 cells to 10(-7) M FMLP for 10 min before membranes were isolated resulted in a 75% reduction in receptor number, without alteration of dissociation constants. The remaining receptors on DM did not interact normally with their G proteins, as demonstrated by 1) the failure of guanine nucleotides to alter FMLP binding, 2) the inability of FMLP to stimulate guanosine-5'-O-(3-thiotriphosphate) binding, and 3) the attenuation of FMLP stimulation of GTPase activity. These results were not due to a reduction in G protein content of DM, as determined by Western blot analysis with an antibody that recognized alpha 40 and by pertussis toxin-catalyzed [32P]ADP-ribosylation of membrane G proteins in NM and DM. The failure of FMLP receptors on DM to interact with their G proteins was not due to differences in receptor number between NM and DM. Increasing the Mg2+ concentration partially restored the FMLP receptor-G protein interaction in DM. We conclude that desensitization of the formyl peptide receptor is associated with both loss of membrane receptors and a functional alteration in the receptor-G protein interaction, which can be partially reversed by increased concentrations of Mg2+.
McLeish
K R
KR
Department of Medicine, University of Louisville, Kentucky.
Gierschik
P
P
Jakobs
K H
KH
D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D008274:Magnesium / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000502:physiology*; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000378:metabolism*; D013873:Thionucleotides / Q000378:metabolism; D014407:Tumor Cells, Cultured
D008565:Membrane Proteins; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D013873:Thionucleotides; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D009240:N-Formylmethionine Leucyl-Phenylalanine; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D008274:Magnesium