Interaction of guanine-nucleotide-binding regulatory proteins with chemotactic peptide receptors in differentiated human leukemic HL-60 cells.
Human leukemic HL-60 cells were differentiated into neutrophil-like cells by treatment with dimethylsulfoxide (Me2SO) or N6,O2'-dibutyryladenosine 3',5'-phosphate (Bt2cAMP), and membrane fractions were prepared from the differentiated cells. Receptors for fMLF (fM,N-formylmethionine) and guanine-nucleotide-binding regulatory proteins (G proteins) serving as the substrate for pertussis toxin (islet-activating protein; IAP) were extracted from cell membranes then reconstituted into phospholipid vesicles. The binding of fMLF to the reconstituted vesicles (or the membranes) was determined with 10 nM [3H] fMLF. In both cases, high-affinity binding to vesicle preparations from the Me2SO- and Bt2cAMP-induced cells was abolished following treatment with IAP, suggesting that fMLF receptors were functionally coupled to IAP-sensitive G proteins in each of the two vesicle types. However, the high-affinity fMLF binding was much higher in vesicle preparations originating from Bt2cAMP-induced cells than in those from Me2SO-induced cells, although the amount of IAP-substrate G protein reconstituted into the each phospholipid vesicles preparation was not significantly different from the other. The G proteins of the two differentiated cells were both identified as inhibitory forms (Gi-2) based on their electrophoretic mobilities and immunoblot analyses. When purified Gi-2 from rat brain was reconstituted into the two IAP-treated vesicles, high-affinity fMLF binding was restored in a similar manner in both. IAP-substrate G proteins partially purified from the two differentiated HL-60 cells were also effective in restoring high-affinity fMLF binding to the IAP-treated vesicles. However, a significant difference was observed that the reconstituted binding was higher with the G-protein-rich fraction from Bt2cAMP-induced cells than with that from Me2SO-induced cells, with each of the two IAP-treated vesicle types. These results suggest that the different high-affinity binding of fMLF observed in the two differentiated HL-60 cells are due to a difference in the property of endogenous G proteins rather than fMLF receptors, though the two G proteins are indistinguishable from each other in terms of the subtype of G protein, Gi-2.
Tohkin
M
M
Department of Life Science, Tokyo Institute of Technology, Yokohama, Japan.
Morishima
N
N
Iiri
T
T
Takahashi
K
K
Ui
M
M
Katada
T
T
D003994:Bucladesine / Q000494:pharmacology; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007938:Leukemia / Q000188:drug therapy / Q000378:metabolism*; D008051:Lipid Bilayers / Q000378:metabolism; D009239:N-Formylmethionine / Q000378:metabolism*; D037342:Pertussis Toxin; D010743:Phospholipids / Q000378:metabolism; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D008051:Lipid Bilayers; D010743:Phospholipids; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D010566:Virulence Factors, Bordetella; D009239:N-Formylmethionine; D003994:Bucladesine; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D004121:Dimethyl Sulfoxide