Evidence that activation of a common G-protein by receptors for leukotriene B4 and N-formylmethionyl-leucyl-phenylalanine in HL-60 cells occurs by different mechanisms.
Differentiated HL-60 cells were found to respond to the chemoattractants leukotriene B4 (LTB4) and N-formylmethionyl-leucyl-phenylalanine (FMLP), in a manner similar to neutrophils. Membranes of myeloid differentiated HL-60 cells were used (a) to examine the ability of LTB4 receptors to interact with a guanine-nucleotide-binding protein (G-protein), and (b) to compare this G-protein with that which is coupled to the FMLP receptor. LTB4 stimulated a dose-dependent increase in GTP hydrolysis and guanosine 5'-[gamma-thio]triphosphate (GTP[S]) binding, demonstrating that LTB4 receptors on HL-60 cells are coupled to a G-protein. Both pertussis toxin and cholera toxin inhibited stimulation of GTPase activity and GTP[S] binding by either LTB4 or FMLP, indicating that both receptors are coupled to a G-protein containing a 40 kDa alpha-subunit. That the two receptors share a common G-protein was shown by FMLP enhancement of cholera-toxin-induced inhibition of GTPase activity stimulated by either FMLP or LTB4. However, LTB4 did not enhance cholera-toxin-induced inhibition of GTPase activity, suggesting that the receptors interacted differently with this G-protein. This difference was confirmed by showing that FMLP, but not LTB4, stimulated receptor-specific [32P]ADP-ribosylation of the 40 kDa alpha-subunit. Concentrations of LTB4 and FMLP which produced maximal responses produced enhanced stimulation in both assays. This additive effect was not abolished by inactivation of up to 80% of G-protein activity by N-ethylmaleimide or cholera toxin. We conclude that LTB4 and FMLP receptors in HL-60 cells are coupled to a common G-protein. The receptor--G-protein interaction is different for the two receptors, and G-proteins not coupled to both receptors may account for the additive response.
McLeish
K R
KR
Department of Medicine, University of Louisville School of Medicine, KY 40292.
Gierschik
P
P
Schepers
T
T
Sidiropoulos
D
D
Jakobs
K H
KH
D002460:Cell Line; 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; D007975:Leukotriene B4 / Q000378:metabolism*; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000378:metabolism*; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000502:physiology*; D018102:Receptors, Leukotriene B4; D013873:Thionucleotides / Q000378:metabolism
D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D018102:Receptors, Leukotriene B4; D013873:Thionucleotides; D007975:Leukotriene B4; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D009240:N-Formylmethionine Leucyl-Phenylalanine; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins