Activation of mitogen-activated protein kinases by formyl peptide receptors is regulated by the cytoplasmic tail.
Wild type formyl peptide receptors (FPRwt) and receptors deleted of the carboxyl-terminal 45 amino acids (FPRdel) were stably expressed in undifferentiated HL-60 promyelocytes. Expression of FPRwt reconstituted N-formylmethionyl-leucyl-phenylalanine (FMLP)-stimulated extracellular signal-regulated kinase (ERK) and p38 kinase activity. Expression of FPRdel resulted in a 2-5-fold increase in basal ERK and p38 kinase activity, whereas FMLP failed to stimulate either mitogen-activated protein kinase (MAPK). Pertussis toxin abolished FMLP stimulation of both MAPKs in FPRwt cells but had no effect on either basal or FMLP-stimulated MAPK activity in FPRdel cells. FMLP stimulated a concentration-dependent increase in guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding in membranes from FPRwt but not FPRdel cells. GTPgammaS inhibited FMLP binding to FPRwt but not FPRdel membranes. Photoaffinity labeling with azidoanilide-[gamma-32P]GTP in the presence or absence of FMLP showed increased labeling only in FPRwt membranes. Immunoprecipitation of alphai2 and alphaq/11 from solubilized, photolabeled membranes showed that FPRwt were coupled to alphai2 but not to alphaq/11. FPRwt cells demonstrated calcium mobilization following stimulation with FMLP, whereas FPRdel cells showed no increase in intracellular calcium. We conclude that the carboxyl-terminal tail of FPRs is necessary for ligand-mediated activation of Gi proteins and MAPK cascades. Deletion of the carboxyl-terminal tail results in constitutive activation of ERK and p38 kinase through a Gi2-independent pathway.
Rane
M J
MJ
Department of Medicine, Louisville, Kentucky 40202, USA.
Arthur
J M
JM
Prossnitz
E R
ER
McLeish
K R
KR
D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002454:Cell Differentiation; D003593:Cytoplasm / Q000378:metabolism*; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism; D018922:HL-60 Cells; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000235:genetics / Q000378:metabolism*; D018000:Receptors, Peptide / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D018000:Receptors, Peptide; D009240:N-Formylmethionine Leucyl-Phenylalanine; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019204:GTP-Binding Proteins; D002118:Calcium