J Biol Chem, Vol. 273, Issue 33, 20916-20923, August 14, 1998

Activation of Mitogen-activated Protein Kinases by Formyl Peptide Receptors Is Regulated by the Cytoplasmic Tail

Madhavi J. Rane, John M. Arthur^§, Eric R. Prossnitz^¶, and Kenneth R. McLeish^§

From the Departments of  Medicine and ^§ Biochemistry and Molecular Biology, University of Louisville Health Science Center and the  Veterans Affairs Medical Center, Louisville, Kentucky 40202 and the ^¶ Department of Cell Biology and Physiology, University of New Mexico, Health Sciences Center, Albuquerque, New Mexico 87131

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 (GTPS) binding in membranes from FPRwt but not FPRdel cells. GTPS inhibited FMLP binding to FPRwt but not FPRdel membranes. Photoaffinity labeling with azidoanilide-[-³²P]GTP in the presence or absence of FMLP showed increased labeling only in FPRwt membranes. Immunoprecipitation of _i2 and _q/11 from solubilized, photolabeled membranes showed that FPRwt were coupled to _i2 but not to _q/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 G_i proteins and MAPK cascades. Deletion of the carboxyl-terminal tail results in constitutive activation of ERK and p38 kinase through a G_i2-independent pathway.