Cellular $~nalllng Vol. 5, No. 6, pp. 735-745, 1993. 0698-6568/93 $6.00 + 0.00 Printed in Great Britain. Perlpunon Press Ltd DESENSITIZATION BY PROTEIN KINASE C ACTIVATION DIFFERENTIALLY UNCOUPLES FORMYL PEPTIDE RECEPTORS FROM EFFECTOR ENZYMES IN HL-60 GRANULOCYTES ELEANOR D. LEVEREt,* ALFRED A. JACOBS* and ~ R. McLmsH*t:~ Departments of *Medicine and i'Biochemistry, University of Louisville and the Veterans Administration Medical Center, Louisville, KY 40292, U.S.A. (Received 1 June 1993; and accepted 30 July 1993) Abstract--The hypothesis that protein kinase C (PKC) participates in agonist-mediated desensitization of formyl peptide receptors in HL-60 granuiocytes was tested, fMet-Leu-Phe and leukotriene B4(LTB0 produced homologous desensitization of agonist-stimulated intracellular calcium transients. Pre-treatment with the PKC activator, phorbol myristate acetate (PMA; 10nM), abolished both fMet-Leu-Phe and LTB4-stimulated calcium transients. Membranes prepared from control HL-60 granulocytes (NM) or cells treated with 10 nM PMA (PMA-M) demonstrated increased formyl peptide receptor and G protein density, as determined by radiofigand binding and pertussis toxin- and cholera toxin-catalysed ADP ribosylation, fMet-Leu-Phe stimula- tion of guanosine 5'-[T-thio]-triphosphate (GTI~S) binding and GTP hydrolysis and GDP inhibition of fMet- Leu-Phe binding were not different between NM and PMA-M. Pre-treatment with 10 nM PMA did not inhibit subsequent fMet-Leu-Phe-stimulated superoxide generation or phospholipase D activation. We conclude that PKC desensitizes fMet-Leu-Phe-stimuiated phospholipase C, but not phospholipase D, responses and that PKC activation does not mediate agonist-induced desensitization of formyl peptide receptors. Key words: Desensitization, protein kinase C, G proteins, intracellular calcium, formyl peptides, HL-60 granulocytes, phospholipase C, phospholipase D. INTRODUCTION DESENSITIZATION is the attenuation or loss of cell responsiveness to receptor activation upon continued or repeated agonist exposure. Homologous desensitization is defined as agonist-specific loss of responsiveness, while heterologous desensitization is a generalized loss of responsiveness following stimulation :~Correspondence to: Kenneth R. McLeish, M.D., Kidney Disease Program, University of Louisville, 500 S Floyd St, Louisville,KY 40292, U.S.A. Abbreviations: fMet-Leu-Phe,N-formylmethionyl-leucyl- phenylalanine; LTB4--1eukotriene B4; PMA--phorbol 12- myristate 13-acetate; PKC--protein kinase C; NM-- plasma membranes from normal differentiated HL-60 cells; PMA-M---plasma membranes prepared from HL-60 cells cultivated with PMA; G protein--guanine-nueleotide-bind- ing protein; G,--stimulatory G protein; GTP~S---guanosine 5'-l~,-thio]-triphosphate; PMN--polymorphonuclear leuko- cytes; flAR--fl-adrcnoceptor; Me2SO--dimethyl sulphox- ide; KRPB--Krebs-Ringers phosphate buffer; HBSS--- Hanks bufferedsalt solution. 735 with a single agonist. Chemoattractant receptor desensitization in polymorphonuclear leuko- cytes (PMNs) serves to limit the extent of tissue damage and contributes to the ability of cells to undergo directed migration. Homologous desensitization of formyl peptide receptors, one of the G protein-coupled chemoattractant receptors, is accompanied by reduced membrane expression of receptors, receptor-G protein uncoupling and suppression of fMet- Leu-Phe-stimulated intraceUular calcium tran- sient and superoxide generation [1-3]. Recently, class desensitization of several chemoattractant receptors following exposure to a single agonist has been reported [1]. Although the mechanism by which formyl peptide receptors undergo desensitization is not known, substantial evidence from other G pro- tein-coupled receptor systems suggests that receptor phosphorylation plays a critical role. The fl-adrenoceptor (tAR) is an extensively