Effects of tunicamycin on the expression and function of formyl peptide chemotactic receptors of differentiated HL-60 cells.
We previously showed that formyl peptide chemotactic receptors (FPCR) of human phagocytic cells contain at least two asparagine-linked oligosaccharide chains located at the distal end of the receptor. The requirement of these N-linked oligosaccharide chains for expression and function of FPCR was investigated in HL-60 cells induced to differentiate by N6,O2-dibutyryladenosine 3',5'-monophosphate (Bt2cAMP) in the presence or absence of 5 micrograms/ml tunicamycin. Tunicamycin did not prevent the changes in morphology associated with Bt2cAMP-induced differentiation of HL-60 cells. Autoradiographic analysis after SDS-PAGE of FPCR affinity labeled with N-formyl-Nle-Leu-Phe-Nle-[125I]iodo-Tyr-Lys (formyl 125I-hexapeptide) and ethylene glycol bis(succinimidyl succinate) demonstrated that greater than 95% of FPCR expressed by tunicamycin-treated cells completely lacked N-linked oligosaccharide (Mr 32,000), and no fully glycosylated FPCR (Mr 62,000 to 85,000) was detectable. Scatchard analysis of formyl 125I-hexapeptide binding indicated the presence of two classes of binding sites for both control and tunicamycin-treated cells (control cells, 82,000 +/- 32,000 sites/cell with Kd 10.0 +/- 4.3 nM and 520,000 +/- 40,000 sites/cell with Kd 250 +/- 80 nM; tunicamycin-treated cells, 11,000 +/- 5000 sites/cell with Kd 3.0 +/- 1.9 nM and 470,000 +/- 70,000 sites/cell with Kd of 500 +/- 140 nM). Both control and tunicamycin-treated cells augmented superoxide anion release, exhibited a migratory response, and showed a transient rise in intracellular free Ca2+ upon stimulation with N-formyl-Nle-Leu-Phe. However, the responses of the tunicamycin-treated cells were less than that of the control cells. The present studies demonstrate that N-glycosylation of FPCR is not essential for cell surface expression or for several FPCR-mediated cell responses.
D002118:Calcium / Q000378:metabolism; D002236:Carbohydrate Conformation; D002454:Cell Differentiation; D002455:Cell Division; D002460:Cell Line; D002633:Chemotaxis; D005944:Glucosamine / Q000031:analogs & derivatives*; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000378:metabolism* / Q000473:pathology; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000378:metabolism* / Q000494:pharmacology; D011869:Radioligand Assay; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000032:analysis / Q000187:drug effects* / Q000502:physiology; D013481:Superoxides / Q000378:metabolism; D014415:Tunicamycin / Q000494:pharmacology*
D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D013481:Superoxides; D014415:Tunicamycin; D009240:N-Formylmethionine Leucyl-Phenylalanine; D005944:Glucosamine; D002118:Calcium