Multiparameter flow cytometric analysis of a pH sensitive formyl peptide with application to receptor structure and processing kinetics.
Environmentally sensitive molecules have many potential cellular applications. We have investigated the utility of a pH sensitive ligand for the formyl peptide receptor, CHO-Met-Leu-Phe-Phe-Lys (SNAFL)-OH (SNAFL-seminaphtho-fluorescein), because in previous studies (Fay et al.: Biochemistry 30:5066-5075, 1991) protonation has been used to explain the quenching when the fluoresceinated formyl pentapeptide ligand binds to this receptor. Moreover, acidification in intracellular compartments is a general mechanism occurring in cells during processing of ligand-receptor complexes. Because the protonated form of SNAFL is excited at 488 nm with emission at 530 nm and the unprotonated form is excited at 568 nm with emission at 650 nm, the ratio of protonated and unprotonated forms can be examined by multiparameter flow cytometry. We found that the receptor-bound ligand is sensitive to both the extracellular and intracellular pH. There is a small increase in the pKa of the ligand upon binding to the receptor consistent with protonation in the binding pocket. Once internalized, spectral changes in the probe consistent with acidification and ligand dissociation from the receptor are observed.
Fay
S P
SP
Department of Cytometry, University of New Mexico School of Medicine, Albuquerque 87131.
Habbersett
R
R
Domalewski
M D
MD
Posner
R G
RG
Houghton
T G
TG
Pierson
E
E
Muthukumaraswamy
N
N
Whitaker
J
J
Haugland
R P
RP
Freer
R J
RJ
D000595:Amino Acid Sequence; D001665:Binding Sites; D005110:Extracellular Space / Q000737:chemistry; D005434:Flow Cytometry / Q000379:methods*; D005452:Fluoresceins / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007424:Intracellular Fluid / Q000737:chemistry; D007700:Kinetics; D015473:Leukemia, Promyelocytic, Acute / Q000378:metabolism / Q000473:pathology; D008024:Ligands; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000378:metabolism; D009842:Oligopeptides / Q000032:analysis* / Q000378:metabolism; D011485:Protein Binding; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000378:metabolism*; D018000:Receptors, Peptide / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014407:Tumor Cells, Cultured
D005452:Fluoresceins; D008024:Ligands; D009363:Neoplasm Proteins; D009842:Oligopeptides; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D018000:Receptors, Peptide; C086983:formylmethionyl-leucyl-phenylalanyl-phenylalanyl-lysine seminaphthofluorescein