Multiparameter flow cytometric analysis of a pH sensitive formyl peptide with application to receptor structure and processing kinetics

Shawn P. Fay, Robert Habbersett, Mark D. Domalewski, Richard G. Posner, Terri Gilbert Houghton, Ed Pierson, Natesa Muthukumaraswamy, James Whitaker, Richard P. Haugland, Richard J. Freer, Larry A. Sklar

Research output: Contribution to journalArticle

1 Scopus citations

Abstract

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‐PheLys (SNAFL)‐OH (SNAFL‐seminaphthofluorescein), 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. © 1994 Wiley‐Liss, Inc.

Original languageEnglish (US)
Pages (from-to)148-153
Number of pages6
JournalCytometry
Volume15
Issue number2
DOIs
StatePublished - Feb 1 1994
Externally publishedYes

Keywords

  • Multiparameter flow cytometry
  • formyl peptide receptor
  • ligand‐receptor interaction
  • pH sensitivity

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Biophysics
  • Hematology
  • Endocrinology
  • Cell Biology

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  • Cite this

    Fay, S. P., Habbersett, R., Domalewski, M. D., Posner, R. G., Houghton, T. G., Pierson, E., Muthukumaraswamy, N., Whitaker, J., Haugland, R. P., Freer, R. J., & Sklar, L. A. (1994). Multiparameter flow cytometric analysis of a pH sensitive formyl peptide with application to receptor structure and processing kinetics. Cytometry, 15(2), 148-153. https://doi.org/10.1002/cyto.990150208