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

S. P. Fay, R. Habbersett, M. D. Domaleski, Richard G Posner, T. G. Houghton, E. Pierson, N. Muthukumaraswamy, J. Whitaker, R. P. Haugland, R. J. Freer, L. 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-Phe-Lys (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 pK(a) 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.

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

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Keywords

  • formyl peptide receptor
  • ligand-receptor interaction
  • multiparameter flow cytometry
  • pH sensitivity

ASJC Scopus subject areas

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

Cite this

Fay, S. P., Habbersett, R., Domaleski, 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.