Related Experiment Video

Updated: Feb 10, 2026

A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry
08:26

A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry

Published on: April 9, 2013

18.4K

Formyl Met-Leu-Phe-Stimulated FPR1 Phosphorylation in Plate-Adherent Human Neutrophils: Enhanced Proteolysis but Lack

Algirdas J Jesaitis1, Jeannie Gripentrog1, Jovanka M Voyich1

  • 1Department of Microbiology and Immunology, Montana State University, Bozeman, MT 59717-3520, USA.

Journal of Immunology Research
|May 23, 2018
PubMed

Insights

Platelet-activating factor (PAF) does not inhibit N-formyl-Met-Leu-Phe (fMLF)-induced G protein-coupled receptor 1 (FPR1) phosphorylation in adherent human neutrophils. This suggests PAF

Area of Science:

  • Immunology
  • Cell Biology
  • Biochemistry

Background:

  • N-formyl-Met-Leu-Phe (fMLF) is a key chemoattractant for human neutrophils (PMN) at sites of injury or infection.
  • fMLF signals through the G protein-coupled receptor, formyl peptide receptor 1 (FPR1), mediating crucial inflammatory responses.
  • Understanding FPR1 phosphorylation dynamics in adherent neutrophils is vital for dissecting inflammatory signaling.

Purpose of the Study:

  • To investigate FPR1 phosphorylation in adherent human neutrophils using a microtiter plate assay.
  • To determine the effect of platelet-activating factor (PAF) on fMLF-induced FPR1 phosphorylation in adherent PMN.
  • To explore the physiological relevance of observed FPR1 phosphorylation fragments and PAF's influence.

Main Methods:

  • Development of a microtiter plate assay for measuring FPR1 phosphorylation in high-density adherent PMN.
  • Analysis of a phosphosensitive FPR1 fragment (25K-FPR1) using SDS-PAGE.
  • Investigation of PAF's effect on fMLF-induced FPR1 phosphorylation and lactate dehydrogenase release in adherent vs. suspension PMN.

Main Results:

  • A phosphosensitive FPR1 fragment (25K-FPR1) was identified in SDS-PAGE extracts of fMLF-stimulated adherent PMN.
  • 25K-FPR1 formation was not significantly affected by ATP depletion, adherence conditions, or mechanism, suggesting it's non-physiologic.
  • PAF did not inhibit fMLF-induced FPR1 phosphorylation in adherent PMN, unlike in suspension, but induced significant PMN lysis.

Conclusions:

  • The observed 25K-FPR1 fragment is likely an artifact of the assay conditions rather than a physiologically relevant marker.
  • PAF-induced inhibition of fMLF-dependent FPR1 phosphorylation seen in suspension PMN does not occur in unlysed adherent PMN.
  • High cell densities in the assay may mimic inflamed tissues, potentially explaining PAF's divergent effects on FPR1 phosphorylation and PMN function.

More Related Videos

Platelet-based Detection of Nitric Oxide in Blood by Measuring VASP Phosphorylation07:13

Platelet-based Detection of Nitric Oxide in Blood by Measuring VASP Phosphorylation

Published on: January 7, 2019

8.5K
Human Peripheral Blood Neutrophil Isolation for Interrogating the Parkinson's Associated LRRK2 Kinase Pathway by Assessing Rab10 Phosphorylation12:49

Human Peripheral Blood Neutrophil Isolation for Interrogating the Parkinson's Associated LRRK2 Kinase Pathway by Assessing Rab10 Phosphorylation

Published on: March 21, 2020

8.0K

Related Concept Videos

Phosphorylation01:02

Phosphorylation

The addition or removal of phosphate groups from proteins is the most common chemical modification that regulates cellular processes. These modifications can affect the structure, activity, stability, and localization of proteins within cells as well as their interactions with other proteins.
During phosphorylation, protein kinases transfer the terminal phosphate group of ATP to specific amino acid side chains of substrate proteins. Serine, threonine, and tyrosine are the most commonly...
54.3K
Inhibition of Cdk Activity02:34

Inhibition of Cdk Activity

The orderly progression of the cell cycle depends on the activation of Cdk protein by binding to its cyclin partner. However, the cell cycle must be restricted when undergoing abnormal changes. Most cancers correlate to the deregulated cell cycle, and since Cdks are a central component of the cell cycle, Cdk inhibitors are extensively studied to develop anticancer agents. For instance, cyclin D associates with several Cdks, such as Cdk 4/6, to form an active complex. The cyclin D-Cdk4/6 complex...
6.0K
Factors Affecting Activity Coefficient01:17

Factors Affecting Activity Coefficient

The extended Debye-Hückel equation indicates that the activity coefficient of an ion in an aqueous solution at 25°C depends on three partially interdependent properties: the ionic strength of the solution, the charge of the ion, and the ion size. 
The activity coefficient value for an ion is close to one when the solution has almost zero ionic strength, i.e., when the solution shows close to ideal behavior. As the ionic strength of the solution increases from 0 to 0.1 mol/L, a...
1.6K
Transcription Factors02:16

Transcription Factors

Tissue-specific transcription factors contribute to diverse cellular functions in mammals. For example, the gene for beta globin, a major component of hemoglobin, is present in all cells of the body. However, it is only expressed in red blood cells because the transcription factors that can bind to the promoter sequences of the beta globin gene are only expressed in these cells. Tissue-specific transcription factors also ensure that mutations in these factors may impair only the function of...
82.9K
Feedback Inhibition00:46

Feedback Inhibition

Biochemical reactions are occurring constantly in cells, converting starting substances to different products, usually with the help of enzymes that speed the reactions. Without enzymes, it would take far too long for most reactions to occur to be useful to the cell!
57.3K
Enzyme Inhibition01:30

Enzyme Inhibition

Inhibitors are molecules that reduce enzyme activity by binding to the enzyme. In a normally functioning cell, enzymes are regulated by a variety of inhibitors. Drugs and other toxins can also inhibit enzymes. Some inhibitors bind to the enzyme’s active site, while others inhibit enzymatic activity by binding to other sites on the protein structure.
92.8K