Full characterization of GPCR monomer-dimer dynamic equilibrium by single molecule imaging

Area of Science:

• Cellular Biology
• Biophysics
• Molecular Signaling

Background:

• Receptor dimerization is critical for signal transduction pathways.
• Previous studies lacked comprehensive characterization of monomer-dimer equilibrium, including 2D equilibrium and rate constants for receptors.
• The N-formyl peptide receptor (FPR), a chemoattractant G protein-coupled receptor (GPCR), plays a vital role in immune cell responses.

Purpose of the Study:

• To quantitatively determine the dynamic monomer-dimer equilibrium of the FPR in live cells.
• To measure the 2D equilibrium constant, dissociation rate constant, and 2D association rate constant for FPR.
• To characterize the kinetics of FPR monomer-dimer interconversion at physiological conditions.

Main Methods:

• Development of a novel single fluorescent-molecule imaging technique.
• Live-cell imaging experiments conducted at physiological temperature (37°C).
• Analysis of receptor dynamics under both basal and liganded conditions.

Main Results:

• The 2D equilibrium constant for FPR monomer-dimerization was determined to be 3.6 copies/µm(2).
• The dissociation rate constant is 11.0 s(-1), and the 2D association rate constant is 3.1 copies/µm(2)s(-1).
• At physiological expression levels (∼2.1 receptor copies/µm(2)), FPR monomers convert to dimers every 150 ms, and dimers dissociate into monomers in 91 ms.

Conclusions:

• FPR dimerization is a highly dynamic process with rapid interconversion between monomeric and dimeric states.
• The equilibrium and kinetics of FPR dimerization are unchanged upon ligand binding.
• These findings provide a comprehensive 'super-quantification' of FPR dynamics, essential for understanding GPCR signaling.