Full characterization of GPCR monomer–dimer dynamic equilibrium by single molecule imaging
نویسندگان
چکیده
Receptor dimerization is important for many signaling pathways. However, the monomer-dimer equilibrium has never been fully characterized for any receptor with a 2D equilibrium constant as well as association/dissociation rate constants (termed super-quantification). Here, we determined the dynamic equilibrium for the N-formyl peptide receptor (FPR), a chemoattractant G protein-coupled receptor (GPCR), in live cells at 37°C by developing a single fluorescent-molecule imaging method. Both before and after liganding, the dimer-monomer 2D equilibrium is unchanged, giving an equilibrium constant of 3.6 copies/µm(2), with a dissociation and 2D association rate constant of 11.0 s(-1) and 3.1 copies/µm(2)s(-1), respectively. At physiological expression levels of ∼2.1 receptor copies/µm(2) (∼6,000 copies/cell), monomers continually convert into dimers every 150 ms, dimers dissociate into monomers in 91 ms, and at any moment, 2,500 and 3,500 receptor molecules participate in transient dimers and monomers, respectively. Not only do FPR dimers fall apart rapidly, but FPR monomers also convert into dimers very quickly.
منابع مشابه
Dynamically varying interactions between heregulin and ErbB proteins detected by single-molecule analysis in living cells.
Heregulin (HRG) belongs to the family of EGFs and activates the receptor proteins ErbB3 and ErbB4 in a variety of cell types to regulate cell fate. The interactions between HRG and ErbB3/B4 are important to the pathological mechanisms underlying schizophrenia and some cancers. Here, we observed the reaction kinetics between fluorescently labeled single HRG molecules and ErbB3/B4 on the surfaces...
متن کاملThermodynamic Characterization of the Aggregation Phenomena of SafranineT by Spectral Titration and Chemometric Analysis
The dimerization constants of Safranine T have been determined by studying the dependence of its absorption spectra on the temperature in the range 30–70 ◦C at different total concentrations of Safranine T (1.03×10−5, 1.44×10−5 and 1.73×10−5 M). The monomer–dimer equilibrium of Safranine T has been determined by applying MCR-ALS method on the absorpti...
متن کاملThermodynamic Characterization of the Aggregation Phenomena of SafranineT by Spectral Titration and Chemometric Analysis
The dimerization constants of Safranine T have been determined by studying the dependence of its absorption spectra on the temperature in the range 30–70 ◦C at different total concentrations of Safranine T (1.03×10−5, 1.44×10−5 and 1.73×10−5 M). The monomer–dimer equilibrium of Safranine T has been determined by applying MCR-ALS method on the absorpti...
متن کاملFolding of Escherichia coli DsbC: characterization of a monomeric folding intermediate.
The homodimeric protein DsbC is a disulfide isomerase and a chaperone located in the periplasm of Escherichia coli. We have studied the guanidine hydrochloride (GdnHCl)-induced unfolding and refolding of DsbC using mutagenesis, intrinsic fluorescence, circular dichroism spectra, size-exclusion chromatography, and sedimentation velocity analysis. The equilibrium refolding and unfolding of DsbC w...
متن کاملX-ray crystallographic and analytical ultracentrifugation analyses of truncated and full-length yeast copper chaperones for SOD (LYS7): a dimer-dimer model of LYS7-SOD association and copper delivery.
Copper-zinc superoxide dismutase (CuZnSOD) acquires its catalytic copper ion through interaction with another polypeptide termed the copper chaperone for SOD. Here, we combine X-ray crystallographic and analytical ultracentrifugation methods to characterize rigorously both truncated and full-length forms of apo-LYS7, the yeast copper chaperone for SOD. The 1.55 A crystal structure of LYS7 domai...
متن کامل