3 research outputs found
Milligram Production and Biological Activity Characterization of the Human Chemokine Receptor CCR3
<div><p>Human chemokine receptor CCR3 (hCCR3) belongs to the G protein-coupled receptors (GPCRs) superfamily of membrane proteins and plays major roles in allergic diseases and angiogenesis. In order to study the structural and functional mechanism of hCCR3, it is essential to produce pure protein with biological functions on a milligram scale. Here we report the expression of hCCR3 gene in a tetracycline-inducible stable mammalian cell line. A cell clone with high hCCR3 expression was selected from 46 stably transfected cell clones and from this cell line pure hCCR3 on a milligram scale was obtained after two-step purification. Circular dichroism spectrum with a characteristic shape and magnitude for α-helix indicated proper folding of hCCR3 after purification. The biological activity of purified hCCR3 was verified by its high binding affinity with its endogenous ligands CCL11 and CCL24, with <i>K</i><sub>D</sub> in the range of 10<sup>−8</sup> M to 10<sup>−6</sup> M.</p></div
Regulation of the Oligomeric Status of CCR3 with Binding Ligands Revealed by Single-Molecule Fluorescence Imaging
The
relationship between the oligomeric status and functions of
chemokine receptor CCR3 is still controversial. We use total internal
reflection fluorescence microscopy at the single-molecule level to
visualize the oligomeric status of CCR3 and its regulation of the
membrane of stably transfected T-REx-293 cells. We find that the population
of the dimers and oligomers of CCR3 can be modulated by the binding
of ligands. Natural agonists can induce an increase in the level of
dimers and oligomers at high concentrations, whereas antagonists do
not have a significant influence on the oligomeric status. Moreover,
monomeric CCR3 exhibits a stronger chemotactic response in the migration
assay of stably transfected CCR3 cells. Together, these data support
the notion that CCR3 exists as a mixture of monomers and dimers under
nearly physiological conditions and the monomeric CCR3 receptor is
the minimal functional unit in cellular signaling transduction. To
the best of our knowledge, these results constitute the first report
of the oligomeric status of CCR3 and its regulation
Single-Molecule Imaging Demonstrates Ligand Regulation of the Oligomeric Status of CXCR4 in Living Cells
The role of dimerization
and oligomerization of G-protein-coupled
receptors in their signal transduction is highly controversial. Delineating
this issue can greatly facilitate rational drug design. With single-molecule
imaging, we show that chemokine receptor CXCR4 exists mainly as a
monomer in normal mammalian living cells and forms dimers and higher-order
oligomers at a high expression level, such as in cancer cells. Chemotaxis
tests demonstrate that the signal transduction activity of CXCR4 does
not depend only on its expression level, indicating a close relation
with the oligomeric status of CXCR4. Moreover, binding ligands can
effectively upregulate or downregulate the oligomeric level of CXCR4,
which suggests that binding ligands may realize their pivotal roles
by regulating the oligomeric status of CXCR4 rather than by simply
inducing conformational changes