High-level expression, purification and characterization of a constitutively active thromboxane A2 receptor...

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• G protein-coupled receptors (GPCRs) can exhibit basal signaling, influencing physiological and pathological processes.
• Constitutively active mutants (CAMs) of GPCRs are crucial for studying basal signaling but often face challenges in expression and purification.
• The thromboxane A2 receptor (TP) is a GPCR involved in vasoconstriction and thrombosis.

Purpose of the Study:

• To develop a system for high-level expression and purification of a TP CAM (A160T mutant).
• To characterize the biophysical properties of the purified TP and TP A160T receptors.

Main Methods:

• Utilized tetracycline-inducible HEK293S-TetR and HEK293S (GnTI⁻)-TetR cell lines for TP and A160T expression.
• Employed single-step affinity purification to isolate the receptors.
• Performed circular dichroism (CD) spectropolarimetry to analyze temperature-dependent secondary structure changes.

Main Results:

• Achieved a 4-fold increase in TP and A160T expression, reaching 15.8 ±0.3 pmol/mg membrane protein.
• Obtained a functional yield of 45 µg/10⁶ cells after single-step purification.
• CD spectra indicated that the reduced activity of the A160T mutant is due to subtle structural effects, not large unfolding.

Conclusions:

• Successfully established a robust system for high-level expression, purification, and biophysical characterization of a TP CAM.
• This work provides a foundation for detailed structural and functional investigations of GPCRs with constitutive activity.
• The findings offer insights into the molecular basis of TP receptor activity and dysfunction.