Ligand activation induces different conformational changes in CXCR3 receptor isoforms as evidenced by plasmon waveguide resonance (PWR)

Abstract

The chemokine receptor CXCR3 plays important roles in angiogenesis, inflammation and cancer. Activation studies and biological functions of CXCR3 are complex due to the presence of spliced isoforms. CXCR3-A is known as a pro-tumor receptor whereas CXCR3-B exhibits anti-tumor properties. Here, we focused on the conformational change of CXCR3-A and CXCR3-B after agonist or antagonist binding using Plasmon Waveguide Resonance (PWR). Agonist stimulation induced an anisotropic response with very distinct conformational changes for the two isoforms. The CXCR3 agonist bound CXCR3-A with higher affinity than CXCR3-B. Using various concentrations of SCH546738, a CXCR3 specific inhibitor, we demonstrated that low SCH546738 concentrations (≤1 nM) efficiently inhibited CXCR3-A but not CXCR3-B’s conformational change and activation. This was confirmed by both, biophysical and biological methods. Taken together, our study demonstrates differences in the behavior of CXCR3-A and CXCR3-B upon ligand activation and antagonist inhibition which may be of relevance for further studies aimed at specifically inhibiting the CXCR3A isoform.

Cholesterol impacts chemokine CCR5 receptor ligand‐binding activity

“…Therefore, the observed spectral changes can only be attributed to ligand‐induced receptor‐conformational changes. Such scenario has already been observed in previous PWR experiments involving ligand interaction with GPCRs . From PWR experiments, it was possible to determine a binding affinity of 0.8 n m between maraviroc and the CCR5 (Fig.…”

“…The present work, performed with wild‐type CCR5 produced via two alternative and completely independent sources, P. pastoris and cell‐free expression, simultaneously reveals Chol to significantly impact maraviroc affinity to the receptor with a 20‐ to 30‐fold decrease in affinity when Chol is present in the membrane. While one can argue that the impact in binding affinity is rather modest, very minor differences in receptor/ligand affinity have been shown to highly impact the selectivity of the activated signaling cascades in the case other chemokine receptors as the CXCR3 . This demonstrates the extremely thin fine‐tuning of GPCR activation and signaling.…”

Innate lymphocyte-induced CXCR3B-mediated melanocyte apoptosis is a potential initiator of T-cell autoreactivity in vitiligo

“…It has previously been demonstrated that CXCL10 has a much greater binding affinity for CXCR3B compared to CXCL9 or CXCL11 while the affinity is similar between the three chemokines for the CXCR3A isoform 35 . This may be attributed to differences in special conformation between CXCR3A and CXCR3B 36 .…”

Improved Detection of Plasmon Waveguide Resonance Using Diverging Beam, Liquid Crystal Retarder, and Application to Lipid Orientation Determination

“…Usually, such approaches are carried out by more than one approach and often rely on the use of a high quantity of material and also require labelled materials. PWR has been successful for studies of ligand-activation and receptor conformational changes [39,40,41,42,43], signaling [44,45], lipid impact [35,46], and domain partitioning [47] in GPCR activity, highly contributing to a better understanding the way such receptors function. The proposed methodology in this work provides higher contrast measurement in high speeds and high resolution, yet maintaining simplicity using a single laser diode and camera.…”