Effect of intracellular loop 1 pepducins on ligand-receptor binding kinetics at the CXCR4 receptor

B. Caspar^1,2, L. A. Stoddart^1,2, J. Goulding^1,2, S. J. Briddon^1,2, S. J. Hill^1,2. ¹Division of Pharmacology, Physiology and Neuroscience, University of Nottingham, Nottingham, United Kingdom, ²Centre of Membrane Proteins and Receptors (COMPARE), Nottingham, United Kingdom.

Introduction: Pepducins are a class of lipidopeptide ligands derived from the intracellular loops of a GPCR and have been reported to act as allosteric modulators, although their exact mechanism remains unclear.¹ Pepducins for the chemokine receptor, CXCR4, have been described to act as positive or negative allosteric modulators and can exhibit agonist activity in the absence of the endogenous ligand CXCL12.² In this study, we investigated the interaction and mechanism of intracellular loop 1 pepducins with CXCR4.

Method: Experiments were performed in HEK293 cells stably expressing human CXCR4 tagged with the luciferase, NanoLuc, on its N-terminus (NL-CXCR4) or C-terminus (CXCR4-NL). The binding and displacement of fluorescently labelled CXCR4 ligands was studied in a NanoBRET assay³ at equilibrium or as kinetic experiments reading once every minute using NL-CXCR4 or CXCR4-NL cells or membranes quantified on a PHERAstar FS plate reader (BMG).

Results: The affinity of fluorescent CXCL12 (CXCL12-red) was determined in NL-CXCR4 cells and membranes using NanoBRET saturation binding (pK_d(Cells)=7.15±0.04, n=5; pK_d(Membranes)=7.61±0.10, n=5) or alternatively the association binding kinetics in cells using 1.5625 to 100nM (pK_d=7.10±0.18, n=7) were analysed. Competition binding experiments in membranes showed that CXCL12-red binding was inhibited by pre-incubation with small molecules AMD3100 (pKi=7.03±0.04, n=5), IT1t (pKi=8.07±0.02, n=5) and CXCR4 pepducin ATI-2341 (pKi<6, maximal inhibition 58.3±2.2% (10μM), n=5). Control pepducins missing a lipid tail or the last three amino acids did not displace CXCL12-red at concentrations up to 10 μM. Displacement of CXCL12-red by ATI-2341 was followed in a kinetic assay in cells. ATI-2341 (10μM) shows displacement of CXCL12-red which reaches a plateau of 53.47±0.98% of the initial signal within 10 minutes (k_off=0.43±0.04 min^-1, n=5). ATI-2341f, a TAMRA-labelled ATI-2341, was used in the membrane binding assay to detect interaction with the Nluc-CXCR4 receptor. ATI-2431f showed no displaceable binding towards the N-terminal labelled NL-CXCR4, but did interact with the C-terminal CXCR4-NL. In CXCR4-NL cells, binding of ATI-2341f reached saturation after 15 min and displacement with high concentrations of ATI-2341 was possible within 10 minutes. AMD3100, IT1t and CXCL12 are not able to displace ATI-2341f binding at concentrations up to 100 or 1 μM, respectively.

Conclusion: These data suggest that ATI-2341 interacts with CXCR4 from an allosteric site on the inner leaflet of the cell membrane. Moreover, ATI-2341 decreases the binding of CXCL12 to CXCR4 suggesting an influence on the orthosteric binding site.

Refrences:

1. Covic et al.(2002). PNAS 99:643-648.

2. Tchernychev et al.(2010). PNAS 107:22255-22259.

3. Stoddart et al.(2015). Nature Methods 12:661-663.