Functional and binding properties of CXCR4 intracellular loop 1 pepducins

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 lipidated peptides derived from the intercellular loop sequences of a GPCR and have been shown to act as allosteric modulators. Their precise mode of action is unclear. Pepducins have been described for the chemokine receptor, CXCR4, which act as positive or negative allosteric modulators and can also exhibit agonist activity in the absence of the endogenous ligand CXCL12.¹ CXCR4 serves as a co-receptor for T-tropic (X4) HIV viruses entry to CD4+ T cells and has the ability to mediate the metastasis of various cancers. In this study, we investigated the interaction of intracellular loop 1 pepducins with CXCR4.

Method: Experiments were performed in HEK293 cells stably expressing the Glosensor^TM cAMP sensor and human CXCR4 tagged with NanoLuc (NL-CXCR4) or SNAP (SNAP-CXCR4) on its N-terminus or NanoLuc (CXCR4-NL) on its C-terminus. For cAMP accumulation, SNAP-CXCR4 cells were studied in a GloSensor^TM assay² recorded on an EnVision® luminescence reader (PerkinElmer). The binding of fluorescently labelled CXCR4 ligands and their displacement was studied with a NanoBRET assay³ using NL-CXCR4 or CXCR4-NL membranes and quantified on a PHERAstar FS plate reader (BMG).

Results: Inhibition of forskolin-stimulated cAMP production was observed upon addition of the endogenous ligand CXCL12 (pEC₅₀=9.81±0.20, n=7) as well as the pepducin ATI-2341 (pEC₅₀=7.93±0.15, n=12). A control pepducin without a lipid tail, as well as a pepducin missing the last three amino acids showed lower potency. This effect was antagonised by the CXCR4 antagonist AMD3100 (pA₂=7.01±0.23, n=5). No response was observed for CXCL12 or the pepducins in HEK293G cells in the absence of SNAP-CXCR4. The affinity of fluorescent CXCL12 (CXCL12-red) was determined through NanoBRET saturation binding (pK_d=7.61±0.10, n=5). Competition binding experiments showed that CXCL12-red binding was inhibited by pre-incubation with AMD3100 (pK_i=7.03±0.04, n=5), IT1t (pK_i=8.07±0.02, n=5) as well as with ATI-2341 (pK_i<6, n=5). The previously mentioned control pepducins were unable to displace CXCL12-red at concentrations up to 10 μM. ATI-2341f, a fluorescent ATI-2341, with an additional TAMRA-tagged lysine on its N-terminus showed no binding towards NL-CXCR4, however binding was observed with CXCR4-NL in the saturation binding assay with membranes.

Conclusion: These data suggests that ATI-2341 signals in a CXCR4-dependent manner. Furthermore, it interacts with the intercellular part of the receptor, and that the lipid tail is crucial for its activity.

Refrences:

1. O’Callaghan K et al. (2012). J Biol Chem 287: 12787-12796.

2. Goulding et al. (2012). Proceeding of the British Pharmacological Society. http://www.pa2online.org/abstracts/1vol10issue4abst038p.pdf

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