主催: The Japanese Pharmacological Society, The Japanese Society of Clinical Pharmacology
会議名: WCP2018 (18th World Congress of Basic and Clinical Pharmacology)
開催地: Kyoto
開催日: 2018/07/01 - 2018/07/06
Introduction
Pepducins are lipid-peptides derived from the intercellular-loop (ICL) sequences of a GPCR and have been shown to act as allosteric modulators. Pepducins, described for the chemokine receptor, C-X-C-Receptor 4 (CXCR4), can exhibit agonist activity in the absence of the endogenous ligand C-X-C-Ligand 12 (CXCL12).(1) To date, their precise mode of action is unclear. In this study, we investigated the mechanism of CXCR4 ICL1 pepducins.
Method
Experiments were performed in HEK293 cells stably expressing the Glosensor cAMP sensor and (a) human CXCR4 tagged with the shrimp luciferase, NanoLuc, on its N-terminus (NL-CXCR4) or C-terminus (CXCR4-NL), (b) human C-C-Receptor 5 (CCR5) or (c) human CXCR4 with ICL1 replaced with the CCR5 sequence (CXCR4_CCR5il1). cAMP inhibition, was studied in a GloSensor assay(2). Binding of fluorescently labelled CXCR4 ligands and their displacement was quantified with a NanoBRET assay(3) using NL-CXCR4 or CXCR4-NL membranes.
Results
The ICL1-based pepducin, ATI-2431 caused CXCR4-dependent activation of Gi mediated signalling and receptor internalisation. Moreover it was able to displace binding of a fluorescently labelled CXCL12 to CXCR4 in a NanoBRET binding assay. Control pepducins with no lipid tail or a modified C termini had no effect in these assays at 10microM.
In NanoBRET assays a TAMRA-labelled ATI-2341 derivative indicated an interaction between the C- but not N-terminal labelled CXCR4 receptor in cell membranes, which could be prevented by excess unlabelled ATI-2341.
ATI-2431 had no functional effect in cells expressing the closely related CCR5, whereas its effect was maintained in cells expressing the hybrid CXCR4_CCR5il1. The location of ATI-2431 interaction with the receptor and the role for individual amino acid residues was further investigated by mutation of ICL1 residues common to both CXCR4 and CCR5.
Conclusion
These data suggest that the ICL1 pepducin ATI-2341f interacts with the intracellular part of the receptor, causes receptor activation, and a change in its conformation which affects the CXCL12 binding site. Further investigation of individual peducin residues in these assays will help determine the exact nature of this interaction.
1 Tchernychev et al (2010). PNAS 107:22255-22259.
2 Goulding et al (2012). Proceeding of the BPS.
3 Stoddart et al (2015). Nature Methods 12:661-663.
