Characterisation of the interaction between the pepducin ATI 2341 and CXCR4 in HEK293 cells stably expressing a SNAP tagged receptor and the GlosensorTM cAMP sensor.

Joelle Goulding1, Leigh A Stoddart1, Kenneth E Carlson2, Stephen Hunt III2, Stephen J Hill1. 1University of Nottingham, Nottingham, UK, 2Anchor Therapeutics, Cambridge, MA, USA

The pepducin ATI 2341, derived from the peptide sequence of intracellular loop 1 of the C-X-C chemokine receptor 4 (CXCR4), has been shown to activate signalling cascades in cells expressing CXCR4 (Tchernychev et al.,2010; Janz et al., 2011). It has been suggested that this pepducin functions via an allosteric mode of action and hence could provide a novel treatment strategy for bone marrow transplantation (Tchernychev et al.,2010). The exact mechanism of action of ATI 2341, however, remains to be determined. Here we explore the effect of ATI 2341 and stromal cell-derived factor-1-alpha (Sdf1α) on cAMP accumulation using a HEK293 cell line expressing CXCR4 and a firefly luciferase-based cAMP sensor (GlosensorTM; Promega). In addition, we examine the effect of ATI 2341 on CXCR4 receptor internalisation and on the specific binding of a fluorescent analogue of Sdf1α (Sdf1αRED; Cisbio) to CXCR4.

A HEK293 GlosensorTM cell line stably expressing CXCR4 containing an N-terminal SNAP-tag was created (HekG_CXCR4SNAP). For the GlosensorTM assay, HekG_CXCR4SNAP cells were seeded in 96-well white-walled plates in growth medium. Prior to assay, cells were incubated for 2hr at 37ºC in 100μL HBSS containing 4% GlosensorTM cAMP reagent (Promega). Cellular luminescence was measured on an Envision plate reader (PerkinElmer) for 1hr at 37ºC in the absence and presence of Sdf1α or ATI 2341 (final concentrations from 10nM to 100μM) in the presence of 30μM forskolin (FSK). Antagonist pre-treatment involved exposure to 1,1′-[1,4-Phenylenebis(methylene)]bis-1,4,8,11-tetraazacyclotetradecane octahydrochloride (AMD3100,10μM) for 30min at 37ºC prior to drug addition. Petussis toxin (PTX) pre-treatment was performed as described previously (Baker and Hill, 2007). For imaging, HekG_CXCR4SNAP cells seeded onto 8-chamber coverslips were labelled with SNAP-surface 488 (0.5μM/ml) and fluorescence measured on a Zeiss LSM 710 laser scanning confocal microscope after 10min pre-incubation in Sdf1αRED (10nM) in HBSS following 30min pre-incubation in the presence or absence of ATI 2341 (1μM), Sdf1α (10-100nM) or AMD3100 (10μM).

Sdf1α and ATI 2341 elicited a concentration dependent inhibition of FSK-mediated GlosensorTM luminescence in HeKG_CXCR4SNAP cells with pEC50 of 8.40 ± 0.21 (n=12) and 6.96 ± 0.13 (n=16) respectively. Both responses were antagonised by AMD3100 with apparent pKb of 6.39 ± 0.53 (n=4) and 6.31 ± 0.14 (n=7) respectively. Pre-incubation with PTX abolished both inhibitory responses without affecting the luminescence output due to 30µM FSK (n=5-6). Following pre-incubation with ATI 2341 and SDF1α, membrane binding of Sdf1αRED was reduced and internalisation of CXCR4 was observed (n=3-4). Pre-treatment with AMD3100 abolished the cell surface binding of Sdf1αRED but did not stimulate receptor internalisation (n=3).

The ability of AMD3100 to antagonise the response of ATI 2341 in HeKG_CXCR4SNAP cells suggests that the inhibition of cAMP production elicited by the pepducin is mediated via the CXCR4 receptor. This conclusion is supported by the PTX sensitivity of the ATI 2341 cAMP responses and the ability of ATI 2341 to both inhibit the binding of Sdf1αRED to CXCR4 and to stimulate CXCR4 receptor internalisation.

Tchernychev et al., Discovery of a CXCR4 agonist pepducin that mobilizes bone marrow hematopoietic cells (2010) PNAS 107(51): 22255–22259

Janz et al., Direct interaction between an allosteric agonist pepducin and the chemokine receptor CXCR4 (2011) JACS 133(40): 15878-15881

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