Baclofen and other GABAB receptor agents are allosteric modulators of the CXCL12 chemokine receptor CXCR4

Abstract

CXCR4, a receptor for the chemokine CXCL12 (stromal-cell derived factor-1α), is a G-protein-coupled receptor (GPCR), expressed in the immune and CNS and integrally involved in various neurological disorders. The GABAB receptor is also a GPCR that mediates metabotropic action of the inhibitory neurotransmitter GABA and is located on neurons and immune cells as well. Using diverse approaches, we report novel interaction between GABAB receptor agents and CXCR4 and demonstrate allosteric binding of these agents to CXCR4. First, both GABAB antagonists and agonists block CXCL12-elicited chemotaxis in human breast cancer cells. Second, a GABAB antagonist blocks the potentiation by CXCL12 of high-threshold Ca(2+) channels in rat neurons. Third, electrophysiology in Xenopus oocytes and human embryonic kidney cell line 293 cells in which we coexpressed rat CXCR4 and the G-protein inward rectifier K(+) (GIRK) channel showed that GABAB antagonist and agonist modified CXCL12-evoked activation of GIRK channels. To investigate whether GABAB ligands bind to CXCR4, we expressed this receptor in heterologous systems lacking GABAB receptors and performed competition binding experiments. Our fluorescent resonance energy transfer experiments suggest that GABAB ligands do not bind CXCR4 at the CXCL12 binding pocket suggesting allosteric modulation, in accordance with our electrophysiology experiments. Finally, using backscattering interferometry and lipoparticles containing only the CXCR4 receptor, we quantified the binding affinity for the GABAB ligands, confirming a direct interaction with the CXCR4 receptor. The effect of GABAergic agents on CXCR4 suggests new therapeutic potentials for neurological and immune diseases.

Figure 1.

GABA_B agents reduce the migration of the breast cancer cells MDA-MB-231 in a chemotactic assay. A, RT-PCR reveals the presence of endogenous mRNAs for CXCR4 and GABA_BR2 in both HEK and MDA-MB-231 cells. B, Western blot reveals the presence of endogenous CXCR4 and GABA_BR in both HEK and MDA-MB-231 cells. HELA cells were used as a positive control. C, Schematic of the agarose spots on the 35 mm Petri dish. −, −−, +, and ++ are markings on the back of the Petri dish: + and ++, agarose drops made with the 10 and 100 nm CXCL12 agarose solution; − and −−, control drops in which equivalent amount of water was added to the agarose solution, respectively, to + and ++. The red inserts correspond to typical fields that were analyzed. D, Images of the agarose spots showing the cells crawling under the spot: Control (no chemoattractant), 100 nm CXCL12, and 100 nm CXCL12 and 5 μm CGP 55845. Scale bar, 100 μm. The dashed line represents the upper limit of the drop. E, Histogram showing the number of MDA-MB-231 cells under the agarose spots. Error bars indicate the mean of n = 18 fields (6 in 3 independent drops). Notice the dose-dependent increase in the migration with CXCL12 concentration and the reduction in migration in the presence of CGP 55845 (5 μm), GABA (100 μm), baclofen (100 μm), AMD3100 (2 μm), and gp120/CD4 (300 nm each). *p < 0.05, ***p < 0.001, t tests after ANOVA against control group. ⁺⁺p < 0.02, ⁺⁺⁺p < 0.01, t test against 100 nm CXCL12 group.

Figure 2.

The facilitator effect of 10 nm CXCL12 on HVA Ca currents recorded in dopaminergic neurons of the substantia nigra in rat brain slices is prevented by 500 nm CGP 55845. A, HVA calcium current recorded in a dopaminergic neuron in response to a voltage step from −60 to −10 mV in the absence (control) or the presence of 10 nm CXCL12. Calcium currents were isolated by the continuous perfusion of 1 μm TTX, 4-aminopyridine, 10 mm tetraethylammonium, 2 mm CsCl, 50 μm APV, 10 μm CNQX, and 1 μm gabazine. B, The effect of 10 nm CXCL12 on HVA Ca currents was significantly blocked by the CXCR4 antagonist AMD3100 (AMD, 200 nm) and by 500 nm CGP 55845, the GABA_B receptor antagonist (applied in a different set of neurons).

Figure 3.

Electrophysiological recordings of oocytes expressing CXCR4, GIRK1, and GIRK2. A, Inward currents recorded in response to various concentrations of CXCL12 as indicated by the bars. B, Concentration–response curve of CXCL12. Currents were measured at the peak and plotted against log[CXCL12]. Values are the mean of several experiments as indicated by the numbers in parenthesis. The curve was fitted to a sigmoid curve using MicroCal Origin software, with EC₅₀ of 0.49 ± 0.02 nm and a Hill number of 1.87. C, Current–voltage relationship obtained in control, in the presence of 1 nm CXCL12 and in the presence of 1 nm CXCL12 + 100 μm Ba²⁺. D, Current–voltage relationship of CXCL12-evoked current (current recorded in 1 nm CXCL12 minus control current). E, The inward current activated by 1 nm CXCL12 was blocked by 100 nm AMD3100, which induced a small inward current by itself. The response to 1 nm CXCL12 partially recovered after washout of AMD3100. F, The inward current activated by 1 nm CXCL12 was blocked by 30 nm gp120 applied together with 30 nm CD4, which induced a small inward current by itself. The response to 1 nm CXCL12 partially recovered after washout of gp120/CD4.

Figure 4.

Dose-dependent effect on CXCR4 receptor of several pharmacological agents acting on GABA_B receptor. A, Inward currents recorded in oocytes expressing CXCR4, GIRK1, and GIRK2 in response to the drugs indicated on the left, at various concentrations as indicated by the bars. B, Concentration–response curves of several pharmacological agents acting on GABA_B receptor as compared with CXCL12. Currents were measured at the peak, normalized to the maximum response, and plotted against log[CXCL12]. Values are the mean of several experiments, which number (n) is indicated in Table 1. The curves were fitted to sigmoid curves using MicroCal Origin software. EC₅₀ and a Hill number are also indicated in Table 1 (mean ± SEM).

Figure 5.

Effects of various pharmacological agents acting on GABA_B receptors on oocytes coinjected with CXCR4 and GIRK1 and GIRK2. A1, B1, CGP 55845 (500 nm) and CGP 54626 (500 nm) both induced an inward current. A2, B2, These currents were reversibly blocked by 200 nm AMD3100 recordings obtained in other oocytes than A1 and B1. C, The inward current elicited by 1 nm CXCL12 was reversibly blocked by 500 nm CGP 55845, which could itself induce an inward current as in the oocyte or have a very small effect by itself. D, The inward current elicited by 1 nm CXCL12 was reversibly blocked by 500 nm CGP 54626, which itself induced an inward current in the oocyte presented in this example. E, The effects of 10 μm baclofen and 10 μm GABA were blocked by perfusion of 200 nm AMD3100. F1, F2, The inward current elicited by 1 nm CXCL12 as blocked by perfusion of both 10 μm baclofen (F1) and 10 μm GABA (F2). G1, G2, The inward currents elicited by 10 μm baclofen (G1) and 10 μm GABA (G2) were blocked by 30 nm gp120. The current even became outward in the presence of 30 nm gp120 suggesting that a proportion of CXCR4 receptors was constitutively activating GIRK in these oocytes, which was blocked by the gp120.

Figure 6.

Effects of various pharmacological agents acting on GABA_B receptors on patch-clamp recordings from HEK293 cells transiently expressing CXCR4 and GIRK channels. A, Current–voltage relationship in control conditions (leak current in the presence of the extracellular solution with elevated KCl), in the presence of CXCL12, or in the presence of CXCL12 + 200 μm of the GIRK channel blocker BaCl₂, as indicated. B, CXCL12-induced current. Leak current has been subtracted. CXCL12 evoked an inwardly rectifying current that had a reversal potential of −24.6 mV. C, Representative traces showing the effect of 1 nm CXCL12 before, during, and after the washout of 500 nm CGP 55845, as indicated by the bars. Cells were held at −25 mV and then stepped to −60 mV for 100 ms every 2 s (see Materials and Methods). D, Representative traces showing the effect of 1 nm CXCL12 before, during, and after the washout of 10 mm GABA, as indicated by the bars. E, Histogram showing the percentage of decrease induced by the various compounds indicated on the GIRK current induced by 1 nm CXCL12 in HEK293 cells transiently expressing the CXCR4 and GIRK channel.

Figure 7.

Representative plots of BSI signal versus ligand concentration for the determination of binding constants for CXCR4 to the following ligands: CXCL12 (A), CGP 55845 (B), CGP 54626 (C), baclofen (D), and GABA (E). Dopamine (tested at 5 different concentrations: 0.02, 2, 200, 2, and 200 nm) was used as a nonbinding control ligand for all five plots. The binding of baclofen to the CXCR4–CXCL12 complex (0.2 nm CXCR4 + 20 nm CXCL12) is represented in F; also shown is the negative control of the interaction of baclofen with CXCL12. For all plots, error bars indicate SDs of the measurements from three independent trials.

Figure 8.

Baclofen and other ligands of GABA_B receptor do not bind at the CXCL12/SDF1 binding site on CXCR4. A, Principle of the Tag-lite receptor-ligand technology. The receptor is expressed at the cell surface with a SNAP tag and then labeled with a fluorescent donor dye (terbium cryptate) through an appropriate substrate. On the other side, the ligand is labeled with a red acceptor dye (d2). If the fluorescent donor dye labeled on the receptor is excited by a nitrogen laser or flash lamp (∼340 nm) when the ligand binds to the receptor, there is a transfer of energy between the donor dye to the acceptor dye, resulting in the latter dye emitting light, in a time-resolved manner, at 665 nm. The receptor-ligand binding can hence be monitored at 665 nm on a time-resolved mode. B, Competition assay run with d2-labeled CXCL12 at K_D (12.5 nm). None of the unlabeled GABA_B ligand was able to compete with the d2-labeled ligand while the d2-labeled CXCL12 was displaced by the unlabeled CXCL12 as expected. C, The specific binding curve was obtained by subtracting the nonspecific signal from the total binding signal. It revealed that the d2-labeled CGP 54626 derivative (used as a GABA_B fluorescent ligand) does not bind to the CXCR4 receptor at the CXCL12 binding pocket.