Signalling by CXC-chemokine receptors 1 and 2 expressed in CHO cells: a comparison of calcium mobilization, inhibition of adenylyl cyclase and stimulation of GTPgammaS binding induced by IL-8 and GROalpha

Abstract

The effect of interleukin-8 (IL-8) and growth-related oncogene alpha (GROalpha) on [35S]-guanosine 5'-O-(3-thiotriphosphate) ([35S]GTPgammaS) binding, forskolin-stimulated cyclic AMP accumulation and cytosolic calcium concentration were determined in recombinant CHO cells expressing HA-tagged CXC-chemokine receptors 1 and 2 (CXCR1 and CXCR2). Radioligand binding assays confirmed that the binding profiles of the recombinant receptors were similar to those of the native proteins. IL-8 displaced [125I]-IL-8 binding to CXCR1 and CXCR2 with pKi values of 8.89+/-0.05 and 9.27+/-0.03, respectively. GROalpha, a selective CXCR2 ligand, had a pKi value of 9.66+/-0.39 at CXCR2 but a pKi>8 at CXCR1. Calcium mobilization experiments were also consistent with previous reports on native receptors. Activation of both receptors resulted in stimulation of [35S]GTPgammaS binding and inhibition of adenylyl cyclase. A comparison of the functional data at CXCRI showed that a similar potency order (IL-8> >GROalpha) was obtained in all three assays. However, at CXCR2 whilst the potency orders for calcium mobilization and inhibition of adenylyl cyclase were similar (IL-8 > or = GROalpha), the order was reversed for stimulation of [35S]GTPgammaS binding (GROalpha > IL-8). All of the functional responses at both receptors were inhibited by pertussis toxin (PTX), suggesting coupling to a Gi/Go protein. However, the calcium mobilization induced by IL-8 at CXCR1 was not fully inhibited by PTX, suggesting an interaction with a G-protein of the Gq family. Our results with pertussis toxin also suggested that, in the [35S]GTPgammaS binding assay, CXCR1 displays some constitutive activity. Thus, we have characterized the binding and several functional responses at HA-tagged CXCRs 1 and 2 and have shown that their pharmacology agrees well with that of the native receptors. We also have preliminary evidence that CXCR1 displays constitutive activity in our cell line and that CXCR2 may traffic between different PTX sensitive G-proteins.

Figure 1

Effect of IL-8 and GROα on cytosolic calcium concentration in CHO-CXCR1 (a) or CHO-CXCR2 (b) cells or of IL-8 in control or PTX-treated CHO-CXCR1 (c) or CHO-CXCR2 (d). Values are the means±s.e.mean of three separate determinations. Vertical bars show the s.e.mean. The curves plotted through the data points are four parameter logistic equations fitted to the mean data.

Figure 2

Effect of IL-8 and GROα on forskolin-stimulated cyclic AMP accumulation in CHO-CXCR1 (a) or CHO-CXCR2 (b) cells or of IL-8 in control or PTX-treated CHO-CXCR1 (c) or CHO-CXCR2 (d). Values are the means±s.e.mean of 3–11 separate determinations in duplicate. Vertical bars show the s.e.mean. The curves plotted through the data points are four parameter logistic equations fitted to the mean data.

Figure 3

Effect of IL-8 and GROα on the binding of [³⁵S]GTPγS to membranes from CHO-CXCR1 (a) or CHO-CXCR2 (b) cells or of IL-8 in control or PTX-treated CHO-CXCR1 (c) or CHO-CXCR2 (d) membranes. Values are the means±s.e.mean of 3–9 separate determinations in duplicate. Vertical bars show the s.e.mean. In (c) and (d) the values are presented as a percentage of the basal in the PTX-treated membranes. The curves plotted through the data points are four parameter logistic equations fitted to the mean data.

Figure 4

Competition binding curves for IL-8 and GROα versus [¹²⁵I]IL-8 in membranes from CHO-CXCR1 (a) or CHO-CXCR2 (b). Values are the means±s.e.mean of three separate determinations. Vertical bars show the s.e.mean. The curves plotted through the data points are four parameter logistic equations fitted to the mean data.