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

1 The effect of interleukin-8 (IL-8) and growth-related oncogene alpha (GRO alpha) on [S-35]-guanosine 5'-O-(3-thiotriphosphate) ([S-35]GTP gamma S) 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). 2 Radioligand binding assays confirmed that the binding profiles of the recombinant receptors were similar to those of the native proteins. IL-8 displaced [I-125]-IL-8 binding to CXCR1 and CXCR2 with pK(1) values of 8.89 +/- 0.05 and 9.27 +/- 0.03, respectively. GRO alpha, a selective CXCR2 ligand, had a pK(1) value of 9.66 +/- 0.39 at CXCR2 but a pK(i) > 8 at CXCR1. Calcium mobilization experiments were also consistent with previous reports on native receptors. 3 Activation of both receptors resulted in stimulation of [S-35]GTP gamma S binding and inhibition of adenylyl cyclase. 4 A comparison of the functional data at CXCR1 showed that a similar potency order (IL-8 > > GRO alpha) 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 greater than or equal to GRO alpha), the order was reversed for stimulation of [S-35]GTP gamma S binding (GRO alpha > IL-8). 5 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 [S-35]GTP gamma S binding assay, CXCR1 displays some constitutive activity. 6 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.