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

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) b inding, 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 rec ombinant receptors were similar to those of the native proteins. IL-8 displ aced [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. Calci um mobilization experiments were also consistent with previous reports on n ative 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 potenc y 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), th e order was reversed for stimulation of [S-35]GTP gamma S binding (GRO alph a > IL-8). 5 All of the functional responses at both receptors were inhibited by pertu ssis toxin (PTX), suggesting coupling to a Gi/Go protein. However, the calc ium mobilization induced by IL-8 at CXCR1 was not fully inhibited by PTX, s uggesting an interaction with a G-protein of the Gq family. Our results wit h pertussis toxin also suggested that, in the [S-35]GTP gamma S binding ass ay, 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 we ll with that of the native receptors. We also have preliminary evidence tha t CXCR1 displays constitutive activity in our cell line and that CXCR2 may traffic between different PTX sensitive G-proteins.