Development of a homogeneous MAP kinase reporter gene screen for the identification of agonists and antagonists at the CXCR1 chemokine receptor

Agonist activity at G protein-coupled receptors (GPCRs) that regulate heter otrimeric G proteins of the G alpha (i/o) or G alphaq families has been sho wn to result in activation of the mitogen-activated protein (MAP) kinase ca scade. To facilitate compound screening for these classes of GPCR, we have developed a reporter gene that detects the activation of the ternary comple x transcription factor Sap1a following MAP kinase activation. In contrast t o other reporter gene assays for G alpha (i/o)-coupled GPCRs, the MAP kinas e reporter generates an increase in signal in the presence of agonist. The reporter gene has been transfected into Chinese hamster ovary cells to gene rate a "host" reporter gene-containing cell line. The G alpha (i)-coupled h uman CXCR1 chemokine receptor was subsequently transfected into this cell l ine in order to develop a 384-well format screen for both agonists and anta gonists of this receptor, Agonists activated the reporter gene with the exp ected rank order of potency and with similar concentration dependence as se en with the regulation of other signal transduction cascades in mammalian c ells: interleukin-8 (IL-8) (pEC(50) = 7.0 +/- 0.1)> GCP-2 (pEC(50) = 6.3 +/ - 0.1)> NAP-2 (pEC(50) < 6), CXCR1-mediated activation of MAP kinase was in hibited by pertussis toxin and the MEK inhibitor PD98059, demonstrating tha t receptor activation of MAP kinase is due to pertussis toxin-sensitive G<a lpha>(i/o)-family G proteins to cause the activation of MEK kinase, Using t he 384-well format, assay performance was unaffected by solvent concentrati ons of 0.5% ethanol, 0.15% glycerol, or 1% DMSO, Signal crosstalk between a djacent wells was less than 1%, The assay exhibited a Z factor of 0.53 and a coefficient of variation of response to repeated application of IL-8 (100 nM) of 15.9%.