Chimeric G proteins allow a high-throughput signaling assay of G(i)-coupled receptors

G-protein-coupled receptors are a major target for potential therapeutics; yet, a large number of these receptors couple to the G(i) pathway, generati ng signals that are difficult to detect. We have combined chimeric G protei ns, automated sample handling, and simultaneous 96-well fluorometric imagin g to develop a high-throughput assay system for G(i) signaling. The chimeri c G proteins alter receptor coupling so that signaling can occur through G( q) and result in mobilization of intracellular calcium stores. An automated signaling assay device, the fluorometric imaging plate reader (FLIPR), can simultaneously measure this response in real time in 96-well microplates, allowing two people to process more than 10,000 points per day. We used the chimeric G protein/FLIPR system to characterize signaling by the G(i)-coup led human opioid receptors. We show that the mu, delta, and kappa opioid re ceptors and the related nociceptin receptor, ORL1, each couple to G alpha(q i5), G alpha(qo5), and G alpha(16) (G alpha(qi5) and G alpha(qo5) refer to G alpha(q) proteins containing the five carboxyl-terminal amino acids from G alpha(i) and G alpha(o), respectively) and that different receptor/G prot ein combinations show different levels of maximal activation. We tested 31 opioid ligands for agonist activity at the opioid receptors (124 ligand-rec eptor combinations); all 31 activated at least one receptor type, and sever al activated multiple receptors with differing potencies. This high-through put assay could be useful for dissecting the complex ligand-receptor relati onships that are common in nature. (C) 1999 Academic Press.