Publications

G-protein-coupled receptors are a major target for potential therapeutics; yet, a large number of these receptors couple to the Gi pathway, generating signals that are difficult to detect. We have combined chimeric G proteins, automated sample handling, and simultaneous 96-well fluorometric imaging to develop a high-throughput assay system for Gi signaling. The chimeric G proteins alter receptor coupling so that signaling can occur through Gq 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 Gi-coupled human opioid receptors. We show that the mu, delta, and kappa opioid receptors and the related nociceptin receptor, ORL1, each couple to Galphaqi5, Galphaqo5, and Galpha16 (Galphaqi5 and Galphaqo5 refer to Galphaq proteins containing the five carboxyl-terminal amino acids from Galphai and Galphao, respectively) and that different receptor/G protein combinations show different levels of maximal activation. We tested 31 opioid ligands for agonist activity at the opioid receptors (124 ligand-receptor combinations); all 31 activated at least one receptor type, and several activated multiple receptors with differing potencies. This high-throughput assay could be useful for dissecting the complex ligand-receptor relationships that are common in nature.

Leishmania major infection of inbred mice leads to a major dichotomous response--death or survival--that depends on the strain of mice. This finding has motivated efforts to locate genetic determinants of disease susceptibility. Genotyping studies have confirmed a complex multilocus trait, but studies directed at the biology of the response suggest identifiable components of susceptibility that may direct the genetic investigations. A confluence of parasite variables--residence in macrophages class II-dependent immunity, and avoidance of early IL-12 induction--with host factors--a prominent helper T-cell precursor frequency to a dominant parasite epitope and a bias in IL-4 gene activation--conspires to drive an aberrant immune response in animals that suffer fatal disease. These insights may lead to an understanding of factors that focus responses on dominant antigens and that mold the naive T-cell repertoire. Collectively, such factors might contribute to the pathogenesis of other infectious and autoimmune diseases.