We describe a procedure for isolating agonists for mammalian G protein-coupled receptors of unknown function. Human formyl peptide receptor like-1 (FPRL-1) receptor, originally identified as an orphan G protein-coupled receptor related to the formyl peptide receptor (FPR1), was expressed in Saccharomyces cells designed to couple receptor activation to histidine prototrophy. Selection for histidine prototrophs among transformants obtained with a plasmid-based library encoding random peptides identified six different agonists, each of whose production yielded autocrine stimulation of the receptor expressed in yeast. A synthetic version of each peptide promoted activation of FPRL-1 expressed in human embryonic kidney (HEK293) cells, and five of the peptides exhibited significant selectivity for activation of FPRL-1 relative to FPR1. One selective peptide was tested and found to mobilize calcium in isolated human neutrophils. This demonstrates that stimulation of FPRL-1 results in neutrophil activation and suggests that the receptor functions as a component of the inflammatory response. This autocrine selection protocol may be a generally applicable method for providing pharmacological tools to evaluate the physiological roles of the growing number of mammalian orphan G protein-coupled receptors.

Localization in human interleukin 2 of the binding site to the a chain (p55) ABSTRACTHuman interleukin 2 (IL-2) analogs with defined amino acid substitutions were used to identify specific residues that interact with the 55-kDa subunit (p55) or a chain ofthe human IL-2 receptor. Analog proteins containing specific substitutions for Lys-35, Arg-38, or Lys-43 were inactive in competitive binding assays for p55. All of these analogs retained substantial competitive binding to the intermediate-affinitj p70 subunit (J3 chain) of the receptor complex.The analogs varied in ability to interact with the high-affinity p55/p70 receptor. Despite the lack of binding to p55, all analogs exhibited significant biological activity, as assayed on the murine CTLL cell line. The dissociation constants ofArg-38 and Phe-42 analogs for p70 were consistent with intermediateaffinity binding; theKd values were not significantly affected by the presence of p55 in binding to the high-affinity IL-2 receptor complex. These results confirm the importance of the B a-helix in IL-2 as the locus for p55-receptor binding and support a revised model of IL-2-IL-2 receptor interaction.Human interleukin 2 (huIL-2) is a potent immunoregulatory cytokine, the biological effects ofwhich are mediated through binding to specific receptors on the surface of target cells. These interleukin 2 (IL-2) receptors (IL-2Rs) are present in at least three forms that bind IL-2 with high (Kd = 10-50 pM), intermediate (Kd = 0.8-2 nM), or low (Kd = 10-30 nM) affinity (1). The high-affinity IL-2R complex consists of at least two receptor subunits, the a chain or p55 (IL-2Ra) and the /3 chain or p70 (IL-2RB). Molecular cloning and expression of these two subunits demonstrate that IL-2Ra corresponds to the low-affinity receptor (2-4), whereas IL-2RP is