1). receptor-agonist relationships involve at least two sites, of which only one is definitely specific for the receptors triggered conformation. is definitely a GPCR that is triggered by binding the 13-residue peptide -element (sequence: WHWLQLKPGQPMY). While Ste2p exhibits little sequence similarity to mammalian GPCRs, it is functionally interchangeable with some mammalian Rabbit Polyclonal to LDLRAD3 receptors 10; 11. Several residues in the receptor that interact with ligand have been recognized by chemical crosslinking and by characterizing amino acid substitutions in the ligand and receptor 12; 13; 14; 15; 16; 17; 18. However, given the size of -element, it is likely that additional residues in Ste2p interact with bound peptide. The recognition of amino acid residues involved in ligand binding can be laborious. Chemical crosslinking approaches require the use of functionalized ligand analogs that retain specific binding and biological activity. Crosslinked products must be fragmented and characterized, either at low resolution, by gel electrophoresis, or at higher resolution by mass spectrometry. Genetic approaches require verification to confirm that mutations that lead to loss of binding or activation result from specific alterations in the binding interface and not from problems in synthesis, folding, intracellular trafficking, or stability of the mutant receptors. The difficulty of these methods offers generally impeded understanding the AZD7687 differential relationships of receptors with specific classes of ligands with different efficacies. We describe here a new method for identifying amino acid residues of a receptor that, when mutated, result in a switch in the chemical environment of a probe attached to bound ligand. This procedure recovers alleles that switch the environment of the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) fluorophore while keeping high affinity binding to a membrane-impermeant ligand in the cell surface. Therefore, it instantly eliminates major classes of undesired loss-of-function alleles such as those affecting overall receptor folding or subcellular trafficking. The approach was developed based on the previously-characterized binding to Ste2p AZD7687 of the AZD7687 AZD7687 (NBD)-tagged agonist analog ([Lys7(NBD),Nle12]-element (see Table 1) 19; 20; 21 but is also applied to receptor-binding of fluorescent antagonist, providing a assessment of the relationships of receptors with the two types of ligands. Table 1 -element analogs gene followed by co-transformation of the PCR products and linearized vector into a in emission of bound [Dap3(NBD),Arg7,Nle12] compared with wild-type receptors, indicative of a decrease in the polarity of the bound Dap3-labeled fluorophore, despite the fact that these same mutations result in Bmax ideals for binding of AZD7687 the [Lys7(NBD),Nle12]-factor. The other mutations in Table 2 exhibit differences in emission intensities for the two different agonists that are more subtle, apparently reflecting differences in the changes in polarity at positions 3 and 7 of the ligand, as well as varying numbers of mutant receptors at the cell surface. Mutations that affect the environment of bound antagonist Several analogs of -factor with truncations or alterations of residues in the N-terminal end of the peptide behave as antagonists toward normal -factor receptors 26; 27; 28. In addition, we report here the use of a new antagonist, [dTyr3,Lys7(NBD),Nle12]-factor, that binds to normal Ste2p with an affinity comparable to that of normal -factor, while exhibiting minimal activation of signaling (Supplementary Fig. 5). We refer to these compounds as antagonists, despite the fact that they exhibit very weak partial agonist activity toward normal receptors and substantial agonist activity toward some mutant receptor alleles, as discussed below. When bound to wild-type receptors, analogs of three antagonists, all modified with NBD at Lys7, [des-Trp1,des-His2, Nle12]-factor, [des-Trp1Ala3, Nle12]-factor, and [dTyr3,Nle12]-factor, all exhibit significant red-shifts compared to the Lys7-labeled agonist, indicating that their NBD groups reside in more polar environments than in the similarly labeled agonist (see Table 1). These red-shifts must reflect the fluorophores environments when.