Differential Interactions of Fluorescent Agonists and Antagonists with the Yeast G Protein Coupled Receptor Ste2p

Abstract

We describe a rapid method to probe for mutations in cell-surface ligand-binding proteins that affect the environment of bound ligand. The method uses fluorescence activated cell sorting to screen randomly-mutated receptors for substitutions that alter the fluorescence emission spectrum of environmentally-sensitive fluorescent ligands. When applied to the yeast α-factor receptor Ste2p, a G protein coupled receptor, the procedure identified 22 substitutions that red-shift the emission of a fluorescent agonist, including substitutions at residues previously implicated in ligand binding and at additional sites. A separate set of substitutions, identified in a screen for mutations that alter the emission of a fluorescent α-factor antagonist, occurs at sites that are unlikely to directly contact the ligand. Instead, they alter receptor conformation to increase ligand-binding affinity and provide signaling in response to antagonists of normal receptors. These results suggest that receptor-agonist interactions involve at least two sites, of which only one is specific for the receptor’s activated conformation.

Highly Active Analogs of α-Factor and Their Activities Against Saccharomyces cerevisiae

“…16,17 Among the many synthetic β-turn peptidomimetics restricted by cyclization, (R)-γ-lactam conformational constraint incorporating [3-(R)-amino-2-oxo-1-pyrrolidineacetamido] in place of ProGly at residues 8 and 9 was reported to have the best activity only equal to that of parent peptide, [Nle ing approach involving the photoactivatable groups attached to the α-factor backbone was developed. 28,30,44,[51][52][53]55,56 pBenzoyl-L-phenylalanine (Bpa) and 3,4-Dihydroxyphenylalanine (DOPA) was reported to have a desirable property for this purpose, although incorporation of these groups at various positions in α-factor have been shown to result in a less than 30-fold decrease in receptor affinity. 44,51,55 Biotin (Bio) tagging modification of DOPA analog (Bio 7…”

“…40,41 On the other hand, those near the N-terminus of α-factor are responsible for triggering cell signaling through Ste2p or stabilizing the activated state of this receptor. 4,10,30,40,57 The two N-terminal Trp residues at position 1 and 3 are the key residues in the interaction between α-factor and the region of the receptor that promotes stimulation of the pheromone-responsive G protein pathway. 30,40,41 In addition, two N-terminal Trp residues preferentially insert into the cell membranes following interaction of α-factor with the phospholipid membrane.…”

“…4,10,30,40,57 The two N-terminal Trp residues at position 1 and 3 are the key residues in the interaction between α-factor and the region of the receptor that promotes stimulation of the pheromone-responsive G protein pathway. 30,40,41 In addition, two N-terminal Trp residues preferentially insert into the cell membranes following interaction of α-factor with the phospholipid membrane. 13,31 Based on the biological activity of lipopeptide conjugates and the combined role of the two Trp residues, 57 C-terminal extended analogs of α-factor with various number of Trp residues can be used to study highly potent α-factor analogs.…”

“…13,18,19,30,32 A study concerning cyclic α-factor analog mimicking β-turn structure in the center of α-factor provided additional proof that the constrained structure of the center of α-factor is deeply correlated with the potency of active analog. [18][19][20]30,32,43 These results show that the central β-turn near the receptor in the lipid membrane promotes interaction of the ligand with two sites in the receptor implicated in ligand binding (C-terminal region) and triggering of G protein signal transduction (Nterminal region). 4,30 Aib is sterically hindered by dialkylated side chains and thus its substitution generates a collapsed turn structure effectively.…”

“…]α-factor) and 7-Nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) 30,53,54 developed for the detection of ligand-receptor conjugates were shown to reduce activity to about 33% that of parent α-factor. 44,56 Although the conjugation strategy of photoactivatable groups is not focused on enhancing potency, all of these analogs display receptor affinities varying from slightly better than that of α-factor to about 6-fold lower.…”