The First Extracellular Loop of the Saccharomyces cerevisiae G Protein-coupled Receptor Ste2p Undergoes a Conformational Change upon Ligand Binding

Abstract

In this study of the Saccharomyces cerevisiae G protein-coupled receptor Ste2p, we present data indicating that the first extracellular loop (EL1) of the ␣-factor receptor has tertiary structure that limits solvent accessibility and that its conformation changes in a ligand-dependent manner. The substituted cysteine accessibility method was used to probe the solvent exposure of single cysteine residues engineered to replace residues Tyr 101 through Gln 135 of EL1 in the presence and absence of the tridecapeptide ␣-factor and a receptor antagonist. Surprisingly, many residues, especially those at the N-terminal region, were not solvent-accessible, including residues of the binding-competent yet signal transduction-deficient mutants L102C, N105C, S108C, Y111C, and T114C. In striking contrast, two N-terminal residues, Y101C and Y106C, were readily solvent-accessible, but upon incubation with ␣-factor labeling was reduced, suggesting a pheromone-dependent conformational change limiting solvent accessibility had occurred. Labeling in the presence of the antagonist, which binds Ste2p but does not initiate signal transduction, did not significantly alter reactivity with the Y101C and Y106C receptors, suggesting that the ␣-factor-dependent decrease in solvent accessibility was not because of steric hindrance that prevented the labeling reagent access to these residues. Based on these and previous observations, we propose a model in which the N terminus of EL1 is structured such that parts of the loop are buried in a solventinaccessible environment interacting with the extracellular part of the transmembrane domain bundle. This study highlights the essential role of an extracellular loop in activation of a G protein-coupled receptor upon ligand binding. G protein-coupled receptors (GPCRs)3 are ubiquitous in eukaryotes and have been found in a diversity of organisms ranging from yeast to humans. In most eukaryotes, GPCRs comprise 1-2% of the total genes in the genome (1) and participate in virtually all aspects of cellular physiology, including hormonal responses, neuronal transmission, and mediation of taste, smell, and vision (2). Modulation of GPCR function is a major pharmaceutical target, currently accounting for Ͼ30% of all drugs prescribed (3-5). Thus a thorough understanding of the nature of the receptor-ligand interaction and subsequent signal transduction is essential for the development of more effective and safer therapeutic agents.The structural hallmarks of this diverse collection of receptors are seven membrane-spanning domains linked by extracellular and intracellular loops, oriented such that the N terminus is external to the cell and the C terminus is internal. Although the structure-function relationships in the membrane-spanning domains and the intracellular loop regions have been well examined in many GPCRs (6 -11), few studies have focused on the importance of the extracellular loop structures and their role in receptor activation. In those studies where the extracellular domains were studied in deta...

Long Range Effect of Mutations on Specific Conformational Changes in the Extracellular Loop 2 of Angiotensin II Type 1 Receptor

“…Induced conformational changes in the ECL2 have been documented in several GPCRs, including the D2 dopamine receptor and C5A complement receptor (44,45) upon ligand occupancy of the orthosteric pocket in the TM domain. Long range conformational changes associated with ligand binding have been shown in ECL1 of glucagon receptor (46); the N terminus, ECL1, and extracellular ends of all TM helices of Saccharomyces cerevisiae G protein-coupled receptor Ste2p are known (47)(48)(49). These observations suggest that a mutation in a particular domain of the receptor causes a local structural perturbation that is eventually transmitted to other domains, leading to a global consequence in terms of affinity toward ligands and G protein.…”

Identification of Residue-to-residue Contact between a Peptide Ligand and Its G Protein-coupled Receptor Using Periodate-mediated Dihydroxyphenylalanine Cross-linking and Mass Spectrometry

“…Saccharomyces cerevisiae strain LM102 (MATa, bar1, leu2, ura3, FUS1-lacZ::URA3, ste2⌬) (17) was used for growth arrest and binding assays and the protease-deficient strain BJS21 (MATa, prc1-407 prb1-1122 pep4-3 leu2 trp1 ura3-52 ste2::Kan R ) (18) was used for protein isolation and immunoblot analysis. The plasmid pBEC1 containing C-terminal FLAG and His-tagged STE2 (17) was transformed by the method of Turcatti et al (19) into LM102 and BJS21 cells.…”

“…The plasmid pBEC1 containing C-terminal FLAG and His-tagged STE2 (17) was transformed by the method of Turcatti et al (19) into LM102 and BJS21 cells. Transformants were selected by growth on yeast medium (20) lacking tryptophan (MLT) to maintain selection for the plasmid.…”

“…Cells (0.5 ml) were combined with ␣-factor pheromone (final concentration of 1 M) and incubated at 30°C for 90 min. The cells were transferred to a 96-well flat bottom plate in triplicates and permeabilized with 0.5% Triton X-100 in 25 mM PIPES buffer, and then ␤-galactosidase assays were carried out using fluorescein di-␤-galactopyranoside (Molecular Probes, Inc., Eugene, OR) as a substrate as described previously (17). The reaction mixtures were incubated at 37°C for 60 min, and 1.0 M Na 2 CO 3 was added to stop the reaction.…”

“…Protein concentration was determined by a Bio-Rad protein assay (17). The membranes were re-suspended in NE-Buffer (20 mM HEPES, 20% glycerol, 100 mM KCl, 12.5 mM EDTA, 0.5 mM DTT) (26) (22), or 2 g of BSA for 120 min at 4°C.…”