Sequences in the intracellular loops of the yeast pheromone receptor Ste2p required for G protein activation.

Celić, A
Martin, NP
Son, Çağdaş Devrim
Becker, JM
Naider, F
Dumont, ME
The α-factor receptor of the yeast Saccharomyces cerevisiae encoded by the STE2 gene is a member of the large family of G protein-coupled receptors (GPCRs) that mediate multiple signal transduction pathways. The third intracellular loop of GPCRs has been identified as a likely site of interaction with G proteins. To determine the extent of allowed substitutions within this loop, we subjected a stretch of 21 amino acids (Leu228−Leu248) to intensive random mutagenesis and screened multiply substituted alleles for receptor function. The 91 partially functional mutant alleles that were recovered contained 96 unique amino acid substitutions. Every position in this region can be replaced with at least two other types of amino acids without a significant effect on function. The tolerance for nonconservative substitutions indicates that activation of the G protein by ligand-bound receptors involves multiple intramolecular interactions that do not strongly depend on particular sequence elements. Many of the functional mutant alleles exhibit greater than normal levels of signaling, consistent with an inhibitory role for the third intracellular loop. Removal of increasing numbers of positively charged residues from the loop by site-directed mutagenesis causes a progressive loss of signaling function, indicating that the overall net charge of the loop is important for receptor function. Introduction of negatively charged residues also leads to a reduced level of signaling. The defects in signaling caused by substitution of charged amino acids are not caused by changes in the abundance of receptors at the cell surface.


Isolation and immunological characterization of theta class glutathione-s-transferase gstt2-2 from bovine liver
İşgör, Sultan Belgin; Çoruh, Nursen; Department of Biochemistry (2004)
The glutathione-S-transferases (GSTs) (EC. are enzymes that participate in cellular detoxification of endogenous as well as foreign electrophilic compounds, function in the cellular detoxification systems and are evolved to protect cells against reactive oxygen metabolites by conjugating the reactive molecules to the nucleophile scavenging tripeptide glutathione (GSH, ?-glu-cys-gly). The GSTs are found in all eukaryotes and prokaryotic systems, in the cytoplasm, on the microsomes, and in the mitoch...
Unnatural amino acid replacement in a yeast G protein-coupled receptor in its native environment.
Huang, LY; Umanah, G; Hauser, M; Son, Çağdaş Devrim; Arshava, B; Naider, F; Becker, JM (American Chemical Society (ACS), 2008-05-20)
Ste2p is the G protein-coupled receptor (GPCR) for the tridecapeptide pheromone cc factor of Saccharomyces cerevisiae. This receptor- pheromone pair has been used extensively as a paradigm for investigating GPCR structure and function. Expression in yeast harboring a cognate tRNA/aminoacyl-tRNA synthetase pair specifically evolved to incorporate p-benzoyl-L-phenylalanine (Bpa) in response to the amber codon allowed the biosynthesis of Bpa-substituted Ste2p in its native cell. We replaced natural amino acid ...
KOCABIVIK, S; PERLIN, MH (Elsevier BV, 1994-01-01)
1. Oligonucleotide-directed mutagenesis of APH(3')-II was used to investigate the functions of key amino acids in the P-loop analogous motif of the enzyme. 2. The mutations of Gly205 --> GIu, Gly210 --> Ala and Arg211 --> Pro considerably reduced the resistance of the resulting strains to KM and to related drugs, e.g. G418. 3. Similarly, enzyme activity in the crude extracts of these mutants was substantially reduced as well as the enzyme's affinity for Mg2+ ATP. 4. Alternatively substitutions at a highly c...
Cross-Linking of a DOPA-Containing Peptide Ligand into its G Protein-Coupled Receptor
Umanah, George E.; Son, Çağdaş Devrim; Ding, FaXiang; Naider, Fred; Becker, Jeffrey M. (American Chemical Society (ACS), 2009-04-01)
The interaction between a 3,4-dihydroxylphenylalanine (DOPA) labeled analog of the tridecapeptide α-factor (W-H-W-L-Q-L-K-P-G-Q-P-M-Y) and Ste2p, a Saccharomyces cerevisiae model G protein-coupled receptor (GPCR), has been analyzed by periodate-mediated cross-linking. Chemically synthesized α-factor with DOPA substituting for tyrosine at position 13 and biotin tagged onto lysine7 ([Lys7 (BioACA),-Nle12,DOPA13]α-factor; Bio-DOPA-α-factor) was used for crosslinking into Ste2p. The biological activity of Bio-D...
Investigation for natural extract inhibitors of bovine lens aldose reductase responsible for the formation of diabetis dependent cataract
Onay, Melih; Çoruh, Nursen; Department of Biochemistry (2008)
In the polyol pathway, Aldose reductase (AR) is an important enzyme in reduction of aldehydes and aldosugars to their suitable alcohols. AR, using NADPH as a coenzyme, has a molecular weight of 37 000 dalton. AR in its activated form, known to increase the sorbitol accumulation in lens, is responsible for the cataract formation in diabetis diseases. Therefore, the inhibition of aldose reductase is important to prevent the incedence of cataract formation in diabetus mellitus. In the treatment of diabetis dep...
Citation Formats
A. Celić, N. Martin, Ç. D. Son, J. Becker, F. Naider, and M. Dumont, “Sequences in the intracellular loops of the yeast pheromone receptor Ste2p required for G protein activation.,” Biochemistry, pp. 3004–17, 2003, Accessed: 00, 2020. [Online]. Available: