Genetic interactions among the transmembrane segments of the G protein coupled receptor encoded by the yeast STE2 gene

Sommers, Christine M.; Dumont, Mark E.

doi:10.1006/jmbi.1996.0816

Cited by 46 publications

(67 citation statements)

References 66 publications

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“…There have been recent advances in membrane protein engineering, including technologies for the screening of highexpressing members in a diverse pool of eukaryotic membrane proteins (24), identification of functionally critical amino acids in a GPCR (25)(26)(27), manual blot screening of randomly mutated membrane proteins for increased expression (28), and introduction of thermostabilizing mutations, individually identified by trial and error, in a GPCR (29). Complementary to such approaches, we present here a powerful, high-throughput platform for the directed evolution of a GPCR to enhance both expression level and stability while retaining function and to tailor ligand selectivity.…”

Section: Resultsmentioning

confidence: 99%

Directed evolution of a G protein-coupled receptor for expression, stability, and binding selectivity

Sarkar

Dodevski

Kenig

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

181

249

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We outline a powerful method for the directed evolution of integral membrane proteins in the inner membrane of Escherichia coli. For a mammalian G protein-coupled receptor, we arrived at a sequence with an order-of-magnitude increase in functional expression that still retains the biochemical properties of wild type. This mutant also shows enhanced heterologous expression in eukaryotes (12-fold in Pichia pastoris and 3-fold in HEK293T cells) and greater stability when solubilized and purified, indicating that the biophysical properties of the protein had been under the pressure of selection. These improvements arise from multiple small contributions, which would be difficult to assemble by rational design. In a second screen, we rapidly pinpointed a single amino acid substitution in wild type that abolishes antagonist binding while retaining agonist-binding affinity. These approaches may alleviate existing bottlenecks in structural studies of these targets by providing sufficient quantities of stable variants in defined conformational states.integral membrane proteins ͉ protein engineering ͉ protein folding

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Section: Resultsmentioning

confidence: 99%

Directed evolution of a G protein-coupled receptor for expression, stability, and binding selectivity

Sarkar

Dodevski

Kenig

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

181

249

View full text Add to dashboard Cite

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“…Studies of the N terminus of ␣-factor indicated a strong preference for a large hydrophobic residue at the Nterminal position of the pheromone (39 -41); analyses of Alascanned ␣-factor analogs and fluorescent ␣-factor analogs indicated that the side chain of Trp 3 is in a hydrophobic pocket when bound to the receptor (14,42) (15). Gln 10 of ␣-factor is suggested to be in close proximity to residues Ser 47 and Thr 48 of Ste2p when ␣-factor is bound to Ste2p (25). When the positions of the loop residues are determined, these constraints may prove useful in attempts to calculate a bound structure of the pheromone.…”

Section: Discussionmentioning

confidence: 99%

“…TM6 of Ste2p, like that of most GPCRs, plays an important role in receptor activation. For example, Ste2p TM6 mutations of P258L and P258L/ S259L resulted in constitutive activation (46), and Y266A and Y266C were deficient in signaling activity (41,47). Moreover, Ste2p Tyr 266 (on the extracellular end of TM6) interacts with Asn 205 (on the extracellular end of TM5) only in the active conformation of the receptor (48), suggesting that these extracellular regions of the Ste2p undergo conformational changes upon ␣-factor binding.…”

Section: Discussionmentioning

confidence: 99%

“…Trp 3 is in a hydrophobic pocket interacting with Asn 205 (TM5) and Tyr 266 (TM6). Gln 10 of ␣-factor is suggested to be in close proximity to residues Ser 47 and Thr 48 (TM1). The Tyr 13 side chain phenyl ring of ␣-factor is proposed to be involved in a cation-interaction with the Arg 58 (TM1) guanidinium moiety, and the phenolic OH (Tyr 13 ) forms a hydrogen bond with the Cys 59 (TM1) sulfhydryl group.…”

Section: Discussionmentioning

confidence: 99%

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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

Umanah

Huang

Ding

et al. 2010

Journal of Biological Chemistry

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G protein-coupled receptors (GPCRs)4 comprise the largest family of cell surface proteins present in the human genome responsible for communication between the internal and external environments in response to signal molecules, including hormones, pheromones, and neurotransmitters. GPCRs are characterized by the presence of seven membrane-spanning helical segments separated by alternating intracellular and extracellular loop regions (1-3). Despite their diverse ligands, all GPCRs perform similar functions, coupling the binding of ligands to the activation of specific heterotrimeric guanine nucleotide-binding proteins (G proteins), leading to the modulation of downstream effector proteins and molecules. Due to their involvement in a multitude of physiological functions, GPCRs are targeted by ϳ50% of the human drugs currently marketed (1,4,5). Detailed information about GPCR-ligand interactions is critical for our understanding of GPCR-ligand binding and function.Despite the differences in the binding mechanisms of various ligand groups in the GPCR family, there are some similarities that span this superfamily of proteins (1, 3, 6 -8). Ligand binding triggers conformational changes at the extracellular and transmembrane regions of the receptor, which are then propagated to the cytoplasmic surfaces, leading to G protein coupling and activation. Thus, ligand binding leads to the alteration of existing interhelical interactions, thereby promoting a set of new interactions that leads to a energetically favorable activated conformational state of the receptor (8, 9). Large ligands, such as proteins, bind to the extracellular loops of GPCRs, whereas small molecules like adrenergic agents bind within the transmembrane region of the receptor. Within the peptide-binding GPCRs, a combination of ligand binding to the extracellular loops followed by ligand penetra-

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“…The only relatively well-conserved negatively charged amino acid in the TM region of Ste2 is Glu143. However, Glu143 is not conserved pairwise with Arg58, and mutational studies also indicate that they are not likely to form a salt-bridge (76). There is some precedent for Arg in the middle of TM helices without a counterion.…”

Section: H1mentioning

confidence: 98%

Comparison of Class A and D G Protein-Coupled Receptors: Common Features in Structure and Activation

et al. 2005

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All G protein-coupled receptors (GPCRs) share a common seven TM helix architecture and the ability to activate heterotrimeric G proteins. Nevertheless, these receptors have widely divergent sequences with no significant homology. We present a detailed structure-function comparison of the very divergent Class A and D receptors to address whether there is a common activation mechanism across the GPCR superfamily. The Class A and D receptors are represented by the vertebrate visual pigment rhodopsin and the yeast α-factor pheromone receptor Ste2, respectively. Conserved amino acids within each specific receptor class and amino acids where mutation alters receptor function were located in the structures of rhodopsin and Ste2 to assess whether there are functionally equivalent positions or regions within these receptors. We find several general similarities that are quite striking. First, strongly polar amino acids mediate helix interactions. Their mutation generally leads to loss of function or constitutive activity. Second, small and weakly polar amino acids facilitate tight helix packing. Third, proline is essential at similar positions in transmembrane helices 6 and 7 of both receptors. Mapping the specific location of the conserved amino acids and sites of constitutively active mutations identified conserved microdomains on transmembrane helices H3, H6, and H7, suggesting that there are underlying similarities in the mechanism of the widely divergent Class A and Class D receptors.G protein-coupled receptors (GPCRs) 1 are the largest and most diverse superfamily of membrane receptors. GPCRs are characterized by their common 7 transmembrane (TM) helix architecture and their ability to activate heterotrimeric G proteins. Surprisingly, there is no significant sequence similarity across the GPCR superfamily. In fact, GPCRs have been grouped into at least five distinct classes (1) with no recognizable sequence similarity between the various classes (1,2). Nevertheless, the working assumption has been that these receptors have a common fold and activation mechanism. For example, the amino acid sequences of the yeast pheromone GPCRs, such as the α-factor receptor, diverge considerably from those of the mammalian hormone GPCRs, such as the β-adrenergic receptor. Yet, in both of these receptors, ligand binding occurs within the core of the 7 TM helices and is, in part, mediated by aromatic amino acids on transmembrane helix H6

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Genetic interactions among the transmembrane segments of the G protein coupled receptor encoded by the yeast STE2 gene

Cited by 46 publications

References 66 publications

Directed evolution of a G protein-coupled receptor for expression, stability, and binding selectivity

Directed evolution of a G protein-coupled receptor for expression, stability, and binding selectivity

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

Comparison of Class A and D G Protein-Coupled Receptors: Common Features in Structure and Activation

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