Random Mutagenesis

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Jurgen Wess - One of the best experts on this subject based on the ideXlab platform.

  • Random Mutagenesis of the m3 muscarinic acetylcholine receptor expressed in yeast identification of second site mutations that restore function to a coupling deficient mutant m3 receptor
    Journal of Biological Chemistry, 2005
    Co-Authors: Nicola M Nowak, Clarice Schmidt, Sookyung Kim, Kenneth A Jacobson, Ali Bagheri, Jurgen Wess
    Abstract:

    Abstract The M3 muscarinic receptor is a prototypical member of the class A family of G protein-coupled receptors (GPCRs). To gain insight into the structural mechanisms governing agonist-mediated M3 receptor activation, we recently developed a genetically modified yeast strain (Saccharomyces cerevisiae) which allows the efficient screening of large libraries of mutant M3 receptors to identify mutant receptors with altered/novel functional properties. Class A GPCRs contain a highly conserved Asp residue located in transmembrane domain II (TM II; corresponding to Asp-113 in the rat M3 muscarinic receptor) which is of fundamental importance for receptor activation. As observed previously with other GPCRs analyzed in mammalian expression systems, the D113N point mutation abolished agonist-induced receptor/G protein coupling in yeast. We then subjected the D113N mutant M3 receptor to PCR-based Random Mutagenesis followed by a yeast genetic screen to recover point mutations that can restore G protein coupling to the D113N mutant receptor. A large scale screening effort led to the identification of three such second-site suppressor mutations, R165W, R165M, and Y250D. When expressed in the wild-type receptor background, these three point mutations did not lead to an increase in basal activity and reduced the efficiency of receptor/G protein coupling. Similar results were obtained when the various mutant receptors were expressed and analyzed in transfected mammalian cells (COS-7 cells). Interestingly, like Asp-113, Arg-165 and Tyr-250, which are located at the cytoplasmic ends of TM III and TM V, respectively, are also highly conserved among class A GPCRs. Our data suggest a conformational link between the highly conserved Asp-113, Arg-165, and Tyr-250 residues which is critical for receptor activation.

  • Random Mutagenesis of the m3 muscarinic acetylcholine receptor expressed in yeast identification of point mutations that silence a constitutively active mutant m3 receptor and greatly impair receptor g protein coupling
    Journal of Biological Chemistry, 2003
    Co-Authors: Clarice Schmidt, Isolde Erlenbach, Lanh M Bloodworth, Fuyue Zeng, Jurgen Wess
    Abstract:

    Abstract The M3 muscarinic receptor is a prototypical member of the class I family of G protein-coupled receptors (GPCRs). To facilitate studies on the structural mechanisms governing M3 receptor activation, we generated an M3 receptor-expressing yeast strain (Saccharomyces cerevisiae) that requires agonist-dependent M3 receptor activation for cell growth. By using receptor Random Mutagenesis followed by a genetic screen in yeast, we initially identified a point mutation at the cytoplasmic end of transmembrane domain (TM) VI (Q490L) that led to robust agonist-independent M3 receptor signaling in both yeast and mammalian cells. To explore further the molecular mechanisms by which point mutations can render GPCRs constitutively active, we subjected a region of the Q490L mutant M3 receptor that included TM V–VII to Random Mutagenesis. We then applied a yeast genetic screen to identify second-site mutations that could suppress the activating effects of the Q490L mutation and restore wild-type receptor-like function to the Q490L mutant receptor. This analysis led to the identification of 12 point mutations that allowed the Q490L mutant receptor to function in a fashion similar to the wild-type receptor. These amino acid substitutions mapped to two distinct regions of the M3 receptor, the exofacial segments of TM V and VI and the cytoplasmic ends of TM V–VII. Strikingly, in the absence of the activating Q490L mutation, all recovered point mutations severely reduced the efficiency of receptor/G protein coupling, indicating that the targeted residues play important roles in receptor activation and/or receptor/G protein coupling. This strategy should be generally applicable to identify sites in GPCRs that are critically involved in receptor function.

  • single amino acid substitutions and deletions that alter the g protein coupling properties of the v2 vasopressin receptor identified in yeast by receptor Random Mutagenesis
    Journal of Biological Chemistry, 2001
    Co-Authors: Isolde Erlenbach, Evi Kostenis, Clarice Schmidt, Claudine Serradeille Gal, Danielle Raufaste, Mark E Dumont, Mark H Pausch, Jurgen Wess
    Abstract:

    Abstract To facilitate structure-function relationship studies of the V2 vasopressin receptor, a prototypical Gs-coupled receptor, we generated V2 receptor-expressing yeast strains (Saccharomyces cerevisiae) that required arginine vasopressin-dependent receptor/G protein coupling for cell growth. V2 receptors heterologously expressed in yeast were unable to productively interact with the endogenous yeast G protein α subunit, Gpa1p, or a mutant Gpa1p subunit containing the C-terminal Gαq sequence (Gq5). In contrast, the V2 receptor efficiently coupled to a Gpa1p/Gαs hybrid subunit containing the C-terminal Gαs sequence (Gs5), indicating that the V2 receptor retained proper G protein coupling selectivity in yeast. To gain insight into the molecular basis underlying the selectivity of V2 receptor/G protein interactions, we used receptor saturation Random Mutagenesis to generate a yeast library expressing mutant V2 receptors containing mutations within the second intracellular loop. A subsequent yeast genetic screen of about 30,000 mutant receptors yielded four mutant receptors that, in contrast to the wild-type receptor, showed substantial coupling to Gq5. Functional analysis of these mutant receptors, followed by more detailed site-directed Mutagenesis studies, indicated that single amino acid substitutions at position Met145 in the central portion of the second intracellular loop of the V2 receptor had pronounced effects on receptor/G protein coupling selectivity. We also observed that deletion of single amino acids N-terminal of Met145 led to misfolded receptor proteins, whereas single amino acid deletions C-terminal of Met145 had no effect on V2 receptor function. These findings highlight the usefulness of combining receptor Random Mutagenesis and yeast expression technology to study mechanisms governing receptor/G protein coupling selectivity and receptor folding.

Loren Miraglia - One of the best experts on this subject based on the ideXlab platform.

  • High throughput Random Mutagenesis and single molecule real time sequencing of the muscle nicotinic acetylcholine receptor
    PLoS ONE, 2016
    Co-Authors: Paul J. Groot-kormelink, Pierre-eloi Imbert, Nicholas Kelley, Felix Freuler, Anthony Marelli, Nicole Gerwin, Lucia G. Sivilotti, Sandrine Ferrand, Anke Bill, Loren Miraglia
    Abstract:

    High throughput Random Mutagenesis is a powerful tool to identify which residues are important for the function of a protein, and gain insight into its structure-function relation. The human muscle nicotinic acetylcholine receptor was used to test whether this technique previously used for monomeric receptors can be applied to a pentameric ligand-gated ion channel. A mutant library for the α1 subunit of the channel was generated by error-prone PCR, and full length sequences of all 2816 mutants were retrieved using single molecule real time sequencing. Each α1 mutant was co-transfected with wildtype β1, δ, and ε subunits, and the channel function characterized by an ion flux assay. To test whether the strategy could map the structure-function relation of this receptor, we attempted to identify mutations that conferred resistance to competitive antagonists. Mutant hits were defined as receptors that responded to the nicotinic agonist epibatidine, but were not inhibited by either α-bungarotoxin or tubocurarine. Eight α1 subunit mutant hits were identified, six of which contained mutations at position Y233 or V275 in the transmembrane domain. Three single point mutations (Y233N, Y233H, and V275M) were studied further, and found to enhance the potencies of five channel agonists tested. This suggests that the mutations made the channel resistant to the antagonists, not by impairing antagonist binding, but rather by producing a gain-of-function phenotype, e.g. increased agonist sensitivity. Our data show that Random high throughput Mutagenesis is applicable to multimeric proteins to discover novel functional mutants, and outlines the benefits of using single molecule real time sequencing with regards to quality control of the mutant library as well as downstream mutant data interpretation.

Miraglia Loren - One of the best experts on this subject based on the ideXlab platform.

  • High Throughput Random Mutagenesis and Single Molecule Real Time Sequencing of the Muscle Nicotinic Acetylcholine Receptor
    PLOS, 2016
    Co-Authors: Groot Kormelink Paul, Ferrand Sandrine, Kelley Nicholas, Bill Anke, Freuler Felix, Imbert Pierre-eloi, Marelli Anthony, Gerwin Nicole, Sivilotti, Lucia G, Miraglia Loren
    Abstract:

    High throughput Random Mutagenesis is a powerful tool to identify important functional residues in the studied protein in an unbiased way, and to gain insight into its structure-function. The human muscle nicotinic acetylcholine receptor was used to test whether high throughput Mutagenesis previously used for monomeric receptors can be applied to a pentameric ligand-gated ion channel. A mutant library for the alpha1 subunit of the channel was generated using error-prone PCR. Novel single molecule real time sequencing was utilized to assess the sequences of the 2816 alpha1 subunit mutants obtained. All alpha1 subunit mutants were co-transfected with wildtype alpha1, beta, delta and epsilon subunits, and functionally studied in an ion flux assay. Mutant hits were extracted based on intact signals for the selective agonist epibatidine, and a loss of either alpha-bungarotoxin or tubocurarine inhibition. Eight alpha1 subunit mutants were identified, and six of them contained mutations of either Y233 or V275. Three mutants with single amino acid changes (Y233N, Y233H, and V275M) were further studied, and demonstrated to enhance the potencies of all five channel agonists tested, suggesting a gain of function and increased ligand sensitivity. This shows that novel single molecule real time sequencing enables to analyze sequences of individual mutants, and that Random high throughput Mutagenesis is applicable to multimeric proteins to discover novel functional mutants

Clarice Schmidt - One of the best experts on this subject based on the ideXlab platform.

  • Random Mutagenesis of the m3 muscarinic acetylcholine receptor expressed in yeast identification of second site mutations that restore function to a coupling deficient mutant m3 receptor
    Journal of Biological Chemistry, 2005
    Co-Authors: Nicola M Nowak, Clarice Schmidt, Sookyung Kim, Kenneth A Jacobson, Ali Bagheri, Jurgen Wess
    Abstract:

    Abstract The M3 muscarinic receptor is a prototypical member of the class A family of G protein-coupled receptors (GPCRs). To gain insight into the structural mechanisms governing agonist-mediated M3 receptor activation, we recently developed a genetically modified yeast strain (Saccharomyces cerevisiae) which allows the efficient screening of large libraries of mutant M3 receptors to identify mutant receptors with altered/novel functional properties. Class A GPCRs contain a highly conserved Asp residue located in transmembrane domain II (TM II; corresponding to Asp-113 in the rat M3 muscarinic receptor) which is of fundamental importance for receptor activation. As observed previously with other GPCRs analyzed in mammalian expression systems, the D113N point mutation abolished agonist-induced receptor/G protein coupling in yeast. We then subjected the D113N mutant M3 receptor to PCR-based Random Mutagenesis followed by a yeast genetic screen to recover point mutations that can restore G protein coupling to the D113N mutant receptor. A large scale screening effort led to the identification of three such second-site suppressor mutations, R165W, R165M, and Y250D. When expressed in the wild-type receptor background, these three point mutations did not lead to an increase in basal activity and reduced the efficiency of receptor/G protein coupling. Similar results were obtained when the various mutant receptors were expressed and analyzed in transfected mammalian cells (COS-7 cells). Interestingly, like Asp-113, Arg-165 and Tyr-250, which are located at the cytoplasmic ends of TM III and TM V, respectively, are also highly conserved among class A GPCRs. Our data suggest a conformational link between the highly conserved Asp-113, Arg-165, and Tyr-250 residues which is critical for receptor activation.

  • Random Mutagenesis of the m3 muscarinic acetylcholine receptor expressed in yeast identification of point mutations that silence a constitutively active mutant m3 receptor and greatly impair receptor g protein coupling
    Journal of Biological Chemistry, 2003
    Co-Authors: Clarice Schmidt, Isolde Erlenbach, Lanh M Bloodworth, Fuyue Zeng, Jurgen Wess
    Abstract:

    Abstract The M3 muscarinic receptor is a prototypical member of the class I family of G protein-coupled receptors (GPCRs). To facilitate studies on the structural mechanisms governing M3 receptor activation, we generated an M3 receptor-expressing yeast strain (Saccharomyces cerevisiae) that requires agonist-dependent M3 receptor activation for cell growth. By using receptor Random Mutagenesis followed by a genetic screen in yeast, we initially identified a point mutation at the cytoplasmic end of transmembrane domain (TM) VI (Q490L) that led to robust agonist-independent M3 receptor signaling in both yeast and mammalian cells. To explore further the molecular mechanisms by which point mutations can render GPCRs constitutively active, we subjected a region of the Q490L mutant M3 receptor that included TM V–VII to Random Mutagenesis. We then applied a yeast genetic screen to identify second-site mutations that could suppress the activating effects of the Q490L mutation and restore wild-type receptor-like function to the Q490L mutant receptor. This analysis led to the identification of 12 point mutations that allowed the Q490L mutant receptor to function in a fashion similar to the wild-type receptor. These amino acid substitutions mapped to two distinct regions of the M3 receptor, the exofacial segments of TM V and VI and the cytoplasmic ends of TM V–VII. Strikingly, in the absence of the activating Q490L mutation, all recovered point mutations severely reduced the efficiency of receptor/G protein coupling, indicating that the targeted residues play important roles in receptor activation and/or receptor/G protein coupling. This strategy should be generally applicable to identify sites in GPCRs that are critically involved in receptor function.

  • single amino acid substitutions and deletions that alter the g protein coupling properties of the v2 vasopressin receptor identified in yeast by receptor Random Mutagenesis
    Journal of Biological Chemistry, 2001
    Co-Authors: Isolde Erlenbach, Evi Kostenis, Clarice Schmidt, Claudine Serradeille Gal, Danielle Raufaste, Mark E Dumont, Mark H Pausch, Jurgen Wess
    Abstract:

    Abstract To facilitate structure-function relationship studies of the V2 vasopressin receptor, a prototypical Gs-coupled receptor, we generated V2 receptor-expressing yeast strains (Saccharomyces cerevisiae) that required arginine vasopressin-dependent receptor/G protein coupling for cell growth. V2 receptors heterologously expressed in yeast were unable to productively interact with the endogenous yeast G protein α subunit, Gpa1p, or a mutant Gpa1p subunit containing the C-terminal Gαq sequence (Gq5). In contrast, the V2 receptor efficiently coupled to a Gpa1p/Gαs hybrid subunit containing the C-terminal Gαs sequence (Gs5), indicating that the V2 receptor retained proper G protein coupling selectivity in yeast. To gain insight into the molecular basis underlying the selectivity of V2 receptor/G protein interactions, we used receptor saturation Random Mutagenesis to generate a yeast library expressing mutant V2 receptors containing mutations within the second intracellular loop. A subsequent yeast genetic screen of about 30,000 mutant receptors yielded four mutant receptors that, in contrast to the wild-type receptor, showed substantial coupling to Gq5. Functional analysis of these mutant receptors, followed by more detailed site-directed Mutagenesis studies, indicated that single amino acid substitutions at position Met145 in the central portion of the second intracellular loop of the V2 receptor had pronounced effects on receptor/G protein coupling selectivity. We also observed that deletion of single amino acids N-terminal of Met145 led to misfolded receptor proteins, whereas single amino acid deletions C-terminal of Met145 had no effect on V2 receptor function. These findings highlight the usefulness of combining receptor Random Mutagenesis and yeast expression technology to study mechanisms governing receptor/G protein coupling selectivity and receptor folding.

Paul J. Groot-kormelink - One of the best experts on this subject based on the ideXlab platform.

  • High throughput Random Mutagenesis and single molecule real time sequencing of the muscle nicotinic acetylcholine receptor
    PLoS ONE, 2016
    Co-Authors: Paul J. Groot-kormelink, Pierre-eloi Imbert, Nicholas Kelley, Felix Freuler, Anthony Marelli, Nicole Gerwin, Lucia G. Sivilotti, Sandrine Ferrand, Anke Bill, Loren Miraglia
    Abstract:

    High throughput Random Mutagenesis is a powerful tool to identify which residues are important for the function of a protein, and gain insight into its structure-function relation. The human muscle nicotinic acetylcholine receptor was used to test whether this technique previously used for monomeric receptors can be applied to a pentameric ligand-gated ion channel. A mutant library for the α1 subunit of the channel was generated by error-prone PCR, and full length sequences of all 2816 mutants were retrieved using single molecule real time sequencing. Each α1 mutant was co-transfected with wildtype β1, δ, and ε subunits, and the channel function characterized by an ion flux assay. To test whether the strategy could map the structure-function relation of this receptor, we attempted to identify mutations that conferred resistance to competitive antagonists. Mutant hits were defined as receptors that responded to the nicotinic agonist epibatidine, but were not inhibited by either α-bungarotoxin or tubocurarine. Eight α1 subunit mutant hits were identified, six of which contained mutations at position Y233 or V275 in the transmembrane domain. Three single point mutations (Y233N, Y233H, and V275M) were studied further, and found to enhance the potencies of five channel agonists tested. This suggests that the mutations made the channel resistant to the antagonists, not by impairing antagonist binding, but rather by producing a gain-of-function phenotype, e.g. increased agonist sensitivity. Our data show that Random high throughput Mutagenesis is applicable to multimeric proteins to discover novel functional mutants, and outlines the benefits of using single molecule real time sequencing with regards to quality control of the mutant library as well as downstream mutant data interpretation.