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Random Mutagenesis of G protein ɑ Subunit G_oɑ. Mutations altering nucleotide binding

Slepak, Vladlen Z. and Quick, Michael W. and Aragay, Anna M. and Davidson, Norman and Lester, Henry A. and Simon, Melvin I. (1993) Random Mutagenesis of G protein ɑ Subunit G_oɑ. Mutations altering nucleotide binding. Journal of Biological Chemistry, 268 (29). pp. 21889-21894. ISSN 0021-9258.

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Nucleotide binding properties of the G protein ɑ subunit G_oɑ were probed by mutational analysis in recombinant Escherichia coli. Thousands of random mutations generated by polymerase chain reaction were screened by in situ [^(35)S]GTPyS (guanosine 5'-(3-O-thio)-triphosphate) binding on the colony lifts following transformation of bacteria with modified G_oɑ cDNA. Clones that did not bind the nucleotide under these conditions were characterized by DNA sequence analysis, and the nucleotide binding properties were further studied in crude bacterial extracts. A number of novel mutations reducing the affinity of G_oɑ for GTPyS or Mg^(2+) were identified. Some of the mutations substitute amino acid residues homologous to those known to interact with guanine nucleotides in p21^(ras) proteins. Other mutations show that previously unstudied residues also participate in the nucleotide binding. Several mutants lost GTPyS binding but retained the capacity to interact with the βy subunit complex as determined by pertussis toxin-mediated ADP-ribosylation. One of these, mutant S47C, was functionally expressed in Xenopus laevis oocytes along with the G protein-coupled thyrotropin-releasing hormone (TRH) receptor. Whereas wild-type G_oɑ increased TRH-promoted chloride currents, S47C significantly decreased the hormone-induced Cl^- response, suggesting that this mutation resulted in a dominant negative phenotype.

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Lester, Henry A.0000-0002-5470-5255
Additional Information:© 1993 by The American Society for Biochemistry and Molecular Biology, Inc. Received for publication, May 29, 1993, and in revised form, July 7, 1993. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Issue or Number:29
Record Number:CaltechAUTHORS:20120307-154341054
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Official Citation:Random mutagenesis of G protein ɑ subunit G_oɑ. Mutations altering nucleotide binding. V Z Slepak, M W Quick, A M Aragay, N Davidson, H A Lester, and M I Simon J. Biol. Chem. 1993 268: 21889-21894.
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:29633
Deposited By: Ruth Sustaita
Deposited On:13 Mar 2012 20:37
Last Modified:03 Oct 2019 03:43

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