Random mutagenesis of G protein alpha subunit G(o)alpha. Mutations altering nucleotide binding.
Nucleotide binding properties of the G protein alpha subunit G(o)alpha were probed by mutational analysis in recombinant Escherichia coli. Thousands of random mutations generated by polymerase chain reaction were screened by in situ [35S]GTP gamma S (guanosine 5'-(3-O-thio)-triphosphate) binding on the colony lifts following transformation of bacteria with modified G(o)alpha 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(o)alpha for GTP gamma S or Mg2+ were identified. Some of the mutations substitute amino acid residues homologous to those known to interact with guanine nucleotides in p21ras proteins. Other mutations show that previously unstudied residues also participate in the nucleotide binding. Several mutants lost GTP gamma S binding but retained the capacity to interact with the beta gamma 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(o)alpha increased TRH-promoted chloride currents, S47C significantly decreased the hormone-induced Cl- response, suggesting that this mutation resulted in a dominant negative phenotype.
Slepak
V Z
VZ
Biology Division, California Institute of Technology, Pasadena 91125.
Quick
M W
MW
Aragay
A M
AM
Davidson
N
N
Lester
H A
HA
Simon
M I
MI
D000818:Animals; D001665:Binding Sites; D004252:DNA Mutational Analysis; D004926:Escherichia coli; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D005799:Genes, Dominant; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism*; D016296:Mutagenesis; D015689:Oncogene Protein p21(ras) / Q000235:genetics; D010641:Phenotype; D018025:Receptors, Thyrotropin-Releasing Hormone / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D014982:Xenopus laevis
D018025:Receptors, Thyrotropin-Releasing Hormone; D011994:Recombinant Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019204:GTP-Binding Proteins; D015689:Oncogene Protein p21(ras)