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. T housands of random mutations generated by polymerase chain reaction we re screened by in situ [S-35]GTPgammaS (guanosine 5'-(3-O-thio)triphos phate) binding on the colony lifts following transformation of bacteri a with modified G(o)alpha cDNA. Clones that did not bind the nucleotid e under these conditions were characterized by DNA sequence analysis, and the nucleotide binding properties were further studied in crude ba cterial extracts. A number of novel mutations reducing the affinity of G(o)alpha for GTPgammaS or Mg2+ were identified. Some of the mutation s substitute amino acid residues homologous to those known to interact with guanine nucleotides in p21ras proteins. Other mutations show tha t previously unstudied residues also participate in the nucleotide bin ding. Several mutants lost GTPgammaS binding but retained the capacity to interact with the betagamma subunit complex as determined by pertu ssis toxin-mediated ADP-ribosylation. One of these, mutant S47C, was f unctionally expressed in Xenopus laevis oocytes along with the G prote in-coupled thyrotropin-releasing hormone (TRH) receptor. Whereas wild- type G(o)alpha increased TRH-promoted chloride currents, S47C signific antly decreased the hormone-induced Cl- response, suggesting that this mutation resulted in a dominant negative phenotype.