THE PRIMARY STRUCTURE OF THE ALPHA SUBUNIT OF A STARFISH GUANOSINE-NUCLEOTIDE-BINDING REGULATORY PROTEIN INVOLVED IN 1-METHYLADENINE-INDUCED OOCYTE MATURATION

Starfish-oocyte maturation induced by 1-methyladenine (MeAde) was inhibited by microinjection of pertussis toxin (PTX). The inhibition appeared to result from PTX-catalyzed ADP-ribosylation of a 39-kDa guanosine-nucleotide-binding regulatory protein (G protein) in the oocyte. These results strongly support the hypothesis that the MeAde-induced signals operate via a membrane receptor and are carried by the PTX-sensitive G protein. When PTX-injected oocytes were treated with dithiothreitol, 85% of them reinitiated meiosis, suggesting that dithiothreitol did not act on the MeAde receptor. We constructed a cDNA library from the immature ovary of starfish, Asterina pectinifera, and screened it with the cDNA of the alpha-subunit of an inhibitory rat G protein (Gi-2). A positive cDNA clone contained an open reading frame of 1062 bases which had 74% identity with the rat G(i-2) cDNA. The deduced amino acid sequence was 85% and 89% identical to rat G(i-1), and rat G(i-1), respectively. The alpha-subunit of the G protein purified from cortices of starfish oocytes was digested by trypsin and the resulting four peptides were microsequenced. Comparison of these amino acid sequences with the predicted one indicated that the isolated cDNA clone encoded the alpha-subunit of the PTX-sensitive G protein in oocytes. The C-terminal sequence, KNNLKDCGLF, was identical to that of G(i), suggesting that the cysteine residue is the site of ADP-ribosylation.