Mutagenesis of the amino terminus of the alpha subunit of the G protein Go. In vitro characterization of alpha o beta gamma interactions.
Heterotrimeric guanine nucleotide-binding proteins are composed of alpha and beta gamma subunits and couple a variety of cell-surface receptors to intracellular enzymes or ion channels. The heterotrimer dissociates into alpha and beta gamma subunits when the alpha subunit is activated by guanine nucleoside triphosphates. Several lines of evidence show that the amino terminus of the alpha subunit is important for the interaction with the beta gamma subunit (Neer, E. J., Pulsifer, L., and Wolf, L. G. (1988) J. Biol. Chem. 263, 8996-9000; Fung, B. K.-K., and Nash, C. R. (1983) J. Biol. Chem. 258, 10503-10510). We have mutagenized the amino terminus of alpha o to dissect the relative contributions of amino-terminal myristoylation and specific amino acid sequences to subunit interaction. Wild-type and mutant alpha o cDNAs were translated in vitro in a rabbit reticulocyte lysate. All proteins were able to bind guanosine 5'-(gamma-thio)triphosphate and to achieve the necessary conformation for protection from tryptic digestion. Two assays of alpha o beta gamma interactions were used: sucrose density gradients to look for stable heterotrimer formation and ADP-ribosylation by pertussis toxin to detect weak or transient alpha o beta gamma interactions. Our results indicate that myristoylation is essential for stable heterotrimer formation, but that nonmyristoylated proteins are also capable of interacting with the beta gamma subunit. Amino acids 7-10 have an important role in alpha o beta gamma interactions whether alpha o is myristoylated or not. Deletion of this region diminishes the ability of alpha o to interact with the beta gamma subunit, but substitutions at this position indicate that other amino acids can be tolerated without affecting subunit interaction.
Denker
B M
BM
Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts.
Neer
E J
EJ
Schmidt
C J
CJ
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002499:Centrifugation, Density Gradient; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D010449:Peptide Mapping; D037342:Pertussis Toxin; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D012156:Reticulocytes / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic; D010566:Virulence Factors, Bordetella / Q000378:metabolism
D046911:Macromolecular Substances; D009227:Myristic Acids; D012333:RNA, Messenger; D010566:Virulence Factors, Bordetella; D019814:Myristic Acid; D000246:Adenosine Diphosphate Ribose; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D037342:Pertussis Toxin; D019204:GTP-Binding Proteins