Amino acids 367-376 of the Gs alpha subunit induce membrane association when fused to soluble amino-terminal deleted Gi1 alpha subunit.
Signal transduction GTP-binding proteins are tightly associated with plasma membrane. In the resting state, the anchorage of the alpha subunit could be indirect by means of the other beta gamma subunits or polydisperse multimers. In the activated state, although the alpha subunit is dissociated from other subunits, it is not released from the membrane and therefore is likely to contain information necessary to remain associated with the plasma membrane. Previous proteolytic experiments suggested that, in contrast to other G proteins alpha subunits, the C-terminal domain of Gs alpha (the G protein involved in adenylate cyclase stimulation) is essential for membrane association of the activated form. To better define the crucial residues involved in membrane attachment, we constructed chimeras between a soluble core and various parts of the Gs alpha C-terminal domain. We first deleted codons 2-6 of Gi1 alpha (the inhibitory G protein of the i1 subtype) to generate a soluble GTP-binding protein, delta N-Gi1 alpha. We then replaced the last 14 C-terminal codons of delta N-Gi1 alpha by different domains of the Gs alpha C terminus and looked for the membrane association of chimeric proteins after in vitro transcription, in vitro translation, and interaction with S49 cyc- membranes (obtained from a mutant cell line that does not express Gs alpha). Our results showed that addition of amino acids 367-376 of Gs alpha is sufficient to promote membrane association of the soluble N-terminal deleted Gi1 alpha.
Journot
L
L
Centre National de la Recherche Scientifique-Institut National de la Santé et de la Recherche Médicale de Pharmacologie-Endocrinologie, Montpellier, France.
Pantaloni
C
C
Poul
M A
MA
Mazarguil
H
H
Bockaert
J
J
Audigier
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology*; D002678:Chimera; D002872:Chromosome Deletion; D003062:Codon / Q000235:genetics; D019204:GTP-Binding Proteins / Q000235:genetics / Q000502:physiology*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D010446:Peptide Fragments / Q000494:pharmacology*; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D015183:Restriction Mapping; D015398:Signal Transduction; D014158:Transcription, Genetic
D003062:Codon; D046911:Macromolecular Substances; D009227:Myristic Acids; D010446:Peptide Fragments; D019814:Myristic Acid; D019204:GTP-Binding Proteins