The carboxy-terminal domain of Gs alpha is necessary for anchorage of the activated form in the plasma membrane.
GTP-binding proteins which participate in signal transduction share a common heterotrimeric structure of the alpha beta gamma-type. In the activated state, the alpha subunit dissociates from the beta gamma complex but remains anchored in the membrane. The alpha subunits of several GTP-binding proteins, such as Go and Gi, are myristoylated at the amino terminus (Buss, J. E., S. M. Mumby, P. J. Casey, A. G. Gilman, and B. M. Sefton. 1987. Proc. Natl. Acad. Sci. USA. 84:7493-7497). This hydrophobic modification is crucial for their membrane attachment. The absence of fatty acid on the alpha subunit of Gs (Gs alpha), the protein involved in adenylate cyclase activation, suggests a different mode of anchorage. To characterize the anchoring domain of Gs alpha, we used a reconstitution model in which posttranslational addition of in vitro-translated Gs alpha to cyc- membranes (obtained from a mutant of S49 cell line which does not express Gs alpha) restores the coupling between the beta-adrenergic receptor and adenylate cyclase. The consequence of deletions generated by proteolytic removal of amino acid sequences or introduced by genetic removal of coding sequences was determined by analyzing membrane association of the proteolyzed or mutated alpha chains. Proteolytic removal of a 9-kD amino-terminal domain or genetic deletion of 28 amino-terminal amino acids did not modify the anchorage of Gs alpha whereas proteolytic removal of a 1-kD carboxyterminal domain abolished membrane interaction. Thus, in contrast to the myristoylated alpha subunits which are tethered through their amino terminus, the carboxy-terminal residues of Gs alpha are required for association of this protein with the membrane.
Audigier
Y
Y
Centre National de la Recherche Scientifique, Montpellier, France.
Journot
L
L
Pantaloni
C
C
Bockaert
J
J
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002474:Cell-Free System; D002872:Chromosome Deletion; D019204:GTP-Binding Proteins / Q000737:chemistry*; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D011233:Precipitin Tests; D012697:Serine Endopeptidases; D013329:Structure-Activity Relationship; D014357:Trypsin
D008565:Membrane Proteins; D010446:Peptide Fragments; D000246:Adenosine Diphosphate Ribose; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin; D019204:GTP-Binding Proteins