Mutagenesis of the amino-terminal glycine to alanine in Gs alpha subunit alters beta gamma-dependent properties and decreases adenylylcyclase activation.
Proteolytic removal and genetic deletion of the amino-terminal domain of G protein alpha subunit have shown that this region is necessary for interaction with beta gamma subunits. In the alpha subunits which undergo myristoylation, myristoylation of the amino-terminal glycine modulates the affinity of alpha subunit for the beta gamma complex. To determine the role of the same glycine in nonmyristoylated alpha subunits, we substituted it for alanine in Gs alpha and characterized the properties of the mutated chain G2A Gs alpha. The mutant could still bind guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) as revealed by its resistance to trypsin proteolysis and was able to interact with the membrane. However, G2A Gs alpha was a poor substrate for cholera toxin-catalyzed ADP-ribosylation either in the soluble form or when membrane-associated. Addition of beta gamma subunits increased the sedimentation rate of G2A Gs alpha in sucrose gradients. Binding experiments performed on cyc- membranes reconstituted by G2A Gs alpha showed that the GTP-induced shift of isoproterenol affinity for the beta-adrenergic receptors was reduced. On the same membranes, isoproterenol, GTP gamma S and NaF were 2-fold less effective for activating adenylylcyclase when compared to cyc- membranes reconstituted by Gs alpha. This differential stimulation of adenylylcyclase was not due to an affinity change for the effector but to a decrease in the maximal activation. Thus the G2A substitution affected beta gamma-dependent properties on reconstituted membranes such as receptor coupling and cholera toxin-catalyzed ADP-ribosylation and we propose that the decreased activation of adenylylcyclase might result from the same defect. Although not essential for association with beta gamma subunits, the amino-terminal glycine of nonmyristoylated Gs alpha might play a modulatory role in this interaction.
Centre National de la Recherche Scientifique-Institut National de la Santé et de la Recherche Médicale de Pharmacologie et Endocrinologie, Montpellier, France.
R
R
van der Neut
C
C
Pantaloni
I
I
Nebout
J
J
Bockaert
Y
Y
Audigier
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000409:Alanine; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D002772:Cholera Toxin / Q000494:pharmacology; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D005998:Glycine; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D007545:Isoproterenol / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000378:metabolism; D011943:Receptors, Adrenergic, beta / Q000378:metabolism; D014158:Transcription, Genetic
D046911:Macromolecular Substances; D009227:Myristic Acids; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D011943:Receptors, Adrenergic, beta; D019814:Myristic Acid; D000246:Adenosine Diphosphate Ribose; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D002772:Cholera Toxin; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D007545:Isoproterenol; D000409:Alanine; D005998:Glycine