Reticulocyte lysates synthesize an active alpha subunit of the stimulatory G protein Gs.
We placed the cDNAs encoding one of the short types of alpha s (alpha s-1) with Asp-Ser in positions 70 and 71 and one of the long types of alpha s (alpha s-2) in which Asp-Ser are substituted with a string of 16 amino acids, into the pGEM-3 transcription vector downstream from its T7 RNA polymerase promoter, obtained transcripts and translated the mRNAs using a rabbit reticulocyte lysate system, to determine if the molecules would be synthesized and, if so, whether they would be active as assessed in cyc- reconstitution assays. The translation products obtained from both alpha s RNAs were a mixture of primarily three polypeptides of which one (approximately 40-50% of total) represented the complete translation product and the other two appeared to be due to internal translation starts at Met60, before the splice difference between the RNAs, and the other at the first Met after the splice difference. Lysates incubated with short or long alpha s RNA when added to cyc- membranes reconstituted fluoride and GTP[gamma S]-stimulated activities. Thus, in vitro synthesized alpha s subunits are active in interacting both with guanine nucleotides and the adenylyl cyclase enzyme. On incubation without and with the receptor agonist isoproterenol, using GTP as sole added guanine nucleotide, both types of alpha s subunits reconstituted the isoproterenol-stimulated adenylyl cyclase activity. Thus, the synthetic alpha s also interact with receptors, and by inference with beta-gamma dimers, shown previously to be needed for activation by receptor. Quantitative assays in which the activity of the synthetic alpha s-1 was compared to that of native purified human erythrocyte type-1 Gs, indicated that the two products are equipotent within a 2-fold margin of error. Thus, the lysate made fully active alpha s subunits, and alpha s subunits require no post-translational modifications dependent on microsomal processes. This approach may be useful in studying biological functions of other cloned alpha subunits of G proteins.
Olate
J
J
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.
Mattera
R
R
Codina
J
J
Birnbaumer
L
L
D000818:Animals; D001483:Base Sequence; D002474:Cell-Free System; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004262:DNA Restriction Enzymes; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000235:genetics*; D005796:Genes; D046911:Macromolecular Substances; D010957:Plasmids; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism*; D014158:Transcription, Genetic
D046911:Macromolecular Substances; D012333:RNA, Messenger; D004247:DNA; D012321:DNA-Directed RNA Polymerases; D004262:DNA Restriction Enzymes; D019204:GTP-Binding Proteins