Promotion of the GTP-liganded state of the Go alpha protein by deletion of the C terminus.
G proteins are active as long as GTP is bound to the alpha subunit. Activation ends when GTP is cleaved to GDP that then stays bound to the active site. Agonist-liganded receptors allow formation of the active state by decreasing the affinity of alpha subunits for GDP allowing exchange of GDP for GTP. Since receptors interact with the C terminus of the alpha subunits, we tested whether deletion of the C terminus could mimic activation by receptors. Three deletions and one point mutation at the C terminus of alpha o were engineered in alpha o cDNA by the polymerase chain reaction, transcribed into RNA, and translated in a rabbit reticulocyte lysate. The ability of in vitro synthesized protein to bind guanine nucleotide was inferred from analysis of native tryptic cleavage patterns, while the ability of the proteins to associate with beta gamma was measured by sucrose density gradient centrifugation. Deletion of 14 amino acids, alpha oD[341], from the C terminus causes a large decrease in GDP affinity, with little or no change in guanosine 5'-3-O-(thio)triphosphate affinity. When GTP is present, alpha oD[341] remains in the activated conformation because exchange of GTP for GDP is rapid. Deletion of 10 amino acids, alpha oD[345], lowers GDP affinity, but less dramatically than in alpha oD[341]. Deletion of 5 amino acids, alpha oD[350], or mutation of Arg-349 to proline alpha oR[349P] has no detectable effects on GDP affinity. Deletion of up to 10 amino acids from the C terminus does not prevent formation of alpha beta gamma heterotrimers. We propose that the C terminus of the alpha subunit is a mobile region that blocks dissociation of GDP. Agonist-liganded receptors may move it aside to allow release of GDP, exchange for GTP, and activation of the alpha subunit.
Denker
B M
BM
Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115.
Schmidt
C J
CJ
Neer
E J
EJ
D000595:Amino Acid Sequence; D000818:Animals; D002499:Centrifugation, Density Gradient; D002872:Chromosome Deletion; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010446:Peptide Fragments / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods; D011485:Protein Binding; D014176:Protein Biosynthesis; D015202:Protein Engineering; D011817:Rabbits; D051381:Rats; D012156:Reticulocytes / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D010446:Peptide Fragments; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D014357:Trypsin; D019204:GTP-Binding Proteins