title: An important functional role of the N terminus domain of type VI adenylyl cyclase in Galphai-mediated inhibition.
abstract: We show herein that removal of the first 86 amino acids (aa) of the N terminus (designated N) of type VI adenylyl cyclase (ACVI) caused the resultant ACVI mutant (ACVI-DeltaA87) to be more greatly inhibited by a Galpha(i)-coupled receptor or activated Galpha(i) protein. Moreover, in vitro binding of the full-length N and C1a domain (designated C1a), which interacts with Galpha(i), was detected. A truncated N terminus (aa 1-86) also interacted with C1a, suggesting that the C1a-interacting region is located within aa 1-86. Mutation analyses further revealed that N might interact with C1a in the region (aa 434-505) where Galpha(i) is bound. Mutations of two residues (Leu-472 and Val-476) located in this N-binding region of C1a suppressed the interaction between recombinant N and C1a and markedly reduced Galpha(i)-mediated inhibition of ACVI-DeltaA87. Further biochemical analyses of the effect of internal mutations of Leu-472/Val-476 on Galpha(i)-mediated inhibition of wild-type ACVI and ACVI-DeltaA87 suggested that N modulates the Galpha(i)-mediated inhibition of ACVI via binding to C1a when the level of Galpha(i) is low (i.e. around the IC(50) value) and that a more complicated interfering mode results when the level of Galpha(i) is high (i.e. approximately 10- to 20-fold of the IC(50) value). Collectively, data presented herein suggest a novel function of the N terminus of ACVI in Galpha(i)-mediated regulation.
authors.affiliation: Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan, ROC.
authors.forename: Yu-Ya
authors.initials: YY
authors.lastname: Kao
authors.forename: Hsing-Lin
authors.initials: HL
authors.lastname: Lai
authors.forename: Ming-Jing
authors.initials: MJ
authors.lastname: Hwang
authors.forename: Yijuang
authors.initials: Y
authors.lastname: Chern
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D020134:Catalytic Domain; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D006801:Humans; D020128:Inhibitory Concentration 50; D007930:Leucine / Q000737:chemistry; D008954:Models, Biological; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011487:Protein Conformation; D020033:Protein Isoforms; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D014633:Valine / Q000737:chemistry
chemical_list: D020033:Protein Isoforms; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; C498015:GNAI2 protein, human; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; C498017:Gnai2 protein, rat; D000262:Adenylyl Cyclases; C121100:adenylyl cyclase 6; D007930:Leucine; D014633:Valine