Isolation and characterization of constitutively active mutants of mammalian adenylyl cyclase.
A genetic screen in Saccharomyces cerevisiae identified mutations in mammalian adenylyl cyclase that activate the enzyme in the absence of G(s)alpha. Thirteen of these mutant proteins were characterized biochemically in an assay system that depends on a mixture of the two cytosolic domains (C(1) and C(2)) of mammalian adenylyl cyclases. Three mutations, I1010M, K1014N, and P1015Q located in the beta4-beta5 loop of the C(2) domain of type II adenylyl cyclase, increase enzymatic activity in the absence of activators. The K1014N mutation displays both increased maximal activity and apparent affinity for the C(1) domain of type V adenylyl cyclase in the absence of activators of the enzyme. The increased affinity of the mutant C(2) domain of adenylyl cyclase for the wild type C(1) domain was exploited to isolate a complex containing VC(1), IIC(2), and G(s)alpha-guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) in the absence of forskolin and a complex of VC(1), IIC(2), forskolin, and P-site inhibitor in the absence of G(s)alpha-GTPgammaS. The isolation of these complexes should facilitate solution of crystal structures of low activity states of adenylyl cyclase and thus determination of the mechanism of activation of the enzyme by forskolin and G(s)alpha.
Hatley
M E
ME
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9041, USA.
Benton
B K
BK
Xu
J
J
Manfredi
J P
JP
Gilman
A G
AG
Sunahara
R K
RK
D000262:Adenylyl Cyclases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019943:Amino Acid Substitution; D000818:Animals; D002118:Calcium / Q000378:metabolism; D003001:Cloning, Molecular; D005576:Colforsin / Q000494:pharmacology; D004926:Escherichia coli; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism; D007700:Kinetics; D008322:Mammals; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000254:growth & development*
D011994:Recombinant Proteins; D005576:Colforsin; D019205:GTP-Binding Protein alpha Subunits, Gs; D000262:Adenylyl Cyclases; D002118:Calcium