Isolation and characterization of constitutively active mutants of mammalian adenylyl cyclase

Mark E. Hatley, Benjamin K. Benton, Jun Xu, John P. Manfredi, Alfred G. Gilman, Roger K. Sunahara

Research output: Contribution to journalArticle

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Abstract

A genetic screen in Saccharomyces cerevisiae identified mutations in mammalian adenylyl cyclase that activate the enzyme in the absence of Gsα. Thirteen of these mutant proteins were characterized biochemically in an assay system that depends on a mixture of the two cytosolic domains (C1 and C2) of mammalian adenylyl cyclases. Three mutations, I1010M, K1014N, and P1015Q located in the β4-β5 loop of the C2 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 C1 domain of type V adenylyl cyclase in the absence of activators of the enzyme. The increased affinity of the mutant C2 domain of adenylyl cyclase for the wild type C1 domain was exploited to isolate a complex containing VC1, IIC2, and Gsα-guanosine 5'-3-0-(thio)triphosphate (GTPγS) in the absence of forskolin and a complex of VC1, IIC2, forskolin, and P-site inhibitor in the absence of Gsα-GTPγS. 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 Gsα.

Original languageEnglish (US)
Pages (from-to)38626-38632
Number of pages7
JournalJournal of Biological Chemistry
Volume275
Issue number49
DOIs
StatePublished - Dec 8 2000

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Adenylyl Cyclases
Colforsin
Mutation
Enzyme Activators
Enzyme Activation
Guanosine
Mutant Proteins
Enzymes
Yeast
Saccharomyces cerevisiae
Assays
Crystal structure
Chemical activation
C2 Domains

ASJC Scopus subject areas

  • Biochemistry

Cite this

Hatley, M. E., Benton, B. K., Xu, J., Manfredi, J. P., Gilman, A. G., & Sunahara, R. K. (2000). Isolation and characterization of constitutively active mutants of mammalian adenylyl cyclase. Journal of Biological Chemistry, 275(49), 38626-38632. https://doi.org/10.1074/jbc.M007148200

Isolation and characterization of constitutively active mutants of mammalian adenylyl cyclase. / Hatley, Mark E.; Benton, Benjamin K.; Xu, Jun; Manfredi, John P.; Gilman, Alfred G.; Sunahara, Roger K.

In: Journal of Biological Chemistry, Vol. 275, No. 49, 08.12.2000, p. 38626-38632.

Research output: Contribution to journalArticle

Hatley, ME, Benton, BK, Xu, J, Manfredi, JP, Gilman, AG & Sunahara, RK 2000, 'Isolation and characterization of constitutively active mutants of mammalian adenylyl cyclase', Journal of Biological Chemistry, vol. 275, no. 49, pp. 38626-38632. https://doi.org/10.1074/jbc.M007148200
Hatley, Mark E. ; Benton, Benjamin K. ; Xu, Jun ; Manfredi, John P. ; Gilman, Alfred G. ; Sunahara, Roger K. / Isolation and characterization of constitutively active mutants of mammalian adenylyl cyclase. In: Journal of Biological Chemistry. 2000 ; Vol. 275, No. 49. pp. 38626-38632.
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