Cyclic AMP (cAMP) is a key second messenger that controls diverse phenomena, including metabolism, gene transcription, olfaction, heart rate, and memory. Cyclic AMP is synthesized by adenylyl cyclases. The modulation of intracellular cAMP concentration is largely at the level of synthesis. The activities of many adenylyl cyclases are controlled dynamically by a variety of hormones, neurotransmitters, and other regulatory molecules. The fundamental components of this pathway are receptors, heterotrimeric G proteins, and adenylyl cyclases. Since defects in the components involved in regulating cAMP concentration are associated with several human diseases, including thyroid and pituitary tumors, it is important to learn more about the regulation of cAMP. Mammalian adenylyl cyclases have a common structure, including two roughly 40 kDa cytoplasmic domains, punctuated by two intensely hydrophobic stretches. Biochemical and genetic analyses are planned to define the regulatory domain(s) of adenylyl cyclase. I have demonstrated that a construct containing two ligated cytoplasmic domains of adenylyl cyclase (IC/1IIC/2) can be expressed as a soluble enzyme in E. coli and it can rescue the catabolic defect of E. coli strains that lack adenylyl cyclase (delta cya). The soluble adenylyl cyclase, IC/1IIC/2, can be regulated by G/s alpha and forskolin. Soluble adenylyl cyclase will be used as a model system to study the adenylyl cyclase site(s) involved in interactions with G/s alpha or forskolin. Complementation of the catabolic defects of E. coli delta cya will be used to screen the randomly mutagenized IC/1IIC/2 expression library for the mutants that gain adenylyl cyclase activity or regulation by G/s alpha or forskolin. Failure of complementation of E. coli delta cya will be used to screen IC/1IIC/2 mutants that lose the adenylyl cyclase activity or regulation by G/s alpha or forskolin. The sensitivity of these mutant enzymes to G/s alpha and forskolin will be assessed and the interesting mutations will be localized by DNA sequencing. The soluble adenylyl cyclases will be purified to homogeneity. The purified enzyme will be labelled with photoaffinity forskolin analogs to determine the forskolin binding site. Determination of the site of adenylyl cyclase that interacts with G/s alpha will allow for manipulation of the interaction between G/s alpha and certain subtypes of adenylyl cyclase, thus providing a strategy to study the physiological role of the G/s alpha mediated signal that is transmitted through a given type of adenylyl cyclase. Determination of the forskolin binding site of adenylyl cyclase will provide crucial information to aid in the design of more specific and potent forskolin analogs to activate adenylyl cyclases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM053459-04
Application #
6019103
Study Section
Pharmacology A Study Section (PHRA)
Project Start
1996-09-01
Project End
2000-08-31
Budget Start
1999-09-01
Budget End
2000-08-31
Support Year
4
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Chicago
Department
Biology
Type
Schools of Medicine
DUNS #
225410919
City
Chicago
State
IL
Country
United States
Zip Code
60637
Hong, Jia; Doebele, Robert C; Lingen, Mark W et al. (2007) Anthrax edema toxin inhibits endothelial cell chemotaxis via Epac and Rap1. J Biol Chem 282:19781-7
Hong, Jia; Beeler, Jeff; Zhukovskaya, Natalia L et al. (2005) Anthrax edema factor potency depends on mode of cell entry. Biochem Biophys Res Commun 335:850-7
Shen, Yuequan; Zhukovskaya, Natalia L; Guo, Qing et al. (2005) Calcium-independent calmodulin binding and two-metal-ion catalytic mechanism of anthrax edema factor. EMBO J 24:929-41
Firoved, Aaron M; Miller, Georgina F; Moayeri, Mahtab et al. (2005) Bacillus anthracis edema toxin causes extensive tissue lesions and rapid lethality in mice. Am J Pathol 167:1309-20
Lee, Young-Sam; Bergson, Pamela; He, Wei Song et al. (2004) Discovery of a small molecule that inhibits the interaction of anthrax edema factor with its cellular activator, calmodulin. Chem Biol 11:1139-46
Shen, Yuequan; Zhukovskaya, Natalia L; Zimmer, Michael I et al. (2004) Selective inhibition of anthrax edema factor by adefovir, a drug for chronic hepatitis B virus infection. Proc Natl Acad Sci U S A 101:3242-7
Beeler, Jeff A; Tang, Wei-Jen (2004) Expression and purification of soluble adenylyl cyclase from Escherichia coli. Methods Mol Biol 237:39-53
Simons, Peter C; Biggs, Sean M; Waller, Anna et al. (2004) Real-time analysis of ternary complex on particles: direct evidence for partial agonism at the agonist-receptor-G protein complex assembly step of signal transduction. J Biol Chem 279:13514-21
Ulmer, Tobias S; Soelaiman, Sandriyana; Li, Shipeng et al. (2003) Calcium dependence of the interaction between calmodulin and anthrax edema factor. J Biol Chem 278:29261-6
Soelaiman, Sandriyana; Wei, Binqing Q; Bergson, Pamela et al. (2003) Structure-based inhibitor discovery against adenylyl cyclase toxins from pathogenic bacteria that cause anthrax and whooping cough. J Biol Chem 278:25990-7

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