Many neurotransmitter receptors that occur in the brain inhibit adenylate cyclase through the mediation of a GTP regulatory complex (Ni). Knowledge of the molecular events associated with the regulation of these receptors by endogenous effectors enhances our understanding of neuronal function. The cerebral cortical adenosine (A1) receptor typifies those neurotransmitter receptors that inhibit adenylate cyclase. As yet information is lacking on its molecular associations in membranes or detergent solution and on its regulation by endogenous modulators. The present proposal seeks to provide a molecular description of the adenosine receptor as it is linked to the inhibition of adenylate cyclase in cortical membranes. The approach taken will be to fractionate, characterize and reconstitute each of the components. The precise components of this study are: i) To identify the adenosine receptor by photolabelling using both agonists and antagonists and verify its identity by analogue specificities, tissue distribution and its hydrodynamic behavior compared with that of the reversibly labelled adenosine A1 receptor. ii) To generate monoclonal antibodies against the receptor, verifying their specificity by a range of tests. iii) To purify the receptor by a combination of class-purification and high-resolution purification techniques in CHAPS. iv) Using purified GTP regulatory proteins from brain, conditions will be devised for the reconstitution of the interaction between solubilized receptor and the N proteins, with particular attention to the role of magnesium. v) The interaction between N proteins and activated forms of C, that is presently observed in detergent solution, will be stabilized in phospholipid micelles. vi) A fully integrated adenosine receptor-N protein and activated catalytic unit from cerebral cortex will be assembled in phospholipid micelles. These strategies aim to provide not only structural information but also insights into the regulatory mechanisms of inhibitory regulation of adenylate cyclase by adenosine A1 receptors.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM032483-04A1
Application #
3281351
Study Section
Neurological Sciences Subcommittee 1 (NLS)
Project Start
1983-08-01
Project End
1990-04-30
Budget Start
1987-05-01
Budget End
1988-04-30
Support Year
4
Fiscal Year
1987
Total Cost
Indirect Cost
Name
University of Colorado Denver
Department
Type
Schools of Medicine
DUNS #
065391526
City
Aurora
State
CO
Country
United States
Zip Code
80045
Martin, Agnes C L; Cooper, Dermot M F (2006) Capacitative and 1-oleyl-2-acetyl-sn-glycerol-activated Ca(2+) entry distinguished using adenylyl cyclase type 8. Mol Pharmacol 70:769-77
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Hu, Biao; Nakata, Hiroko; Gu, Chen et al. (2002) A critical interplay between Ca2+ inhibition and activation by Mg2+ of AC5 revealed by mutants and chimeric constructs. J Biol Chem 277:33139-47
Gu, Chen; Cali, James J; Cooper, Dermot M F (2002) Dimerization of mammalian adenylate cyclases. Eur J Biochem 269:413-21
Fagan, K A; Smith, K E; Cooper, D M (2000) Regulation of the Ca2+-inhibitable adenylyl cyclase type VI by capacitative Ca2+ entry requires localization in cholesterol-rich domains. J Biol Chem 275:26530-7
Gu, C; Cooper, D M (2000) Ca(2+), Sr(2+), and Ba(2+) identify distinct regulatory sites on adenylyl cyclase (AC) types VI and VIII and consolidate the apposition of capacitative cation entry channels and Ca(2+)-sensitive ACs. J Biol Chem 275:6980-6

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