Dr Margery Barrand

Prof Eric Barnard, FRS

Dr Brian Billups

Dr Denis Burdakov

Dr Sangeeta Chawla

Prof. Dermot Cooper

Prof. Michael Edwardson

Dr Tai-Ping Fan

Dr Lora Heisler

Dr Robert Henderson

Dr Robin Hiley

Dr Stephen Hladky

Prof. Robin Irvine, FRS

Dr Lesley MacVinish

Prof. Jenny Morton

Dr Ruth Murrell-Lagnado

Prof. Peter McNaughton

Dr Llewelyn Roderick

Dr Zoltan Sarnyai

Prof. Colin Taylor

Dr Rik van Veen

Dr. Rietie Venter

Dr. Xuming Zhang

Cyclic AMP

Every aspect in the life of mammalian cells is affected to some extent by cAMP-signaling, from very acute events like regulation of cardiac contraction or neurotransmitter release, to long-term events such as cell growth or development. It is being increasingly recognized that control over so many diverse processes is mediated by a combination of a variety of differently-regulated adenylyl cyclases and phosphodiesterases (the degraders of cAMP) and subcellular targeting of the interacting components. The recognition of this level of organization is now making cAMP signaling a target for very discerning pharmaceutical strategies, which promise far greater specificity than the global strategies of the past. This lab is pitched at the forefront of this compartmentalization arena, developing new tools and perspectives. Techniques in use include a broad range of contemporary molecular and cell biological approaches, as described under the headings below. Four interactive projects are ongoing.

1 - Structure-function studies of adenylyl cyclases

We want to know which structural features render individual adenylyl cyclases susceptible to Ca²⁺-stimulation or inhibition. We manipulate cyclase cDNAs and express the proteins in either mammalian or insect cells to examine the functional consequences. We are also probing intermolecular associations of the transmembrane-spanning segments of adenylyl cyclases in mediating oligomerization and association with other proteins by the use of CFP- and YFP-tagged constructs, coupled with FRET analysis.

Structure of the catalytic domain of adenylyl cyclase and location of amino acid mutations. Left panel, the forskolin binding site and active site of adenylyl cyclase. The active site occurs at the interface of the C1a and C2a subunits of adenylyl cyclase, which are represented in dark and light blue cartoon format, respectively. The structure is that of the complex of ACVC1a and ACIIC2a (Protein Data Bank code 1CJT) (8). Forskolin, shown in stick representation and in red, binds in the cleft that contains the active site. The ATP analog (DAD, in yellow) and two Mg2+ ions (represented as orange spheres) are bound in the adenylyl cyclase active site in this crystal structure. Right panel, location of the mutated amino acids. The panel shows ribbon representation of the active site and location of the four active site mutations. The two Mg2+ ions are displayed as orange spheres, whereas DAD and the amino acids which are mutated are shown in stick representation. Each residue is coloured by atom (carbon, yellow; nitrogen, blue; oxygen, red; sulphur, pink;). Each of the four mutations occurs proximate to the Mg2+ binding sites. Cys441 and Tyr442 are the two residues just after Asp440, which is of prime importance to Mg2+ binding and adenylyl cyclase activity. The Arg434 and Phe423 are located more distally from the active site. Both residues contact Tyr442, suggesting that mutations of these amino acids might transmit their effects on Mg2+ activation through this residue. Both panels were generated with Visual Molecular Dynamics software (34) and POVRAY (www.povray.org)

2 - Local cAMP and [Ca²⁺]i signals

We wish to know what interdependent changes occur in these signals in the microenvironment in which they are generated. We use mutated cyclic nucleotide gated ion channels to measure cAMP either by electrophysiological measurements, or fluorimetrically (exploiting the Ca²⁺-conducting properties of these channels). We also use aequorin-modified adenylyl cyclases to measure Ca²⁺ in the microdomain of adenylyl cyclase. An extension of these studies is our prediction that cAMP levels may, like [Ca²⁺]i, oscillate and we are trying to devise methods to determine how targets may respond to cAMP oscillations.

See Willoughby et al., 2006

3 - Factors that cause the association of adenylyl cyclases with Ca2^+-entry channels

We already know that Ca^+--sensitive adenylyl cyclases are exquisitely sensitive to capacitative Ca^+--entry in non-excitable cells in the face of much larger elevations in [Ca^+-]i arising from either release or ionophore-mediated entry. We are trying to determine by cell biological and molecular biological approaches how this intimate association is maintained.

See Smith et al., 2002

4 - The cAMP microdomain

Apart from immediate factors promoting the association of Ca2+-channels with adenylyl cyclases, other proteins and enzymes also contribute to the environment where cAMP originates at the plasma membrane; the role of lipid rafts, calmodulin recruitment, protein kinases, phosphodiesterases and phosphatases, and other factors controlling the macro-environment are also explored.

See Simpson et al., 2006

Please click on the link if you would like to know more about Cyclic AMP - the rich vein to the Nobel motherlode

Joining the Lab

Graduate Students (Ph.D or M.Phil.) are admitted via the Department. Details of application procedures are found at http://www.phar.cam.ac.uk/phd/index.html Please feel free to contact Prof. Dermot Cooper with informal enquiries about joining the lab before submitting a full application.

Postdoctoral positions are advertised on www.jobs.ac.uk when they arise, but informal enquiries can be considered at any time.

Selected Publications

Capacitative Ca2+-entry exclusively inhibits cAMP synthesis in C6-2B glioma cells; evidence that physiologically evoked Ca2+ entry regulates Ca2+-inhibitable adenylyl cyclase in non-excitable cells. M. Chiono, R. Mahey, G. Tate and D.M.F. Cooper. J Biol Chem 270: 1149-1155 (1995). 

Ca2+-inhibitable adenylyl cyclase modulates pulmonary artery endothelial cell cAMP content and barrier function. T. Stevens, Nakahashi, Y., Cornfield, D., McMurtry, I.F., Cooper, D.M.F., and Rodman, D.M. Proc Nat Acad Sci USA 92: 2696-2700 (1995). [Medline]

Adenylyl cyclases and the interaction between calcium and cAMP signalling. D.M.F. Cooper, N. Mons and J.W. Karpen. Nature 374: 421-424 (1995). [Medline]

The characterization of a novel human adenylyl cyclase which is present in brain and other tissues. Hellevuo, K., Yoshimura, M., Mons, N., Hoffman, P.L., Cooper, D.M.F. and Tabakoff, B. J Biol Chem 270: 11581-11589 (1995). 

Immunohistochemical localization of adenylyl cyclase in rat brain indicates a highly selective concentration at synapses. N. Mons, A. Harry, P. Dubourg, R.T. Premont, R. Iyengar and D.M.F. Cooper. Proc Nat Acad Sci USA 92: 8473-8477 (1995). [Medline]

Adenylate cyclases; critical foci in neuronal signalling. N. Mons and D.M.F. Cooper. Trends in Neurosciences 18: 536-542 (1995).[Medline]

Functional co-localization of transfected Ca2+-stimulable adenylyl cyclases with capacitative Ca2+-entry sites. K. Fagan, Mahey, R. and D.M.F. Cooper. J Biol Chem 271: 12438-12444 (1996). 

Construction of a full-length Ca2+-sensitive adenylyl cyclase/aequorin chimera. Y. Nakahashi, E. Nelson, K. Fagan, E. Gonzales, J.-L. Guillou and D.M.F. Cooper. J Biol Chem 272: 18093-18097 (1997). 

Dependence of the Ca2+-inhibitable adenylyl cyclase of C6-2B glioma cells on capacitative Ca2+-entry. K. Fagan, N. Mons, and D.M.F. Cooper. J Biol Chem 273: 9297-9305 (1998). 

Regulation of adenylyl cyclase by membrane potential. D.M.F. Cooper, M.J. Schell, P. Thorn and R.F. Irvine. J Biol Chem 273: 27703-27707 (1998). 

Calmodulin binding sites of adenylyl cyclase type VIII. C. Gu and D.M.F. Cooper. J Biol Chem 274: 8012-8021 (1999). 

Adenovirus-mediated expression of an olfactory cyclic necleotide-gated channel regulates the endogenous Ca2+-inhibitude adenylyl cyclase in C6-2B glioma cells. K.A. Fagan, T.C. Rich, S. Tolman, J. Schaack, J.W. Karpen and D.M.F. Cooper. J Biol Chem 274: 12445-12453 (1999). 

Differential Activation of Adenylyl Cyclases by Spatial and Procedural Learning. J-L. Guillou, G.M. Rose and D.M.F. Cooper. J Neuroscience 19: 6183-6190 (1999). 

Inhibition by Calcium of mammalian adenylyl cyclases. J-L. Guillou, H. Nakata and D.M.F. Cooper. J Biol Chem 274: 35539-35345 (1999). 

Ca²⁺, Sr²⁺ and Ba²⁺ identify distinct regulatory sites on adenylyl cyclase (AC) types VI and VIII and consolidate the apposition of capacitative cation entry channels and Ca²⁺-sensitive ACs. C. Gu and D.M.F. Cooper. J. Biol. Chem. 275, 6980-6986 (2000).

Sustained endothelial nitric oxide synthase activation requires capacitative Ca²⁺-entry. S. Lin, K.A. Fagan, K.-X. Li, P.W. Shaul, D.M.F. Cooper and D.M. Rodman J. Biol. Chem. 275,17979-17985 (2000).

Characteristics of the Calcium-dependent inhibition of cyclic AMP accumulation by histamine and thaspsigargin in human U373 MG astrocytoma cells. M.P.M. Wong, D.M.F. Cooper, K.W. Young, and J.M. Young. British Journal of Pharmacology 130 1021-1030 (2000)

Cyclic nucleotide-gated channels colocalize with adenylyl cyclase in regions of restricted cAMP diffusion. T.C. Rich, K.A. Fagan, H. Nakata, Schaack, J., D.M.F. Cooper and Karpen, J.W. J. Gen. Physiol. 116, 147-161 (2000).

Regulation of the Ca²⁺-inhibitable adenylyl cyclase type VI by capacitative Ca²⁺-entry requires localization in cholesterol-rich domains. K.A. Fagan, K.E. Smith and D.M.F. Cooper J. Biol. Chem. 275, 26530-26537 (2000)

Regulation of a Ca²⁺-sensitive adenylyl cyclase in an excitable cell; role of voltage-gated versus capacitative Ca²⁺-entry. KA Fagan, RA Graf, S Tolman, J Schaack and DMF Cooper J. Biol. Chem. 275 40187-40194 (2000).

The two transmembrane clusters of adenylyl cyclase interact tightly to govern the plasma membrane location and function of the enzyme. C. Gu, A. Sorkin and DMF Cooper. Current Biology 11,185-190 (2001).

A uniform stimulus triggers distinct cAMP signals in different compartments of a simple cell. TC Rich, KA Fagan, TE Tse, J Schaack, DMF Cooper and J Karpen Proc. Natl. Acad. Sci. USA  98, 13049-13054 (2001).

Ca²⁺-sensitive adenylyl cyclase/aequorin chimerae as sensitive probes for discrete modes of elevation of cytosolic Ca²⁺. DMF Cooper. Methods in Enzymology 345, 105-112 (2002).

Dimerization of Mammalian Adenylyl Cyclases; Functional, Biochemical and FRET studies. C Gu, JJ Cali and DMF Cooper. European Journal of Biochemistry 269, 412-421 (2002).

Residence of adenylyl cyclase type 8 in caveolae is necessary but not sufficient for regulation by capacitative Ca²⁺ entry. KE Smith, C Gu, KA Fagan, B Hu and DMF Cooper. J. Biol. Chem. 277, 6025-6031 (2002).

Dominant regulation of interendothelial cell gap formation by calcium-inhibited type 6 adenylyl cyclase. D Cioffi, TM Moore, J Schaack, JR Creighton, DMF Cooper and TR Stevens. J. Cell Biology 157,1267-1278 (2002)

A critical interplay between Ca²⁺-inhibition and activation by Mg²⁺ of AC5 revealed by mutants and chimeric constructs. B Hu, H Nakata, C Gu, T De Beer and DMF Cooper. J. Biol. Chem. 277, 33139-33147 (2002)

Regulation and organization of adenylyl cyclases and cyclic AMP. DMF Cooper. Biochem. J. 375, 517-529 (2003)

Sustained entry of Ca²⁺ is required to activate Ca²⁺-calmodulin-dependent phosphodiesterase 1A. TA Goraya, N Masada, A Ciruela and DMF Cooper. J. Biol. Chem. 279, 40494-40504 (2004)

Negative feedback exerted by PKA and cAMP phosphodiesterase on subsarcolemmal cAMP signals in intact cardiac myocytes. An in vivo study using adenovirus-mediated expression of CNG channels. F Rochais, G Vandecasteele, F Lefebvre, C Lugnier , H Lum, J-L Mazet, DMF Cooper and R Fischmeister J. Biol. Chem. 279, 52095-52105 (2004)

The cytosolic domains of Ca²⁺-sensitive adenylyl cyclases dictate their targeting to plasma membrane lipid rafts. AJ Crossthwaite, T Seebacher, N Masada, A Ciruela, K Dufraux, JE Schultz and DMF Cooper J. Biol. Chem. 280, 6380-6391 (2005)

Ca²⁺/calmodulin-dependent phosphodiesterase (PDE1): Current perspectives. TA Goraya and DMF Cooper Cellular Signalling 17, 789-797 (2005)

Localized Na+/H+ Exchanger 1 expression protects Ca²⁺-regulated adenylyl cyclases from changes in intracellular pH. D Willoughby, N Masada, A Ciruela, AJ Crossthwaite and DMF Cooper. J. Biol. Chem. 280, 30864-30872 (2005)

Mapping Protein Interfaces by Chemical Cross-Linking and FTICR Mass Spectrometry: Application to a Calmodulin / Adenylyl Cyclase 8 Peptide Complex. A Schmidt, S Kalkhof, C Ihling, D M F Cooper, and A Sinz. Eur. J. Mass Spectrom. 11, 525-534 (2005)

Cooper, DMF and Dessauer, C. Adenylyl cyclase type 6. AfCS-Nature Molecule Pages (2005). (doi:10.1038/mp.a000138.01)

A direct interaction between the N-terminus of adenylyl cyclase AC 8 and the catalytic subunit of protein phosphatase 2A. AJ Crossthwaite, A Ciruela, TF Rayner and DMF Cooper. Mol. Pharmacol. 69, 608-617 (2006)

Ca2+ stimulation of adenylyl cyclase generates dynamic oscillations in cyclic AMP. D Willoughby and DMF Cooper. J Cell Science 119, 828-836 (2006)

A specific pattern of phosphodiesterases controls the cAMP signals generated by different Gs-coupled receptors in adult rat ventricular myocytes. F Rochais, A Abi-Gerges, K Horner, F Lefebvre, DMF Cooper, M Conti, R Fischmeister and G Vandecasteele. Circulation Research 98, 1081-1088 (2006)

Cyclic guanosine monophosphate compartmentation in rat cardiac myocytes. LRV Castro, I Verde, DMF Cooper and R Fischmeister. Circulation 113, 2221-2228 (2006)

An anchored PKA and PDE4 complex regulates subplasmalemmal cAMP dynamics. D Willoughby, W Wong, J Schaack, JD Scott and DMF Cooper. EMBO J 25, 2051-2061 (2006)

The role of calmodulin recruitment in Ca2+-stimulation of adenylyl cyclase type 8. RE Simpson, A Ciruela and DMF Cooper. J Biol Chem 281, 17379-17389 (2006)

Capacitative and OAG-activated Ca2+ entry distinguished using adenylyl cyclase type 8. ACL Martin and DMF Cooper Mol. Pharmacol 70, 769-777 (2006)

Higher order organization and regulation of adenylyl cyclases. DMF Cooper and AJ Crossthwaite Trends in Pharmacological Sciences 27, 426-431(2006)

Organization and Ca²⁺ regulation of adenylyl cyclases in cAMP microdomains. Willoughby, D and Cooper, DMF. Physiological Reviews 87, 965-1010 (2007)

Dynamic regulation, desensitization and crosstalk in discrete sub-cellular microdomains during β₂‑adrenoceptor and prostanoid receptor cAMP signalling. Willoughby, D., Baillie, G., Lynch, MJ., Ciruela, A., Houslay, MD and Cooper, DMF. J. Biol. Chem. 282 34235-34249 (2007)

Live cell imaging of cAMP dynamics. Review. D Willoughby & DMF Cooper Nature Methods 5, 29 -36 (2008)

Kinetic properties of Ca²⁺/Calmodulin-dependent phosphodiesterase isoforms dictate intracellular cAMP dynamics in response to elevation of cytosolic Ca²⁺. Goraya, TA, Masada, N., Ciruela, A., Willoughby, D., Clynes, MJ and Cooper, DMF Cellular Signalling 20, 359-374 (2008)

Distinct mechanisms of regulation by Ca²⁺/calmodulin of type 1 and 8 adenylyl cyclases support their different physiological roles. N Masada, A Ciruela, DA MacDougall, and DMF Cooper J. Biol. Chem. 284, 4451-4463 (2009)

Insights into the residence in lipid rafts of adenylyl cyclase AC8 and its regulation by capacitative Calcium entry. M Pagano, MA. Clynes, N Masada, A Ciruela, LJ Ayling, S Wachten and DMF Cooper. Am. J. Phys. 296, C607-C619 (2009)

Capacitative Ca²⁺ entry via Orai1 and STIM1 regulates adenylyl cyclase type 8. ACL Martin, D Willoughby, A Ciruela, LJ Ayling M Pagano, S Wachten, A Tengholm and DMF Cooper. Mol. Pharmacol. 75, 830-842 (2009)

Structural basis for inhibition of mammalian adenylyl cyclase by Calcium. Mou, TC, Masada, N, Cooper, DMF and Sprang, SS. Biochemistry (in press)

Separate Elements within a Single IQ-like Motif in Adenylyl Cyclase Type 8 Impart Ca²⁺/CaM Binding and Autoinhibition. DA. MacDougall, S Wachten, A Ciruela, A Sinz, and DMF Cooper. J. Biol. Chem. (in press)

© 2009 University of Cambridge
Department of Pharmacology

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