title: Pharmacological characterization of the putative cADP-ribose receptor.
abstract: cADP-ribose (cADPR), a naturally occurring metabolite of NAD(+), has been shown to be an important regulator of intracellular Ca(2+) release. Considerable evidence suggests that cADPR is the endogenous modulator of the ryanodine receptor (RyR), which mediates Ca(2+)-induced Ca(2+) release (CICR). Indeed, cADPR-mediated Ca(2+) release is subject to functional regulation by other modulators of CICR, including Ca(2+), caffeine and calmodulin. However, the underlying basis behind the effect of such agents on cADPR activity (in particular whether they regulate cADPR binding), as well as the precise nature of the cADPR receptor remains unclear. In the present study, use of (32)P-radiolabelled cADPR has enabled a detailed pharmacological characterization of cADPR-binding sites in sea urchin egg homogenates. We report that cADPR binds specifically to a single class of high affinity receptor. Retainment of binding to membranes after a high-salt wash suggests the involvement of either an integral membrane protein (possibly the RyR itself) or a peripheral protein tightly associated to the membrane. Insensitivity of [(32)P]cADPR binding to either FK506 or rapamycin suggests that this does not concern the FK506-binding protein. Significantly, binding is highly robust, being relatively insensitive to both endogenous and pharmacological modulators of RyR-mediated CICR. In turn, this suggests that such agents modulate cADPR-mediated Ca(2+) release primarily by tuning the 'gain' of the CICR system, upon which cADPR acts, rather than influencing the interaction of cADPR with its target receptor. The exception to this is calmodulin, for which our results indicate an additional role in facilitating cADPR binding.
authors.affiliation: Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, UK. justyn.thomas@pharmacology.oxford.ac.uk
authors.forename: J M
authors.initials: JM
authors.lastname: Thomas
authors.forename: R
authors.initials: R
authors.lastname: Masgrau
authors.forename: G C
authors.initials: GC
authors.lastname: Churchill
authors.forename: A
authors.initials: A
authors.lastname: Galione
mesh_terms: D000246:Adenosine Diphosphate Ribose / Q000031:analogs & derivatives* / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D020013:Calcium Signaling; D002147:Calmodulin / Q000378:metabolism; D002413:Cations, Divalent / Q000494:pharmacology; D036563:Cyclic ADP-Ribose; D005260:Female; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D010063:Ovum / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D011956:Receptors, Cell Surface / Q000378:metabolism*; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism; D012617:Sea Urchins; D020123:Sirolimus / Q000494:pharmacology; D022021:Tacrolimus Binding Proteins / Q000037:antagonists & inhibitors
chemical_list: D002147:Calmodulin; D002413:Cations, Divalent; D010761:Phosphorus Radioisotopes; D011956:Receptors, Cell Surface; D019837:Ryanodine Receptor Calcium Release Channel; C067025:cyclic ADP-ribose receptor; D036563:Cyclic ADP-Ribose; D000246:Adenosine Diphosphate Ribose; D022021:Tacrolimus Binding Proteins; D020123:Sirolimus