Pharmacological characterization of the putative cADP-ribose receptor

cADP-ribose (cADPR), a naturally occurring metabolite of NAD(+), has been s hown to be an important regulator of intracellular Ca2+ release. Considerab le evidence suggests that cADPR is the endogenous modulator of the ryanodin e receptor (RyR), which mediates Ca2+-induced Ca2+ release (CICR). Indeed, cADPR-mediated Ca2+ release is subject to functional regulation by other mo dulators of CICR, including Ca2+, caffeine and calmodulin. However, the und erlying basis behind the effect of such agents on cADPR activity (in partic ular whether they regulate cADPR binding), as well as the precise nature of the cADPR receptor remains unclear. In the present study, use of P-32-radi olabelled cADPR has enabled a detailed pharmacological characterization of cADPR-binding sites in sea urchin egg homogenates. We report that cADPR bin ds specifically to a single class of high affinity receptor. Retainment of binding to membranes after a high-salt wash suggests the involvement of eit her an integral membrane protein (possibly the RyR itself) or a peripheral protein tightly associated to the membrane. Insensitivity of [P-32]cADPR bi nding to either FK506 or rapamycin suggests that this does not concern the FK506-binding protein. Significantly, binding is highly robust, being relat ively insensitive to both endogenous and pharmacological modulators of RyR- mediated CICR. In turn, this suggests that such agents modulate cADPR-media ted Ca2+ release primarily by tuning the 'gain' of the CICR system, upon wh ich cADPR acts, rather than influencing the interaction of cADPR with its t arget receptor. The exception to this is calmodulin, for which our results indicate an additional role in facilitating cADPR binding.