Identification of cyclic ADP-ribose-binding proteins by photoaffinity labeling.
We have synthesized 8-azido-cyclic ADP-ribose (8N3-cADPR) and [32P]8-azido-cyclic ADP-ribose ([32P]8N3-cADPR) in order to characterize cyclic ADP-ribose-(cADPR) binding sites in sea urchin egg homogenates. 8N3-cADPR was an antagonist of cADPR since it did not induce Ca2+ release from egg microsomes but did inhibit the ability of cADPR to do so. The effect of 8N3-cADPR was reversible and could be overcome by high concentrations of cADPR, suggesting that both were acting on the same site. This was supported by the fact that 8N3-cADPR effectively competed for [32P]cADPR binding to microsomes. Reciprocally, binding of [32P]8N3-cADPR could also be selectively displaced by cADPR and 8N3-cADPR, but not by ADP-ribose. These results indicate that 8N3-cADPR binds specifically to the cADPR-binding sites and inhibits cADPR from releasing Ca2+. Photolysis of microsomes preincubated with [32P]8N3-cADPR resulted in specific labeling of proteins of 140 and 100 kDa, which could be prevented by 8N3-cADPR or nanomolar concentrations of cADPR, but not by micromolar concentrations of ADP-ribose, AMP, ADP, ATP, cyclic AMP or inositol 1,4,5-trisphosphate. Caffeine, an agonist of Ca(2+)-induced Ca2+ release, preferentially inhibited the labeling of the 100 kDa as compared to the 140-kDa protein. These results suggest that cADPR may not interact directly with the ryanodine receptor, but may instead, exert its effect through intermediate proteins.
Walseth
T F
TF
Department of Pharmacology, University of Minnesota, Minneapolis 55455.
Aarhus
R
R
Kerr
J A
JA
Lee
H C
HC
D000246:Adenosine Diphosphate Ribose / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D000345:Affinity Labels; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D036563:Cyclic ADP-Ribose / Q000037:antagonists & inhibitors; D010063:Ovum; D010777:Photochemistry; D011506:Proteins / Q000032:analysis*; D012617:Sea Urchins
D000345:Affinity Labels; D011506:Proteins; D036563:Cyclic ADP-Ribose; C084796:8-azido-cyclic adenosine diphosphate ribose; D000246:Adenosine Diphosphate Ribose; D002118:Calcium