title: Adenine-nucleotide binding sites on the inositol 1,4,5-trisphosphate receptor bind caffeine, but not adenophostin A or cyclic ADP-ribose.
abstract: Binding of ATP to the inositol 1,4,5-trisphosphate receptor (IP3R) results in a more pronounced Ca2+ release in the presence of inositol 1,4,5-trisphosphate (IP3). We have expressed the cDNAs encoding two putative adenine-nucleotide binding sites of the neuronal form of IP3R-1 as glutathione S-transferase (GST)-fusion proteins in bacteria. Specific [alpha-32P]ATP binding was observed for the two GST-fusion proteins, representing aa 1710-1850 and aa 1944-2040 of IP3R-1. The ATP-binding sites in both fusion proteins had the same nucleotide specificity as found for the intact IP3R (ATP > ADP > AMP > GTP). Smaller GST-fusion proteins (aa 1745-1792 and aa 2005-2023) displayed a much weaker ATP-binding activity. CoA, which also potentiated IP3-induced Ca2+ release in A7r5 cells, interacted with the ATP-binding sites on the fusion proteins. Such interaction was not observed for 1,N6-etheno CoA and 3'-dephospho-CoA, which are much less effective in potentiating IP3-induced Ca2+ release. Since the adenine-containing compounds adenophostin A, caffeine and cyclic ADP-ribose modulate IP3-induced Ca2+ release, a possible effect of these compounds on the ATP-binding sites was examined. ATP stimulated adenophostin A- and IP3-induced Ca2+ release in A7r5 cells with an EC50 of respectively 21 and 20 microM. Also the threshold concentration of ATP for stimulating the release was similar for the two agonists. Adenophostin A (100 microM) and cyclic ADP-ribose (100 microM) were ineffective in displacing [alpha-32P]ATP from the binding sites of both GST-fusion proteins. Caffeine (50 mM), however, inhibited [alpha-32P]ATP binding to both fusion proteins by more than 50%. These data provide evidence for a direct interaction of caffeine but not of adenophostin A or cyclic ADP-ribose with the adenine-nucleotide binding sites of the IP3R.
mesh_terms: D000225:Adenine / Q000378:metabolism*; D000241:Adenosine / Q000031:analogs & derivatives* / Q000378:metabolism; D000246:Adenosine Diphosphate Ribose / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002110:Caffeine / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism*; D036563:Cyclic ADP-Ribose; D004305:Dose-Response Relationship, Drug; D020128:Inhibitory Concentration 50; D053496:Inositol 1,4,5-Trisphosphate Receptors; D051379:Mice; D008957:Models, Genetic; D011485:Protein Binding; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D013997:Time Factors