A novel recombinant hyperaffinity inositol 1,4,5-trisphosphate (IP(3)) absorbent traps IP(3), resulting in specific inhibition of IP(3)-mediated calcium signaling.
We have developed a novel recombinant hyperaffinity inositol 1,4,5-trisphosphate (IP(3)) absorbent, called the "IP(3) sponge," which we constructed on the basis of the ligand-binding site of the mouse type 1 IP(3) receptor (IP(3)R1). The IP(3) sponge exhibited approximately 1000-fold higher affinity for IP(3) than the parental IP(3)R1 and specifically competed with the endogenous IP(3)R for binding to IP(3). Trapping IP(3) with the IP(3) sponge inhibited IP(3)-induced Ca(2+) release (IICR) from cerebellar microsomes in a dose-dependent manner. The IP(3) sponge expressed in HEK293 cells also inhibited IICR in response to stimulation with carbachol or ATP. Its inhibitory effects were dependent upon the level of its expression over the increased IP(3) contents. Moreover, the IP(3) sponge significantly reduced the carbachol-induced phosphorylation of cAMP-response element-binding protein in HEK293 cells, indicating that the activation of cAMP-response element-binding protein by Ca(2+)-dependent phosphorylation may be partly attributable to IICR.
Uchiyama
Tsuyoshi
T
Department of Basic Medical Sciences, Division of Molecular Neurobiology, the Institute of Medical Science, the University of Tokyo, Tokyo 108-8639, Japan.
Yoshikawa
Fumio
F
Hishida
Akira
A
Furuichi
Teiichi
T
Mikoshiba
Katsuhiko
K
D000255:Adenosine Triphosphate / Q000378:metabolism; D001671:Biochemistry / Q000379:methods*; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002217:Carbachol / Q000378:metabolism; D002460:Cell Line; D002531:Cerebellum / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D007700:Kinetics; D008861:Microsomes / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence; D013752:Tetracycline / Q000494:pharmacology; D013997:Time Factors
D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D015544:Inositol 1,4,5-Trisphosphate; D000255:Adenosine Triphosphate; D002217:Carbachol; D000242:Cyclic AMP; D005982:Glutathione Transferase; D013752:Tetracycline; D002118:Calcium