Determination of specificity of a high-affinity inositol 1,3,4,5-tetrakisphosphate binding site at a 42 kDa receptor protein, p42IP4: comparison of affinities of all inositoltris-,-tetrakis-, and -pentakisphosphate regioisomers.
The specificity of the binding site of p42IP4, a high-affinity 42 kDa Ins (1,3,4,5)P4 receptor protein identified by photoaffinity labelling (Reiser et al., Biochem. J. 280, 533, 1991) was analyzed by determining the affinities for all possible inositoltris-, -tetrakis-, and -pentakisphosphate regioisomers. We tested the purified receptor protein displaying a Kd of 2.2 nM for Ins (1,3,4,5)P4 which was unequalled by any of the other inositoltetrakis- and -trisphosphate regioisomers. The affinities of inositoltetrakisphosphates were 25 to 150 times lower, with a substitution at C-2 having the largest effect in reducing the affinity. The inositoltrisphosphate isomers were three orders of magnitude less potent than Ins (1,3,4,5)P4, apart from D/L-Ins (3,4,5)P3. The pentakisphosphate Ins(1,3,4,5,6)P5 had an affinity for the solubilized and purified receptor comparable to that of D-Ins (1,3,4,5)P4. This lack of discrimination was unique for the solubilized receptor, since it was not observed with the membrane-associated receptor protein. Most importantly, D-Ins (1,3,4,5)P4 and D-Gro PtdIns (3,4,5)P3 had identical affinities with the 42 kDa protein. Thus, this protein p42IP4 selectively recognizes two potential second messengers.
Stricker
R
R
Institut für Neurobiochemie, Medizinische Fakultät der Otto-von-Guericke-Universität Magdeburg, Germany.
Chang
Y T
YT
Chung
S K
SK
Reiser
G
G
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D015153:Blotting, Western; D002531:Cerebellum / Q000378:metabolism*; D007295:Inositol Phosphates / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D018160:Receptors, Cytoplasmic and Nuclear / Q000302:isolation & purification / Q000378:metabolism*; D013237:Stereoisomerism; D013379:Substrate Specificity; D013552:Swine
D007295:Inositol Phosphates; D018160:Receptors, Cytoplasmic and Nuclear; C061107:inositol-1,3,4,5-tetrakisphosphate receptor; C081655:inositol-1,3,4,5-tetrakisphosphate