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Stimulation of inositol 1,4,5-trisphosphate (IP3) receptor subtypes by adenophostin A and its analogues.

Saleem H, Tovey SC, Riley AM, Potter BV, Taylor CW - PLoS ONE (2013)

Bottom Line: The two complementary contacts between AdA and the α-domain (cation-π interaction and 3″-phosphate) allow activation of IP3R by an analogue of AdA (3″-dephospho-AdA) that lacks a phosphate group equivalent to the essential 5-phosphate of IP3.These data provide the first structure-activity analyses of key AdA analogues using homogenous populations of all mammalian IP3R subtypes.They demonstrate that differences in the Ca(2+) signals evoked by AdA analogues are unlikely to be due to selective regulation of IP3R subtypes.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Cambridge, United Kingdom.

ABSTRACT
Inositol 1,4,5-trisphosphate receptors (IP3R) are intracellular Ca(2+) channels. Most animal cells express mixtures of the three IP3R subtypes encoded by vertebrate genomes. Adenophostin A (AdA) is the most potent naturally occurring agonist of IP3R and it shares with IP3 the essential features of all IP3R agonists, namely structures equivalent to the 4,5-bisphosphate and 6-hydroxyl of IP3. The two essential phosphate groups contribute to closure of the clam-like IP3-binding core (IBC), and thereby IP3R activation, by binding to each of its sides (the α- and β-domains). Regulation of the three subtypes of IP3R by AdA and its analogues has not been examined in cells expressing defined homogenous populations of IP3R. We measured Ca(2+) release evoked by synthetic adenophostin A (AdA) and its analogues in permeabilized DT40 cells devoid of native IP3R and stably expressing single subtypes of mammalian IP3R. The determinants of high-affinity binding of AdA and its analogues were indistinguishable for each IP3R subtype. The results are consistent with a cation-π interaction between the adenine of AdA and a conserved arginine within the IBC α-domain contributing to closure of the IBC. The two complementary contacts between AdA and the α-domain (cation-π interaction and 3″-phosphate) allow activation of IP3R by an analogue of AdA (3″-dephospho-AdA) that lacks a phosphate group equivalent to the essential 5-phosphate of IP3. These data provide the first structure-activity analyses of key AdA analogues using homogenous populations of all mammalian IP3R subtypes. They demonstrate that differences in the Ca(2+) signals evoked by AdA analogues are unlikely to be due to selective regulation of IP3R subtypes.

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The 2′-phosphate of AdA is not the primary cause of its increased potency.(A) Effects of 2′-dephospho AdA on Ca2+ release via each IP3R subtype. (B) The same analogue compared with AdA. Results are means ± S.E.M. from 3–4 independent experiments.
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pone-0058027-g005: The 2′-phosphate of AdA is not the primary cause of its increased potency.(A) Effects of 2′-dephospho AdA on Ca2+ release via each IP3R subtype. (B) The same analogue compared with AdA. Results are means ± S.E.M. from 3–4 independent experiments.

Mentions: It has been suggested that the 2′-phosphate of AdA interacts with the IBC in a manner that allows it to behave as a super-optimal mimic of the 1-phosphate of IP3[44], [45]. However, our recent study combining structure-activity analyses with mutagenesis of the binding site suggest that the 1-phosphate of IP3 is more important for binding than is the 2′-phosphate of AdA [12]. Removal of the 1-phosphate from IP3 (to give (4,5)IP2) caused its potency and affinity for IP3R1 to decrease by ∼100-fold [12], whereas removal of the 2′-phosphate from AdA (2′-dephospho AdA) causes a decrease in potency of ∼17-fold in IP3R1 (Figure 5) and ∼40-fold decreases in potency were obtained with 2′-dephospho AdA and IP3R2 and IP3R3 (Figure 5, Table 1 and 2). These results establish that for all three IP3R subtypes, the enhanced affinity of AdA is not due to its 2′-phosphate interacting more effectively than the 1-phosphate of IP3 with the IBC.


Stimulation of inositol 1,4,5-trisphosphate (IP3) receptor subtypes by adenophostin A and its analogues.

Saleem H, Tovey SC, Riley AM, Potter BV, Taylor CW - PLoS ONE (2013)

The 2′-phosphate of AdA is not the primary cause of its increased potency.(A) Effects of 2′-dephospho AdA on Ca2+ release via each IP3R subtype. (B) The same analogue compared with AdA. Results are means ± S.E.M. from 3–4 independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585173&req=5

pone-0058027-g005: The 2′-phosphate of AdA is not the primary cause of its increased potency.(A) Effects of 2′-dephospho AdA on Ca2+ release via each IP3R subtype. (B) The same analogue compared with AdA. Results are means ± S.E.M. from 3–4 independent experiments.
Mentions: It has been suggested that the 2′-phosphate of AdA interacts with the IBC in a manner that allows it to behave as a super-optimal mimic of the 1-phosphate of IP3[44], [45]. However, our recent study combining structure-activity analyses with mutagenesis of the binding site suggest that the 1-phosphate of IP3 is more important for binding than is the 2′-phosphate of AdA [12]. Removal of the 1-phosphate from IP3 (to give (4,5)IP2) caused its potency and affinity for IP3R1 to decrease by ∼100-fold [12], whereas removal of the 2′-phosphate from AdA (2′-dephospho AdA) causes a decrease in potency of ∼17-fold in IP3R1 (Figure 5) and ∼40-fold decreases in potency were obtained with 2′-dephospho AdA and IP3R2 and IP3R3 (Figure 5, Table 1 and 2). These results establish that for all three IP3R subtypes, the enhanced affinity of AdA is not due to its 2′-phosphate interacting more effectively than the 1-phosphate of IP3 with the IBC.

Bottom Line: The two complementary contacts between AdA and the α-domain (cation-π interaction and 3″-phosphate) allow activation of IP3R by an analogue of AdA (3″-dephospho-AdA) that lacks a phosphate group equivalent to the essential 5-phosphate of IP3.These data provide the first structure-activity analyses of key AdA analogues using homogenous populations of all mammalian IP3R subtypes.They demonstrate that differences in the Ca(2+) signals evoked by AdA analogues are unlikely to be due to selective regulation of IP3R subtypes.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Cambridge, United Kingdom.

ABSTRACT
Inositol 1,4,5-trisphosphate receptors (IP3R) are intracellular Ca(2+) channels. Most animal cells express mixtures of the three IP3R subtypes encoded by vertebrate genomes. Adenophostin A (AdA) is the most potent naturally occurring agonist of IP3R and it shares with IP3 the essential features of all IP3R agonists, namely structures equivalent to the 4,5-bisphosphate and 6-hydroxyl of IP3. The two essential phosphate groups contribute to closure of the clam-like IP3-binding core (IBC), and thereby IP3R activation, by binding to each of its sides (the α- and β-domains). Regulation of the three subtypes of IP3R by AdA and its analogues has not been examined in cells expressing defined homogenous populations of IP3R. We measured Ca(2+) release evoked by synthetic adenophostin A (AdA) and its analogues in permeabilized DT40 cells devoid of native IP3R and stably expressing single subtypes of mammalian IP3R. The determinants of high-affinity binding of AdA and its analogues were indistinguishable for each IP3R subtype. The results are consistent with a cation-π interaction between the adenine of AdA and a conserved arginine within the IBC α-domain contributing to closure of the IBC. The two complementary contacts between AdA and the α-domain (cation-π interaction and 3″-phosphate) allow activation of IP3R by an analogue of AdA (3″-dephospho-AdA) that lacks a phosphate group equivalent to the essential 5-phosphate of IP3. These data provide the first structure-activity analyses of key AdA analogues using homogenous populations of all mammalian IP3R subtypes. They demonstrate that differences in the Ca(2+) signals evoked by AdA analogues are unlikely to be due to selective regulation of IP3R subtypes.

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