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Selective coupling of type 6 adenylyl cyclase with type 2 IP3 receptors mediates direct sensitization of IP3 receptors by cAMP.

Tovey SC, Dedos SG, Taylor EJ, Church JE, Taylor CW - J. Cell Biol. (2008)

Bottom Line: Human embryonic kidney cells express several isoforms of adenylyl cyclase (AC) and IP(3)R, but IP(3)R2 and AC6 are specifically associated, and inhibition of AC6 or IP(3)R2 expression by small interfering RNA selectively attenuates potentiation of Ca(2+) signals by PTH.We define two modes of cAMP signaling: binary, where cAMP passes directly from AC6 to IP(3)R2; and analogue, where local gradients of cAMP concentration regulate cAMP effectors more remote from AC.Binary signaling requires localized delivery of cAMP, whereas analogue signaling is more dependent on localized cAMP degradation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Univesrsity of Cambridge, Cambridge, England, UK.

ABSTRACT
Interactions between cyclic adenosine monophosphate (cAMP) and Ca(2+) are widespread, and for both intracellular messengers, their spatial organization is important. Parathyroid hormone (PTH) stimulates formation of cAMP and sensitizes inositol 1,4,5-trisphosphate receptors (IP(3)R) to IP(3). We show that PTH communicates with IP(3)R via "cAMP junctions" that allow local delivery of a supramaximal concentration of cAMP to IP(3)R, directly increasing their sensitivity to IP(3). These junctions are robust binary switches that are digitally recruited by increasing concentrations of PTH. Human embryonic kidney cells express several isoforms of adenylyl cyclase (AC) and IP(3)R, but IP(3)R2 and AC6 are specifically associated, and inhibition of AC6 or IP(3)R2 expression by small interfering RNA selectively attenuates potentiation of Ca(2+) signals by PTH. We define two modes of cAMP signaling: binary, where cAMP passes directly from AC6 to IP(3)R2; and analogue, where local gradients of cAMP concentration regulate cAMP effectors more remote from AC. Binary signaling requires localized delivery of cAMP, whereas analogue signaling is more dependent on localized cAMP degradation.

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IP3R2 and AC6 are specifically required for PTH to potentiate IP3-evoked Ca2+ release. (A) WB (representative of five experiments) shows the effects of siRNA (boxed in red) on the expression of IP3R1 (260 kD), IP3R2 (260 kD), and β-adaptin (100 kD; protein loadings are given in μg; molecular weight standards are indicated in kD). Each block shows IP3R above and β-adaptin below. (B and C) Effects of reducing IP3R1 or IP3R2 expression on CCh-evoked Ca2+ signals (B) and the potentiation of CCh (1 mM)-evoked Ca2+ signals by PTH (C). The latter were measured as in Fig. 1 A. Results are means ± SEM of triplicate determinations from a single experiment. Insets show the maximal responses (means ± SEM) from four to five independent siRNA treatments. (D) The Ca2+ release evoked by CCh alone requires IP3R1, whereas the Ca2+ release evoked by CCh acting in concert with cAMP specifically requires IP3R2. (E and F) Effects of reducing AC3 or AC6 expression on PTH-evoked cAMP formation (E) and on the potentiation of CCh-evoked Ca2+ signals by PTH (F). Results are means ± SEM from at least five independent siRNA treatments; the main panel in F shows means ± SEM of triplicate determinations from a single experiment. (G) Summary showing that uniform inhibition (by ∼60–70%) of AC by SQ/DDA massively inhibits PTH-evoked cAMP formation, whereas a comparable (∼60–70%) loss of AC6 (using siRNA) has no detectable effect on cAMP formation but attenuates PTH-mediated Ca2+ signaling. Results indicate means ± SEM, n ≥ 3.
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fig9: IP3R2 and AC6 are specifically required for PTH to potentiate IP3-evoked Ca2+ release. (A) WB (representative of five experiments) shows the effects of siRNA (boxed in red) on the expression of IP3R1 (260 kD), IP3R2 (260 kD), and β-adaptin (100 kD; protein loadings are given in μg; molecular weight standards are indicated in kD). Each block shows IP3R above and β-adaptin below. (B and C) Effects of reducing IP3R1 or IP3R2 expression on CCh-evoked Ca2+ signals (B) and the potentiation of CCh (1 mM)-evoked Ca2+ signals by PTH (C). The latter were measured as in Fig. 1 A. Results are means ± SEM of triplicate determinations from a single experiment. Insets show the maximal responses (means ± SEM) from four to five independent siRNA treatments. (D) The Ca2+ release evoked by CCh alone requires IP3R1, whereas the Ca2+ release evoked by CCh acting in concert with cAMP specifically requires IP3R2. (E and F) Effects of reducing AC3 or AC6 expression on PTH-evoked cAMP formation (E) and on the potentiation of CCh-evoked Ca2+ signals by PTH (F). Results are means ± SEM from at least five independent siRNA treatments; the main panel in F shows means ± SEM of triplicate determinations from a single experiment. (G) Summary showing that uniform inhibition (by ∼60–70%) of AC by SQ/DDA massively inhibits PTH-evoked cAMP formation, whereas a comparable (∼60–70%) loss of AC6 (using siRNA) has no detectable effect on cAMP formation but attenuates PTH-mediated Ca2+ signaling. Results indicate means ± SEM, n ≥ 3.

Mentions: We used siRNA to inhibit selectively the expression of IP3R2, IP3R1, AC3, and AC6 (Fig. 9 A and Table III). Although siRNA also reduced IP3R3 expression, these cells failed to attach to the plates used for Ca2+ and cAMP assays, preventing further analysis. Loss of IP3R1 decreased the Ca2+ signals evoked by CCh without affecting their potentiation by PTH. In contrast, loss of IP3R2 had no affect on the Ca2+ signals evoked by CCh alone but attenuated their potentiation by PTH (Fig. 9, B and C). These results suggest that the muscarinic receptors that alone evoke Ca2+ release are distributed differently to those that release Ca2+ in synergy with cAMP. IP3R1 mediates the Ca2+ release evoked by CCh alone, whereas IP3R2 mediates the response to CCh with cAMP (Fig. 9 D). Loss of AC3, the major AC isoform, reduced PTH-evoked cAMP formation, though without affecting the ability of PTH to potentiate CCh-evoked Ca2+ signals. Conversely, loss of AC6 had no detectable effect on PTH-evoked cAMP formation but attenuated PTH-evoked Ca2+ signals (Fig. 9, E and F). The Ca2+ release evoked by CCh alone was unaffected by loss of AC3 or AC6 (Fig. S4, E and F). These results confirm that the specific association between IP3R2 and AC6 revealed by IP (Fig. 7) underlies the ability of PTH to potentiate CCh-evoked Ca2+ signals.


Selective coupling of type 6 adenylyl cyclase with type 2 IP3 receptors mediates direct sensitization of IP3 receptors by cAMP.

Tovey SC, Dedos SG, Taylor EJ, Church JE, Taylor CW - J. Cell Biol. (2008)

IP3R2 and AC6 are specifically required for PTH to potentiate IP3-evoked Ca2+ release. (A) WB (representative of five experiments) shows the effects of siRNA (boxed in red) on the expression of IP3R1 (260 kD), IP3R2 (260 kD), and β-adaptin (100 kD; protein loadings are given in μg; molecular weight standards are indicated in kD). Each block shows IP3R above and β-adaptin below. (B and C) Effects of reducing IP3R1 or IP3R2 expression on CCh-evoked Ca2+ signals (B) and the potentiation of CCh (1 mM)-evoked Ca2+ signals by PTH (C). The latter were measured as in Fig. 1 A. Results are means ± SEM of triplicate determinations from a single experiment. Insets show the maximal responses (means ± SEM) from four to five independent siRNA treatments. (D) The Ca2+ release evoked by CCh alone requires IP3R1, whereas the Ca2+ release evoked by CCh acting in concert with cAMP specifically requires IP3R2. (E and F) Effects of reducing AC3 or AC6 expression on PTH-evoked cAMP formation (E) and on the potentiation of CCh-evoked Ca2+ signals by PTH (F). Results are means ± SEM from at least five independent siRNA treatments; the main panel in F shows means ± SEM of triplicate determinations from a single experiment. (G) Summary showing that uniform inhibition (by ∼60–70%) of AC by SQ/DDA massively inhibits PTH-evoked cAMP formation, whereas a comparable (∼60–70%) loss of AC6 (using siRNA) has no detectable effect on cAMP formation but attenuates PTH-mediated Ca2+ signaling. Results indicate means ± SEM, n ≥ 3.
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fig9: IP3R2 and AC6 are specifically required for PTH to potentiate IP3-evoked Ca2+ release. (A) WB (representative of five experiments) shows the effects of siRNA (boxed in red) on the expression of IP3R1 (260 kD), IP3R2 (260 kD), and β-adaptin (100 kD; protein loadings are given in μg; molecular weight standards are indicated in kD). Each block shows IP3R above and β-adaptin below. (B and C) Effects of reducing IP3R1 or IP3R2 expression on CCh-evoked Ca2+ signals (B) and the potentiation of CCh (1 mM)-evoked Ca2+ signals by PTH (C). The latter were measured as in Fig. 1 A. Results are means ± SEM of triplicate determinations from a single experiment. Insets show the maximal responses (means ± SEM) from four to five independent siRNA treatments. (D) The Ca2+ release evoked by CCh alone requires IP3R1, whereas the Ca2+ release evoked by CCh acting in concert with cAMP specifically requires IP3R2. (E and F) Effects of reducing AC3 or AC6 expression on PTH-evoked cAMP formation (E) and on the potentiation of CCh-evoked Ca2+ signals by PTH (F). Results are means ± SEM from at least five independent siRNA treatments; the main panel in F shows means ± SEM of triplicate determinations from a single experiment. (G) Summary showing that uniform inhibition (by ∼60–70%) of AC by SQ/DDA massively inhibits PTH-evoked cAMP formation, whereas a comparable (∼60–70%) loss of AC6 (using siRNA) has no detectable effect on cAMP formation but attenuates PTH-mediated Ca2+ signaling. Results indicate means ± SEM, n ≥ 3.
Mentions: We used siRNA to inhibit selectively the expression of IP3R2, IP3R1, AC3, and AC6 (Fig. 9 A and Table III). Although siRNA also reduced IP3R3 expression, these cells failed to attach to the plates used for Ca2+ and cAMP assays, preventing further analysis. Loss of IP3R1 decreased the Ca2+ signals evoked by CCh without affecting their potentiation by PTH. In contrast, loss of IP3R2 had no affect on the Ca2+ signals evoked by CCh alone but attenuated their potentiation by PTH (Fig. 9, B and C). These results suggest that the muscarinic receptors that alone evoke Ca2+ release are distributed differently to those that release Ca2+ in synergy with cAMP. IP3R1 mediates the Ca2+ release evoked by CCh alone, whereas IP3R2 mediates the response to CCh with cAMP (Fig. 9 D). Loss of AC3, the major AC isoform, reduced PTH-evoked cAMP formation, though without affecting the ability of PTH to potentiate CCh-evoked Ca2+ signals. Conversely, loss of AC6 had no detectable effect on PTH-evoked cAMP formation but attenuated PTH-evoked Ca2+ signals (Fig. 9, E and F). The Ca2+ release evoked by CCh alone was unaffected by loss of AC3 or AC6 (Fig. S4, E and F). These results confirm that the specific association between IP3R2 and AC6 revealed by IP (Fig. 7) underlies the ability of PTH to potentiate CCh-evoked Ca2+ signals.

Bottom Line: Human embryonic kidney cells express several isoforms of adenylyl cyclase (AC) and IP(3)R, but IP(3)R2 and AC6 are specifically associated, and inhibition of AC6 or IP(3)R2 expression by small interfering RNA selectively attenuates potentiation of Ca(2+) signals by PTH.We define two modes of cAMP signaling: binary, where cAMP passes directly from AC6 to IP(3)R2; and analogue, where local gradients of cAMP concentration regulate cAMP effectors more remote from AC.Binary signaling requires localized delivery of cAMP, whereas analogue signaling is more dependent on localized cAMP degradation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Univesrsity of Cambridge, Cambridge, England, UK.

ABSTRACT
Interactions between cyclic adenosine monophosphate (cAMP) and Ca(2+) are widespread, and for both intracellular messengers, their spatial organization is important. Parathyroid hormone (PTH) stimulates formation of cAMP and sensitizes inositol 1,4,5-trisphosphate receptors (IP(3)R) to IP(3). We show that PTH communicates with IP(3)R via "cAMP junctions" that allow local delivery of a supramaximal concentration of cAMP to IP(3)R, directly increasing their sensitivity to IP(3). These junctions are robust binary switches that are digitally recruited by increasing concentrations of PTH. Human embryonic kidney cells express several isoforms of adenylyl cyclase (AC) and IP(3)R, but IP(3)R2 and AC6 are specifically associated, and inhibition of AC6 or IP(3)R2 expression by small interfering RNA selectively attenuates potentiation of Ca(2+) signals by PTH. We define two modes of cAMP signaling: binary, where cAMP passes directly from AC6 to IP(3)R2; and analogue, where local gradients of cAMP concentration regulate cAMP effectors more remote from AC. Binary signaling requires localized delivery of cAMP, whereas analogue signaling is more dependent on localized cAMP degradation.

Show MeSH
Related in: MedlinePlus