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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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Potentiation of IP3-evoked Ca2+ release by cAMP in permeabilized cells. (A) Ca2+ release from permeabilized HEK-PR1 cells evoked by IP3 alone, after treatment with the catalytic subunit of PKA (100 units per milliliter for 10 min) or 30 mM cAMP. Results are shown as a percentage of the ionomycin-releasable Ca2+ store. (B) Concentration-dependent effect of cAMP on the EC50 for IP3-evoked Ca2+ release from permeabilized HEK-PR1 cells. (C) Concentration-dependent stimulation of Ca2+ release by IP3 alone and with 3 mM 8-Br-cAMP. (D) Concentration-dependent effect of 8-Br-cAMP on the EC50 for IP3-evoked Ca2+ release. These results allow direct comparison with the effects of 8-Br-cAMP in intact cells (Fig. 2 C). The latter measurements were made after a 20-min preincubation with 8-Br-cAMP, and we have shown that although shorter preincubations cause lesser potentiation of Ca2+ signals, by 20 min, the response has reached its maximum. We conclude that 8-Br-cAMP equilibrates across the plasma membrane within 20 min. Results are means ± SEM, n ≥ 3.
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fig4: Potentiation of IP3-evoked Ca2+ release by cAMP in permeabilized cells. (A) Ca2+ release from permeabilized HEK-PR1 cells evoked by IP3 alone, after treatment with the catalytic subunit of PKA (100 units per milliliter for 10 min) or 30 mM cAMP. Results are shown as a percentage of the ionomycin-releasable Ca2+ store. (B) Concentration-dependent effect of cAMP on the EC50 for IP3-evoked Ca2+ release from permeabilized HEK-PR1 cells. (C) Concentration-dependent stimulation of Ca2+ release by IP3 alone and with 3 mM 8-Br-cAMP. (D) Concentration-dependent effect of 8-Br-cAMP on the EC50 for IP3-evoked Ca2+ release. These results allow direct comparison with the effects of 8-Br-cAMP in intact cells (Fig. 2 C). The latter measurements were made after a 20-min preincubation with 8-Br-cAMP, and we have shown that although shorter preincubations cause lesser potentiation of Ca2+ signals, by 20 min, the response has reached its maximum. We conclude that 8-Br-cAMP equilibrates across the plasma membrane within 20 min. Results are means ± SEM, n ≥ 3.

Mentions: All three subtypes of IP3R can be phosphorylated by PKA, although at different sites and with different consequences (Bruce et al., 2003; Soulsby and Wojcikiewicz, 2007). IP3R2 is the major (46%) IP3R subtype in HEK cells (Table I), and in other cells expressing mainly IP3R2, PKA potentiates IP3-evoked Ca2+ release (Burgess et al., 1991; Bruce et al., 2003). In HEK-PR1 cells, PTH caused modest phosphorylation of IP3R (Figs. 3 A and S2 B, available at http://www.jcb.org/cgi/content/full/jcb.200803172/DC1), and PKA very slightly (though not to a statistically significant degree) increased the sensitivity of the intracellular Ca2+ stores to IP3 (see Fig. 4 A).


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)

Potentiation of IP3-evoked Ca2+ release by cAMP in permeabilized cells. (A) Ca2+ release from permeabilized HEK-PR1 cells evoked by IP3 alone, after treatment with the catalytic subunit of PKA (100 units per milliliter for 10 min) or 30 mM cAMP. Results are shown as a percentage of the ionomycin-releasable Ca2+ store. (B) Concentration-dependent effect of cAMP on the EC50 for IP3-evoked Ca2+ release from permeabilized HEK-PR1 cells. (C) Concentration-dependent stimulation of Ca2+ release by IP3 alone and with 3 mM 8-Br-cAMP. (D) Concentration-dependent effect of 8-Br-cAMP on the EC50 for IP3-evoked Ca2+ release. These results allow direct comparison with the effects of 8-Br-cAMP in intact cells (Fig. 2 C). The latter measurements were made after a 20-min preincubation with 8-Br-cAMP, and we have shown that although shorter preincubations cause lesser potentiation of Ca2+ signals, by 20 min, the response has reached its maximum. We conclude that 8-Br-cAMP equilibrates across the plasma membrane within 20 min. Results are means ± SEM, n ≥ 3.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2568025&req=5

fig4: Potentiation of IP3-evoked Ca2+ release by cAMP in permeabilized cells. (A) Ca2+ release from permeabilized HEK-PR1 cells evoked by IP3 alone, after treatment with the catalytic subunit of PKA (100 units per milliliter for 10 min) or 30 mM cAMP. Results are shown as a percentage of the ionomycin-releasable Ca2+ store. (B) Concentration-dependent effect of cAMP on the EC50 for IP3-evoked Ca2+ release from permeabilized HEK-PR1 cells. (C) Concentration-dependent stimulation of Ca2+ release by IP3 alone and with 3 mM 8-Br-cAMP. (D) Concentration-dependent effect of 8-Br-cAMP on the EC50 for IP3-evoked Ca2+ release. These results allow direct comparison with the effects of 8-Br-cAMP in intact cells (Fig. 2 C). The latter measurements were made after a 20-min preincubation with 8-Br-cAMP, and we have shown that although shorter preincubations cause lesser potentiation of Ca2+ signals, by 20 min, the response has reached its maximum. We conclude that 8-Br-cAMP equilibrates across the plasma membrane within 20 min. Results are means ± SEM, n ≥ 3.
Mentions: All three subtypes of IP3R can be phosphorylated by PKA, although at different sites and with different consequences (Bruce et al., 2003; Soulsby and Wojcikiewicz, 2007). IP3R2 is the major (46%) IP3R subtype in HEK cells (Table I), and in other cells expressing mainly IP3R2, PKA potentiates IP3-evoked Ca2+ release (Burgess et al., 1991; Bruce et al., 2003). In HEK-PR1 cells, PTH caused modest phosphorylation of IP3R (Figs. 3 A and S2 B, available at http://www.jcb.org/cgi/content/full/jcb.200803172/DC1), and PKA very slightly (though not to a statistically significant degree) increased the sensitivity of the intracellular Ca2+ stores to IP3 (see Fig. 4 A).

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