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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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Binary and analogue signaling by cAMP. (A) Comparison of the effects of cAMP on IP3-evoked Ca2+ release from permeabilized cells with estimates of the global cAMP concentration associated with agonist-mediated potentiation of Ca2+ signals in intact cells. A typical HEK cell has a radius of 8.4 μm and thus a total volume of ∼2.5 pl (Rich et al., 2000). Each well used for cAMP assays contained a mean of 4.8 × 105 cells, and our results show cAMP levels determined in 20% of the sample taken from each well, equivalent therefore to 96,000 cells and a total cytosolic volume of 0.24 μl. Maximal stimulation with each agonist would generate a uniformly distributed cytosolic cAMP concentration of up to ∼28 μM (PTH), ∼1 μM (FK), and ∼380 nM (isoproterenol). Means ± SEM, n = 3–6. (B) AC6 and IP3R2 form an intimate junction that allows cAMP to pass directly to IP3R2, enhancing its activity. Ca2+ released by the IP3R may then locally inhibit AC6. The interplay may allow local oscillations in both cAMP and Ca2+ concentration. (C) AC, IP3R, Gs, and a G-protein-coupled receptor (GPCR) are each drawn to approximate scale, with the ER and plasma membrane (PM) separated by 10–15 nm (Treves et al., 2004; Luik et al., 2006). (D) The IP3R–AC junction allows a low-affinity cAMP-sensor (IP3R2) to respond robustly to saturating concentrations of cAMP delivered directly to it by AC. Higher affinity cAMP sensors (e.g., PKA) can be more remote from AC and respond to local gradients of cAMP. Although the binary coding requires local delivery of cAMP, the analogue coding requires its local degradation. The two modes of cAMP signaling invite comparison with the evolution of excitation–contraction coupling in striated muscle, where coupling between the PM voltage-sensor (DHPR) and ryanodine receptor (RyR) in the SR of striated muscle probably evolved from chemical coupling mediated by Ca2+ (cardiac muscle) to conformational coupling (skeletal muscle; Di Biase and Franzini-Armstrong, 2005).
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fig10: Binary and analogue signaling by cAMP. (A) Comparison of the effects of cAMP on IP3-evoked Ca2+ release from permeabilized cells with estimates of the global cAMP concentration associated with agonist-mediated potentiation of Ca2+ signals in intact cells. A typical HEK cell has a radius of 8.4 μm and thus a total volume of ∼2.5 pl (Rich et al., 2000). Each well used for cAMP assays contained a mean of 4.8 × 105 cells, and our results show cAMP levels determined in 20% of the sample taken from each well, equivalent therefore to 96,000 cells and a total cytosolic volume of 0.24 μl. Maximal stimulation with each agonist would generate a uniformly distributed cytosolic cAMP concentration of up to ∼28 μM (PTH), ∼1 μM (FK), and ∼380 nM (isoproterenol). Means ± SEM, n = 3–6. (B) AC6 and IP3R2 form an intimate junction that allows cAMP to pass directly to IP3R2, enhancing its activity. Ca2+ released by the IP3R may then locally inhibit AC6. The interplay may allow local oscillations in both cAMP and Ca2+ concentration. (C) AC, IP3R, Gs, and a G-protein-coupled receptor (GPCR) are each drawn to approximate scale, with the ER and plasma membrane (PM) separated by 10–15 nm (Treves et al., 2004; Luik et al., 2006). (D) The IP3R–AC junction allows a low-affinity cAMP-sensor (IP3R2) to respond robustly to saturating concentrations of cAMP delivered directly to it by AC. Higher affinity cAMP sensors (e.g., PKA) can be more remote from AC and respond to local gradients of cAMP. Although the binary coding requires local delivery of cAMP, the analogue coding requires its local degradation. The two modes of cAMP signaling invite comparison with the evolution of excitation–contraction coupling in striated muscle, where coupling between the PM voltage-sensor (DHPR) and ryanodine receptor (RyR) in the SR of striated muscle probably evolved from chemical coupling mediated by Ca2+ (cardiac muscle) to conformational coupling (skeletal muscle; Di Biase and Franzini-Armstrong, 2005).

Mentions: We can compare quantitatively the relationships between cAMP and Ca2+ because each was measured at the same time under identical conditions, all cells responded similarly, and cAMP alone mediates the effects of PTH (Figs. 1–4). The similar sensitivity of intact and permeabilized cells to 8-Br-cAMP (Figs. 2 C and 4 D) confirms the fact that permeabilized cells retain their sensitivity to cAMP. This allows comparisons of the cAMP sensitivity of IP3R in permeabilized cells with the calculated concentrations of cAMP evoked by PTH if cAMP were uniformly distributed through the cytosol. For PTH, which produces more cAMP than other stimuli (Fig. 1, C and D), the calculated cAMP concentrations are ∼1,000-fold too low to modulate IP3R. The disparity is even greater when AC is inhibited and the AC–IP3R junctions operate with a reduced safety margin. Therefore, the half-maximal Ca2+ signal evoked by PTH is associated with an estimated global cAMP concentration of ∼500 nM (Fig. 10 A), whereas the half-maximal response in permeabilized cells requires a 5,400-fold higher concentration of cAMP (2.7 mM; Fig. 4 B).


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)

Binary and analogue signaling by cAMP. (A) Comparison of the effects of cAMP on IP3-evoked Ca2+ release from permeabilized cells with estimates of the global cAMP concentration associated with agonist-mediated potentiation of Ca2+ signals in intact cells. A typical HEK cell has a radius of 8.4 μm and thus a total volume of ∼2.5 pl (Rich et al., 2000). Each well used for cAMP assays contained a mean of 4.8 × 105 cells, and our results show cAMP levels determined in 20% of the sample taken from each well, equivalent therefore to 96,000 cells and a total cytosolic volume of 0.24 μl. Maximal stimulation with each agonist would generate a uniformly distributed cytosolic cAMP concentration of up to ∼28 μM (PTH), ∼1 μM (FK), and ∼380 nM (isoproterenol). Means ± SEM, n = 3–6. (B) AC6 and IP3R2 form an intimate junction that allows cAMP to pass directly to IP3R2, enhancing its activity. Ca2+ released by the IP3R may then locally inhibit AC6. The interplay may allow local oscillations in both cAMP and Ca2+ concentration. (C) AC, IP3R, Gs, and a G-protein-coupled receptor (GPCR) are each drawn to approximate scale, with the ER and plasma membrane (PM) separated by 10–15 nm (Treves et al., 2004; Luik et al., 2006). (D) The IP3R–AC junction allows a low-affinity cAMP-sensor (IP3R2) to respond robustly to saturating concentrations of cAMP delivered directly to it by AC. Higher affinity cAMP sensors (e.g., PKA) can be more remote from AC and respond to local gradients of cAMP. Although the binary coding requires local delivery of cAMP, the analogue coding requires its local degradation. The two modes of cAMP signaling invite comparison with the evolution of excitation–contraction coupling in striated muscle, where coupling between the PM voltage-sensor (DHPR) and ryanodine receptor (RyR) in the SR of striated muscle probably evolved from chemical coupling mediated by Ca2+ (cardiac muscle) to conformational coupling (skeletal muscle; Di Biase and Franzini-Armstrong, 2005).
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fig10: Binary and analogue signaling by cAMP. (A) Comparison of the effects of cAMP on IP3-evoked Ca2+ release from permeabilized cells with estimates of the global cAMP concentration associated with agonist-mediated potentiation of Ca2+ signals in intact cells. A typical HEK cell has a radius of 8.4 μm and thus a total volume of ∼2.5 pl (Rich et al., 2000). Each well used for cAMP assays contained a mean of 4.8 × 105 cells, and our results show cAMP levels determined in 20% of the sample taken from each well, equivalent therefore to 96,000 cells and a total cytosolic volume of 0.24 μl. Maximal stimulation with each agonist would generate a uniformly distributed cytosolic cAMP concentration of up to ∼28 μM (PTH), ∼1 μM (FK), and ∼380 nM (isoproterenol). Means ± SEM, n = 3–6. (B) AC6 and IP3R2 form an intimate junction that allows cAMP to pass directly to IP3R2, enhancing its activity. Ca2+ released by the IP3R may then locally inhibit AC6. The interplay may allow local oscillations in both cAMP and Ca2+ concentration. (C) AC, IP3R, Gs, and a G-protein-coupled receptor (GPCR) are each drawn to approximate scale, with the ER and plasma membrane (PM) separated by 10–15 nm (Treves et al., 2004; Luik et al., 2006). (D) The IP3R–AC junction allows a low-affinity cAMP-sensor (IP3R2) to respond robustly to saturating concentrations of cAMP delivered directly to it by AC. Higher affinity cAMP sensors (e.g., PKA) can be more remote from AC and respond to local gradients of cAMP. Although the binary coding requires local delivery of cAMP, the analogue coding requires its local degradation. The two modes of cAMP signaling invite comparison with the evolution of excitation–contraction coupling in striated muscle, where coupling between the PM voltage-sensor (DHPR) and ryanodine receptor (RyR) in the SR of striated muscle probably evolved from chemical coupling mediated by Ca2+ (cardiac muscle) to conformational coupling (skeletal muscle; Di Biase and Franzini-Armstrong, 2005).
Mentions: We can compare quantitatively the relationships between cAMP and Ca2+ because each was measured at the same time under identical conditions, all cells responded similarly, and cAMP alone mediates the effects of PTH (Figs. 1–4). The similar sensitivity of intact and permeabilized cells to 8-Br-cAMP (Figs. 2 C and 4 D) confirms the fact that permeabilized cells retain their sensitivity to cAMP. This allows comparisons of the cAMP sensitivity of IP3R in permeabilized cells with the calculated concentrations of cAMP evoked by PTH if cAMP were uniformly distributed through the cytosol. For PTH, which produces more cAMP than other stimuli (Fig. 1, C and D), the calculated cAMP concentrations are ∼1,000-fold too low to modulate IP3R. The disparity is even greater when AC is inhibited and the AC–IP3R junctions operate with a reduced safety margin. Therefore, the half-maximal Ca2+ signal evoked by PTH is associated with an estimated global cAMP concentration of ∼500 nM (Fig. 10 A), whereas the half-maximal response in permeabilized cells requires a 5,400-fold higher concentration of cAMP (2.7 mM; Fig. 4 B).

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