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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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Local cAMP signaling to IP3R via hyperactive cAMP junctions. (A–C) The relationship between the increase (Δ) in cAMP and [Ca2+]i for each stimulus is shown (B is an enlargement of A to show FK and isoproterenol). The changes in cAMP associated with an increase in [Ca2+]i of 30 nM evoked by each stimulus are shown in C. (D and E) The effects of inhibitors of AC (SQ/DDA: 1 mM SQ 22536; 200 μM DDA for 20 min) and PDE (IBMX: 1 mM for 20 min) on responses to PTH. Results are means ± SEM from four to five experiments. Similar results with FK and isoproterenol are shown in Fig. S3 (available at http://www.jcb.org/cgi/content/full/jcb.200803172/DC1). (F) Concentration-dependent phosphorylation of the 75-kD band (Fig. 3 B, arrow) after a 30-s treatment with PTH alone, and after pretreatment with SQ/DDA or H89 (means ± SEM, n = 11). (G) Targets of the inhibitors. (H) Sensitization of IP3R in signaling complexes by receptors that generate supramaximal local concentrations of cAMP. The concentration-dependent effects of PTH (bottom) are proposed to arise from recruitment of these all-or-nothing junctions. (I) Steps preceding activation of AC might contribute to the surplus cAMP that allows communication between PTH and IP3R to survive massive inhibition of AC, but for FK, only cAMP separates it from IP3R. The AC–IP3R interaction thus defines the minimal signaling complex, the “AC–IP3R junction” (boxed).
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fig5: Local cAMP signaling to IP3R via hyperactive cAMP junctions. (A–C) The relationship between the increase (Δ) in cAMP and [Ca2+]i for each stimulus is shown (B is an enlargement of A to show FK and isoproterenol). The changes in cAMP associated with an increase in [Ca2+]i of 30 nM evoked by each stimulus are shown in C. (D and E) The effects of inhibitors of AC (SQ/DDA: 1 mM SQ 22536; 200 μM DDA for 20 min) and PDE (IBMX: 1 mM for 20 min) on responses to PTH. Results are means ± SEM from four to five experiments. Similar results with FK and isoproterenol are shown in Fig. S3 (available at http://www.jcb.org/cgi/content/full/jcb.200803172/DC1). (F) Concentration-dependent phosphorylation of the 75-kD band (Fig. 3 B, arrow) after a 30-s treatment with PTH alone, and after pretreatment with SQ/DDA or H89 (means ± SEM, n = 11). (G) Targets of the inhibitors. (H) Sensitization of IP3R in signaling complexes by receptors that generate supramaximal local concentrations of cAMP. The concentration-dependent effects of PTH (bottom) are proposed to arise from recruitment of these all-or-nothing junctions. (I) Steps preceding activation of AC might contribute to the surplus cAMP that allows communication between PTH and IP3R to survive massive inhibition of AC, but for FK, only cAMP separates it from IP3R. The AC–IP3R interaction thus defines the minimal signaling complex, the “AC–IP3R junction” (boxed).

Mentions: PKA is not required for potentiation of CCh-evoked Ca2+ mobilization. (A) IP3R phosphorylation (AbP/AbIP3R3) is expressed relative to levels detected in unstimulated cells (means ± SEM, n = 3–4). AbP is an anti-phospho Ser/Thr antibody (Table S1, available at http://www.jcb.org/cgi/content/full/jcb.200803172/DC1). (B) The effects of 8-Br-cAMP (10 mM for 20 min), FK (100 μM for 30 s), and PTH (100 nM for 30 s) on protein phosphorylation (assessed using AbP) alone or in the presence of H89 (89; 10 μM for 20 min) or st-ht31 (ht; 100 μM for 30 min). The arrow indicates the band used for quantification of protein phosphorylation in Fig. 5 F. Mr markers (kD) are indicated on the left. (C and D) The effect of H89 (C; 10 μM for 20 min) and st-ht31 (D; 100 μM for 30 min) on the ability of PTH to potentiate CCh-evoked Ca2+ mobilization. The targets of the inhibitors are shown in the insets. (E) The effect of H89 (10 μM for 20 min) on the ability of FK to potentiate CCh-evoked Ca2+ mobilization. (F) The effect of RpcAMPS (50 μM for 20 min) on the ability of isoproterenol to potentiate CCh-evoked Ca2+ signals. RpcAMPS was used to inhibit PKA because H89 decreased the sensitivity to isoproterenol by competing with it for occupancy of β-adrenoceptors (Fig. S2 E; Penn et al., 1999). Results (C–F) are means ± SEM, n ≥ 3.


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)

Local cAMP signaling to IP3R via hyperactive cAMP junctions. (A–C) The relationship between the increase (Δ) in cAMP and [Ca2+]i for each stimulus is shown (B is an enlargement of A to show FK and isoproterenol). The changes in cAMP associated with an increase in [Ca2+]i of 30 nM evoked by each stimulus are shown in C. (D and E) The effects of inhibitors of AC (SQ/DDA: 1 mM SQ 22536; 200 μM DDA for 20 min) and PDE (IBMX: 1 mM for 20 min) on responses to PTH. Results are means ± SEM from four to five experiments. Similar results with FK and isoproterenol are shown in Fig. S3 (available at http://www.jcb.org/cgi/content/full/jcb.200803172/DC1). (F) Concentration-dependent phosphorylation of the 75-kD band (Fig. 3 B, arrow) after a 30-s treatment with PTH alone, and after pretreatment with SQ/DDA or H89 (means ± SEM, n = 11). (G) Targets of the inhibitors. (H) Sensitization of IP3R in signaling complexes by receptors that generate supramaximal local concentrations of cAMP. The concentration-dependent effects of PTH (bottom) are proposed to arise from recruitment of these all-or-nothing junctions. (I) Steps preceding activation of AC might contribute to the surplus cAMP that allows communication between PTH and IP3R to survive massive inhibition of AC, but for FK, only cAMP separates it from IP3R. The AC–IP3R interaction thus defines the minimal signaling complex, the “AC–IP3R junction” (boxed).
© Copyright Policy
Related In: Results  -  Collection

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fig5: Local cAMP signaling to IP3R via hyperactive cAMP junctions. (A–C) The relationship between the increase (Δ) in cAMP and [Ca2+]i for each stimulus is shown (B is an enlargement of A to show FK and isoproterenol). The changes in cAMP associated with an increase in [Ca2+]i of 30 nM evoked by each stimulus are shown in C. (D and E) The effects of inhibitors of AC (SQ/DDA: 1 mM SQ 22536; 200 μM DDA for 20 min) and PDE (IBMX: 1 mM for 20 min) on responses to PTH. Results are means ± SEM from four to five experiments. Similar results with FK and isoproterenol are shown in Fig. S3 (available at http://www.jcb.org/cgi/content/full/jcb.200803172/DC1). (F) Concentration-dependent phosphorylation of the 75-kD band (Fig. 3 B, arrow) after a 30-s treatment with PTH alone, and after pretreatment with SQ/DDA or H89 (means ± SEM, n = 11). (G) Targets of the inhibitors. (H) Sensitization of IP3R in signaling complexes by receptors that generate supramaximal local concentrations of cAMP. The concentration-dependent effects of PTH (bottom) are proposed to arise from recruitment of these all-or-nothing junctions. (I) Steps preceding activation of AC might contribute to the surplus cAMP that allows communication between PTH and IP3R to survive massive inhibition of AC, but for FK, only cAMP separates it from IP3R. The AC–IP3R interaction thus defines the minimal signaling complex, the “AC–IP3R junction” (boxed).
Mentions: PKA is not required for potentiation of CCh-evoked Ca2+ mobilization. (A) IP3R phosphorylation (AbP/AbIP3R3) is expressed relative to levels detected in unstimulated cells (means ± SEM, n = 3–4). AbP is an anti-phospho Ser/Thr antibody (Table S1, available at http://www.jcb.org/cgi/content/full/jcb.200803172/DC1). (B) The effects of 8-Br-cAMP (10 mM for 20 min), FK (100 μM for 30 s), and PTH (100 nM for 30 s) on protein phosphorylation (assessed using AbP) alone or in the presence of H89 (89; 10 μM for 20 min) or st-ht31 (ht; 100 μM for 30 min). The arrow indicates the band used for quantification of protein phosphorylation in Fig. 5 F. Mr markers (kD) are indicated on the left. (C and D) The effect of H89 (C; 10 μM for 20 min) and st-ht31 (D; 100 μM for 30 min) on the ability of PTH to potentiate CCh-evoked Ca2+ mobilization. The targets of the inhibitors are shown in the insets. (E) The effect of H89 (10 μM for 20 min) on the ability of FK to potentiate CCh-evoked Ca2+ mobilization. (F) The effect of RpcAMPS (50 μM for 20 min) on the ability of isoproterenol to potentiate CCh-evoked Ca2+ signals. RpcAMPS was used to inhibit PKA because H89 decreased the sensitivity to isoproterenol by competing with it for occupancy of β-adrenoceptors (Fig. S2 E; Penn et al., 1999). Results (C–F) are means ± SEM, n ≥ 3.

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