Abstract
Ca(2+)-sensitive adenylyl cyclases may act as early integrators of the two major second messenger-signaling pathways mediated by Ca(2+) and cAMP. Ca(2+) stimulation of adenylyl cyclase type I (ACI) and adenylyl cyclase type VIII (ACVIII) is mediated by calmodulin and the site on these adenylyl cyclases that interacts with calmodulin has been defined. By contrast, the mechanism whereby Ca(2+) inhibits adenylyl cyclase type V (ACV) and adenylyl cyclase type VI (ACVI) is unknown. In this study, Ca(2+), Sr(2+), and Ba(2+) were compared to probe the involvement of E-F hand-like domains in both Ca(2+) stimulation and inhibition of ACVIII and ACVI, respectively. HEK 293 cells transfected with ACVIII cDNA and C6-2B glioma cells (where the endogenous adenylyl cyclases is predominantly ACVI) were used to compare the effects of these three cations in in vitro and in vivo measurements. The in vitro data identified two Ca(2+) regulatory sites for both ACVIII and ACVI. Strikingly different potency series for these cations at mediating high affinity stimulation and inhibition of ACVIII and ACVI, respectively, effectively rule out the possibility that calmodulin or proteins utilizing similar Ca(2+)-binding motifs mediate inhibition of ACVI. On the other hand, the low affinity inhibition that is common to both ACVIII and ACVI showed virtually identical potency profiles for the IIa cation series, indicating a common site of action. Remarkably, whereas Sr(2+) was rather ineffective at regulating these cyclases (particularly ACVI) in vitro, adequate concentrations accumulated in the vicinity of these enzymes as a consequence of capacitative cation entry to partially regulate both of these activities in vivo. This latter finding consolidates earlier observations that Ca(2+)-sensitive adenylyl cyclases detect and respond to capacitative cation entry rather than global cytosolic cation concentrations.
Highlights
Ca2؉-sensitive adenylyl cyclases may act as early integrators of the two major second messenger-signaling pathways mediated by Ca2؉ and cAMP
Whereas Sr2؉ was rather ineffective at regulating these cyclases ( adenylyl cyclase type VI (ACVI)) in vitro, adequate concentrations accumulated in the vicinity of these enzymes as a consequence of capacitative cation entry to partially regulate both of these activities in vivo
Distinct and Common Ca2ϩ Regulatory Sites of adenylyl cyclase type VIII (ACVIII) and ACVI—The Ca2ϩ stimulation of ACVIII is mediated through loosely bound calmodulin
Summary
Materials—Thapsigargin, forskolin, and Ro 20 –1724 were from Calbiochem (San Diego, CA). [2-3H]Adenine, [3H]cAMP, and [␣-32P]ATP were obtained from Amersham Pharmacia Biotech. Adenylyl Cyclase Activity Measurements—The adenylyl cyclase activity of the C6 –2B glioma cell membranes and transfected HEK 293 cell membranes was measured in the presence of the following components: 12 mM phosphocreatine, 2.5 units of creatine phosphokinase, 0.1 mM cAMP, 1 mM MgCl2, 0.1 mM ATP, 70 mM Tris buffer, pH 7.4, 0.04 mM GTP, 1 Ci of [␣-32P]ATP, 3-isobutyl-1-methylxanthine (500 M), and 20 M forskolin. The Sr2ϩ and Ba2ϩ data from C6 –2B glioma cells and N⌬1–106-transfected cells fit better to single-site competition curves. Assays were terminated by addition of 5% (w/v final) trichloroacetic acid and the percentage of conversion of [3H]ATP to [3H]cAMP was measured as described previously [22]. [Ca2ϩ]i, [Sr2ϩ]i, and [Ba2ϩ]i Measurements Using fura-2—[Ca2ϩ]i was measured in populations of HEK 293 cells and C6 –2B glioma cells, using fura-2 as the Ca2ϩ indicator, exactly as described previously [15]. The Kd values, 224 nM for Ca2ϩ, 2.62 M for Sr2ϩ, and 780 nM for Ba2ϩ were used in the calculation [25]
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