Distinct Mechanisms of Regulation by Ca2+/Calmodulin of Type 1 and 8 Adenylyl Cyclases Support Their Different Physiological Roles

Nanako Masada,Antonio Ciruela,David A Macdougall,Dermot M.F Cooper

doi:10.1074/jbc.m807359200

Nanako Masada, Antonio Ciruela + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m807359200

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Abstract

Nine membrane-bound mammalian adenylyl cyclases (ACs) have been identified. Type 1 and 8 ACs (AC1 and AC8), which are both expressed in the brain and are stimulated by Ca(2+)/calmodulin (CaM), have discrete neuronal functions. Although the Ca(2+) sensitivity of AC1 is higher than that of AC8, precisely how these two ACs are regulated by Ca(2+)/CaM remains elusive, and the basis for their diverse physiological roles is quite unknown. Distinct localization of the CaM binding domains within the two enzymes may be essential to differential regulation of the ACs by Ca(2+)/CaM. In this study we compare in detail the regulation of AC1 and AC8 by Ca(2+)/CaM both in vivo and in vitro and explore the different role of each Ca(2+)-binding lobe of CaM in regulating the two enzymes. We also assess the relative dependence of AC1 and AC8 on capacitative Ca(2+) entry. Finally, in real-time fluorescence resonance energy transfer-based imaging experiments, we examine the effects of dynamic Ca(2+) events on the production of cAMP in cells expressing AC1 and AC8. Our data demonstrate distinct patterns of regulation and Ca(2+) dependence of AC1 and AC8, which seems to emanate from their mode of regulation by CaM. Such distinctive properties may contribute significantly to the divergent physiological roles in which these ACs have been implicated.

Highlights

Lin (FSK; except AC9) and Gs␣, they are distributed and regulated differently [1, 3, 4]
Four adenylyl cyclases (ACs) are regulated by physiological concentrations of Ca2ϩ and thereby provide a critical link between the Ca2ϩ- and cAMP-signaling pathways [3, 5]; AC5 and AC6 are directly inhibited by Ca2ϩ, whereas AC1 and AC8 are stimulated by Ca2ϩ in a calmodulin (CaM)dependent manner [5]
Ca2؉/Calmodulin Regulation of AC1 and AC8 caused by capacitative Ca2ϩ entry (CCE), the mode of Ca2ϩ entry triggered by emptying Ca2ϩ from internal stores [23], preferentially stimulates AC1 and AC8 [21]

Summary

EXPERIMENTAL PROCEDURES

Materials—FSK and thapsigargin (TG) were purchased from Merck (Nottingham, UK). [2-3H]Adenine, [2,8-3H]cAMP, ECL Western blotting analysis system, HyperfilmTM, and horseradish peroxidase-conjugated goat anti-rabbit IgG were obtained from GE Healthcare (Little Chalfont, UK). [␣-32P]ATP was from PerkinElmer Life Sciences. To establish stable cell lines expressing AC8, AC8M1 (AC8⌬1–106), AC1, AC1M1, AC1F503A, or vector (pcDNA3.1; pcD), HEK 293 cells were transfected with 2 ␮g of cDNA according to the calcium phosphate method described previously [33]. Transfected HEK 293 cells were incubated in minimum essential medium with [2-3H]adenine (1.5 ␮Ci/well in 24-well plates) at 37 °C for 90 min to label the ATP pool. Cells were plated onto poly-L-lysine-coated coverslips 1 day before experiments and loaded with 2 ␮M fura-2/AM plus 0.02% Pluronic F-127 for 40 min at room temperature in HBS buffer (140 mM NaCl, 4 mM KCl, 0.2 mM MgCl2, 11 mM D-glucose, 10 mM HEPES, pH 7.4) with 1 mM CaCl2. Statistical significance was assessed by using one-way analysis of variance followed by Newman-Keuls multiple comparisons tests, where p Ͻ 0.05 was considered significant

RESULTS

DISCUSSION

ADDITIONS AND CORRECTIONS