Molecular biological approaches to unravel adenylyl cyclase signaling and function

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ACs 1 catalyze the conversion of ATP into the second messenger cAMP, PP i being the second product of the cyclase reaction. Mammals express nine membranous ACs (ACs 1-9) (1, 2) and a sAC that is predominantly expressed in testis (3). Bacillus anthracis and Bacillus pertussis produce the AC toxins EF and ACT, respectively, that are activated by Ca 2ϩ /calmodulin and act through excessive cAMP accumulation in host cells (4,5). sGC is structurally related to ACs 1-9 in the catalytic site and is activated by [6][7][8]. sGC catalyzes the formation of the second messenger cGMP from GTP. ACs 1-9 contain a tandem repeat structure with two transmembrane domains and two cytosolic domains (1, 2). The cytosolic domains are referred to as C1 and C2, respectively. Together, C1 and C2 form the catalytic site of AC. C1 and C2 also contain the regulatory sites for the stimulatory G-protein, G␣ s , for the inhibitory G-protein, G␣ i , and for the diterpene, forskolin. Catalytic activity of all AC isoforms depends on the presence of divalent cations (Mg 2ϩ or Mn 2ϩ ). Membranous ACs possess two Me 2ϩ -binding sites (9 -11). When mixed together, purified C1 and C2 form a functional AC that is efficiently activated by forskolin and G␣ s -GTP␥S (12, 13).AC isoforms differ from each other in their regulation (1, 2). ACs 1-9 are all activated by G␣ s , whereas sAC is activated by HCO 3 Ϫ (14). Forskolin activates ACs 1-8 but not AC9 or sAC. G␣ i inhibits ACs 1, 5, and 6. G-protein ␤␥ subunits exhibit stimulatory or inhibitory effects on AC isoforms. Ca 2ϩ /calmodulin stimulates ACs 1, 3, and 8. In addition, Mg 2ϩ and Mn 2ϩ show differential stimulatory effects on AC isoforms (15). More-

“…ACs 5 and 6 are structurally closely related to each other (1,71). Thus, it was not surprising that their overall inhibitor profiles were also similar.…”

Section: Potent and Selective Ac Inhibition By Mant-itp␥s Relative Tomentioning

confidence: 90%

Differential Inhibition of Adenylyl Cyclase Isoforms and Soluble Guanylyl Cyclase by Purine and Pyrimidine Nucleotides

Gille

Lushington

Mou

et al. 2004

215

“…Regulation of AC activities by the G i and G q /Ca 2ϩ pathways has been well studied both in vitro and in vivo (11,14,15). Recently, regulation of cAMP responses by the G 13 pathway was also reported (16), thereby establishing that cAMP can be regulated by all major groups of heterotrimeric G proteins.…”

mentioning

confidence: 99%

“…There are nine membrane-bound mammalian ACs that can be divided into four subgroups based on their sequence similarities and regulatory properties (11,14,15). AC1, AC3, and AC8 can be stimulated by Ca 2ϩ /calmodulin upon elevation of intracellular free Ca 2ϩ through voltage-gated Ca 2ϩ channels or capacitative entry.…”

mentioning

confidence: 99%

Regulation of cAMP Responses by the G12/13 Pathway Converges on Adenylyl Cyclase VII

Jiang

Collins

Davis

et al. 2008

Regulation of intracellular cAMP by multiple pathways enables differential function of this ubiquitous second messenger in a context-dependent manner. Modulation of G s -stimulated intracellular cAMP has long been known to be modulated by the G i and G q /Ca 2؉ pathways. Recently, the G 13 pathway was also shown to facilitate cAMP responses in murine macrophage cells. We report here that this synergistic regulation of cAMP synthesis by the G s and G 13 pathways is mediated by a specific isoform of adenylyl cyclase, AC7. Furthermore, this signaling paradigm exists in several hematopoietic lineages and can be recapitulated by exogenous expression of AC7 in HEK 293 cells. Mechanistic characterization of this synergistic interaction indicates that it occurs downstream of receptor activation and it can be mediated by the ␣ subunit of either G 12 or G 13 . Our results demonstrate that AC7 is a specific downstream effector of the G 12/13 pathway.

“…All are stimulated by G␣ s and, with the exception of type IX, also by forskolin. The different AC isoforms also share similar structural features consisting of a cytosolic N-terminal region, two sets of six transmembrane spans separated by a cytosolic loop (C1), and a cytosolic tail (C2) (1)(2)(3)(4). The N termini of the two cytosolic domains (C1a and C2a) are highly conserved among nine types of AC and form the catalytic core (5)(6)(7)(8)(9).…”

mentioning

confidence: 99%

Conditional Stimulation of Type V and VI Adenylyl Cyclases by G Protein βγ Subunits

Gao

Sadana

Dessauer

et al. 2007

Self Cite

In a yeast two-hybrid screen of mouse brain cDNA library, using the N-terminal region of human type V adenylyl cyclase (hACV) as bait, we identified G protein ␤2 subunit as an interacting partner. Additional yeast two-hybrid assays showed that the G␤ 1 subunit also interacts with the N-terminal segments of hACV and human type VI adenylyl cyclase (hACVI). In vitro adenylyl cyclase (AC) activity assays using membranes of Sf9 cells expressing hACV or hACVI showed that G␤␥ subunits enhance the activity of these enzymes provided either G␣ s or forskolin is present. Deletion of residues 77-151, but not 1-76, in the N-terminal region of hACVI obliterated the ability of G␤␥ subunits to conditionally stimulate the enzyme. Likewise, activities of the recombinant, engineered, soluble forms of ACV and ACVI, which lack the N termini, were not enhanced by G␤␥ subunits. Transfection of the C terminus of G protein receptor kinase 2 to sequester endogenous G␤␥ subunits attenuated the ability of isoproterenol to increase cAMP accumulation in COS-7 cells overexpressing hACVI even when G i was inactivated by pertussis toxin. Therefore, we conclude that the N termini of human hACV and hACVI are necessary for interactions with, and regulation by, G␤␥ subunits both in vitro and in intact cells. Moreover, G␤␥ subunits derived from a source(s) other than G i are necessary for the full activation of hACVI by isoproterenol in intact cells.