Kinetic Properties of “Soluble” Adenylyl Cyclase

Abstract

"Soluble" adenylyl cyclase (sAC) is a widely expressed source of cAMP in mammalian cells that is evolutionarily, structurally, and biochemically distinct from the G protein-responsive transmembrane adenylyl cyclases. In contrast to transmembrane adenylyl cyclases, sAC is insensitive to heterotrimeric G protein regulation and forskolin stimulation and is uniquely modulated by bicarbonate ions. Here we present the first report detailing kinetic analysis and biochemical properties of purified recombinant sAC. We confirm that bicarbonate regulation is conserved among mammalian sAC orthologs and demonstrate that bicarbonate stimulation is consistent with an increase in the V max of the enzyme with little effect on the apparent K m for substrate, ATPMg 2؉ . Bicarbonate can further increase sAC activity by relieving substrate inhibition. We also identify calcium as a direct modulator of sAC activity. In contrast to bicarbonate, calcium stimulates sAC activity by decreasing its apparent K m for ATP-Mg 2؉ . Because of their different mechanisms, calcium and bicarbonate synergistically activate sAC; therefore, small changes of either calcium or bicarbonate will lead to significant changes in cellular cAMP levels. Two types of adenylyl cyclase (AC)1 are ubiquitously expressed in mammalian cells, a well characterized gene family of transmembrane ACs (tmACs) and the recently discovered "soluble" AC (sAC). The tmACs are plasma membrane bound, and their activities are regulated by G proteins in response to extracellular stimuli such as neurotransmitters and hormones (reviewed in Ref. 1). In contrast, sAC is associated with various intracellular organelles, including mitochondria, centrioles, mitotic spindle, mid-bodies, and nuclei (2). sAC activity is modulated by bicarbonate (3) and, as shown in this report, by Ca 2ϩ ; regulation by these intracellular signaling molecules suggests that sAC mediates cAMP-dependent responses to intrinsic cellular changes (4, 5).The catalytic mechanism of tmACs has been determined from biochemical and crystallographic studies. tmACs convert ATP to cAMP using two-metal catalysis where one ion acts as a free metal and the other coordinates ATP in the active site (6, 7). Its activators, G␣ s subunit or forskolin, stimulate tmACs by allosteric modulation of the active site (8, 9). More than 25 years ago, when soluble AC activity was first discovered, it was predicted to be molecularly distinct from tmACs because its activity appeared to be dependent on the presence of the divalent cation, Mn 2ϩ , and it was insensitive to forskolin and G protein regulation (10 -12). These differential properties enabled purification (13) and cloning of sAC from rat testis (14). The sAC gene is indeed molecularly distinct from tmACs; it possesses no transmembrane domains, and its catalytic domains are more closely related to those of cyanobacterial ACs than to those from other eukaryotic ACs. The purified soluble AC exhibited ϳ10-fold lower affinity for substrate ATP relative to tmACs (tmAC K m for ATP-Mn 2ϩ is ϳ 100...

Moving to the beat: a review of mammalian sperm motility regulation

“…Therefore, either CaM is upstream of PKA in the sperm motility regulation pathway or the two pathways are independent but redundant. Interestingly, the effects of calcium on sAC are independent of CaM (Jaiswal and Conti 2003;Litvin et al 2003). Therefore, although it is known that sAC is required for sperm motility (Esposito et al 2004;Hess et al 2005), and although sAC is regulated by Ca, the effect of CaM on motility is not achieved via this cyclase.…”

“…One mechanism by which calcium is directly linked to flagellar function is through its regulation of the atypical 'soluble' adenylyl cyclase (sAC), which generates cAMP to activate PKA. The sAC is required for sperm motility and is molecularly and biochemically distinct from the transmembrane adenylyl cyclases (tmACs), in part because sAC is uniquely sensitive to both bicarbonate and calcium (Buck et al 1999;Chen et al 2000;Wuttke et al 2001;Jaiswal and Conti 2003;Litvin et al 2003;Esposito et al 2004;Liguori et al 2004;Hess et al 2005). Thus, through sAC, calcium signalling can be linked to PKA signalling as parts of a single pathway.…”

Soluble Adenylyl Cyclase Mediates Nerve Growth Factor-induced Activation of Rap1

“…Previously, calcium had been identified as an activator of sAC (20,21,28,41). On the other hand, calcium is also known to regulate several types of tmACs, some of which are expressed in PC12 cells (31).…”

“…Among mammalian adenylyl cyclases, sAC is uniquely bicarbonate-stimulated (19,21,36,37), and cAMP production via sAC represents the only known signaling effect initiated by bicarbonate (24). Generation of cAMP can lead to activation of Rap1 (5, 11, 38, 39), and we tested whether bicarbonate could stimulate Rap1 activation in PC12 cells.…”

“…We have identified an additional, ubiquitously expressed (17,18) source of cAMP in mammalian cells, "soluble" adenylyl cyclase (sAC) (16). In contrast to tmACs, sAC is not regulated by heterotrimeric G proteins but is, instead, regulated by bicarbonate (19) and calcium (20,21) ions. Also, in contrast to tmACs, sAC is not bound to the plasma membrane but is localized to various intracellular compartments (22), where it is thought to be the source of local pools of cAMP (23,24).…”