Mammalian soluble adenylyl cyclase

Details Mammalian soluble adenylyl cyclase Mammalian soluble adenylyl cyclase

2000-05-11

2003-04-08

Monshipouri, M. (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Lyase

C435S325000, C435S320100, C435S252300, C435S006120, C536S023200

Reexamination Certificate

active

06544768

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to isolated animal soluble adenylyl cyclase and its role in the regulation of the cAMP signaling pathway.
BACKGROUND OF THE INVENTION
Adenylyl cyclase (AC) is the effector molecule of one of the most widely used signal transduction pathways. Its product, cyclic AMP (cAMP), is a nearly universally utilized second messenger molecule, which mediates cellular responses to nutritional conditions and extracellular signals in organisms from prokaryotes to higher eukaryotes. cAMP has long been known to exert both stimulatory and inhibitory effects on cell growth and proliferation (Dumont, J. E., et al., Trends Biochem. Sci., 1989, 14:67-71; Rozengurt, E., Science, 1986, 234:161-6). In metazoans, a seemingly ubiquitous membrane-associated AC activity is encoded by a family of transmembrane adenylyl cyclases (tmACs) that mediate cellular responses to external stimuli.
Throughout the animal kingdom members of the transmembrane adenylyl cyclase (tmAC) superfamily synthesize cAMP to mediate communication between cells (Sunahara, R. K., et al., Annu. Rev. Pharmaco. Toxicol., 1996, 36: 461-80; Taussig, R., et al., J. Biol. Chem., 1995, 270:1-4). For example, in mammals, signals arising from other cells such as hormones, neurotransmitters, and olfactants, modulate tmAC activity via cell surface receptors and G proteins (Taussig, R., et al., Adv. Second Messenger Phosphoprotein Res., 1998, 32:81-98). A similar cAMP signaling cascade is present in other multicellular organisms, including Drosophila (Cann, M. J. et al., Adenylyl Cyclases, 32. Lippincott-Raven, 1998; Cann, M. J. et al., Adenylyl Cyclase, 32. Lippincott-Raven, 1999; lourgenko, V., et al, FEBS Lett., 1997, 413:104-8; lourgenko, V. et al., “A calcium inhibited Drosophila adenylyl cyclase (submitted); Levin, L. R., et al., Cell, 1992, 68:479-89), C. elegans (Bargmann, C. I., et al., Science, 1998, 282:2028-33; Berger, A. J., et al., J. Neurosci., 1998, 18:2871-80-; Korswagen, H. C. et al., Embo J., 1998, 17:5059-65), and Dictyostelium (Pitt, G. S. et al., Cell, 1991, 69:305-15). In contrast, the ACs found in unicellular eukaryotes and bacteria transmit nutritional information to the inside of the cell (Danchin, A., Adv. Second Messenger Phosphoprotein Res., 1991, 27:109-62).
Current models for cAMP signal transduction in mammals involve only transmembrane adenylyl cyclases (tmACs), which generate cAMP near the plasma membrane (Hempel, C. M., et al., Nature, 1996, 384:166-9; Sunahara, R. K., et al., Annu. Rev. Pharmacol. Toxicol., 1996, 36:461-80; Taussig, R., et al., J. Biol. Chem., 1995, 270:1-4). With the major effector of cAMP, the cAMP-dependent protein kinase (PKA), tethered to intracellular sites often far removed from the plasma membrane by a family of
A
Kinase
A
nchoring
P
roteins (AKAP) (Lester, L. B. et al., Recent Prog. Horm. Res., 1997, 52:409-29; Pawson, T., et al., Science, 1997, 278:2075-80) these models depend upon diffusion of cAMP past membrane-proximal targets to activate intracellular PKA at more distal sites. Furthermore, it must survive in a cytoplasm filled with phosphodiesterases (Beavo, J. A., et al., Mol. Pharmacol., 1994, 46:399-405; Bushnik, T., et al., Biochem. Soc. Trans., 1996, 24:1014-9). However the evidence for cAMP diffusion is based on exogenous addition of millimolar concentrations of cAMP (Bacskai, B. J., et al., Science, 1993, 260:222-6), and experiments which demonstrate diffusion of liberated PKA catalytic subunit (Bacskai, B. J., et al., Science, 1993, 260:222-6; Hempel, C. M., et al., Nature, 1996, 384:166-9). Thus there has not been a satisfactory explanation for the problems associated with how these models operate.
Soluble Adenylyl Cyclase
In addition to tmACs, another type of AC activity has been described in mammals, that of soluble adenylyl cyclase (sAC), which is thought to be expressed only in testis and sperm (Ahn, S., et al., Mol. Cell Biol., 1998, 18:967-77; Bacskai, B. J., et al., Science, 1993, 260:222-6). sAC activity appears to be biochemically and chromatographically different from tmACs, particularly a genetically engineered tmAC which is soluble, and soluble guanylyl cyclases previously described in testis (Neer, E. J., J. Biol. Chem., 1978, 253:5808-5812; Neer, E. J. et al., Biochim. Biophys. Acta, 1979, 583:531-534; Braun, T. et al., Biochim. Biophys. Acta, 1977,481:227-235). Unlike the known tmACs, sAC biochemical activity has been shown to depend on the divalent cation Mn2
+
(Braun, T and Dods, R. F., Proc. Natl. Acad. Sci. USA, 1975, 72:1097-1101), sAC is insensitive to G protein regulation (Braun, T. et al., Biochim. Biophys. Acta, 1977, 481:227-235), and sAC displays approximately 10-fold lower affinity for the substrate ATP (Km approximately equal to 1 mM) (Neer, E. J., J. Biol. Chem., 1978, 253:5808-5812; Gordeladze, J. O. et al., Mol. Cell Endocrinol., 1981, 23:125-136; Braun T., Methods Enzymol., 1991, 195:130-136) than the tmACs (Km approximately equal to 100 &mgr;M) (Johnson, R. A. et al., Methods Enzymol., 1994, 238:56-71). Based on these studies, this soluble form of AC was thought to be molecularly distinct from tmACs (Beltran, C. et al., Biochemistry, 1996, 35:7591-8; Berkowitz, L. A., et al., Mol. Cell Biol., 1989, 9:4272-81).
Semipurified soluble adenylyl cyclase activity is inhibited by submicromolar amounts of catechol estrogens (Braun, T., Proc. Soc. Exp. Biol. Med., 1990, 194:58-63). Braun demonstrated that the two hydroxyls of the catechol moiety were essential for the inhibitory interaction, estradiol and estrone were completely inactive. Catechols with aliphatic side chain like dopamine, L-dopa, and norepinephrine were able to inhibit sAC activity, but were 1,000 fold less potent.
Molecular evidence confirming that soluble AC represents a distinct form of adenylyl cyclase is lacking. Thus a need remains for the identification, cloning, characterization and purification of the signaling molecule having soluble adenylyl cyclase activity. There is a further need to modulate sAC activity in order to affect cell function.
Carbon Dioxide and Bicarbonate
Carbon dioxide (CO
2
) is the end product of metabolism in animals. It is normally released into the atmosphere via breathing, but is also soluble in cell membranes. CO
2
combines with water in the presence of carbonic anhydrase (CA) to form carbonic acid (H
2
CO
3
) which dissociates to liberate a proton and bicarbonate ion (HCO
3
−
).
CO
2
+H
2
O⇄H
2
CO
3
⇄HCO
3
−
+H
+
CA
By itself, this reaction reaches equilibrium after about 4 minutes. However, in most biological systems, due to the ubiquitous presence of carbonic anhydrase, bicarbonate/CO
2
equilibrium is reached nearly instantaneously (Johnson, L. R.
Essential Medical Physiology
, 1998, Phila. Lippincott-Raven).
In mammals, blood is a bicarbonate/CO
2
buffer system, and the relationship between blood pH, bicarbonate and CO
2
partial pressure can be described by the Henderson-Hasselbach equation:
pH=6.1+log([HCO
3
−
]/0.03P
CO2
)
This equilibrium in serum is tightly controlled in two ways; the kidneys regulate the bicarbonate concentration and the breathing frequency determines the concentration of carbon dioxide.
Bicarbonate is the carbon source for the initial reactions of gluconeogenesis and ureagenesis (Henry, Annu. Rev. Physiol., 1996, 58:523-538). Additionally, CO
2
and/or bicarbonate have been shown to modulate a number of physiological processes (i.e., diuresis, breathing, blood flow, cerebrospinal fluid formation, aqueous humor formation, and spermatocyte development). In most cases, the effects of CO
2
have been ascribed to as yet undescribed chemoreceptors, and the effects of bicarbonate are usually thought to be mediated by changes in cellular pH (Johnson, 1998).
Measurement of physiological levels of bicarbonate is typically determined indirectly by calculation from the direct measurement of carbon dioxide and pH using the Henderson Hasselbalch equation. However, a certain degree of error is inherent

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