21529, a novel adenylate cyclase

Details 21529, a novel adenylate cyclase 21529, a novel adenylate cyclase

1999-10-05

2002-06-11

Slobodyansky, Elizabeth (Department: 1652)

Chemistry: molecular biology and microbiology

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

Lyase

C435S252300, C435S320100, C435S325000, C536S023200

Reexamination Certificate

active

06403358

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to novel adenylate cyclase nucleic acid sequences and proteins. Also provided are vectors, host cells, and recombinant methods for making and using the novel molecules.
BACKGROUND OF THE INVENTION
Adenylate cyclase is a membrane-bound enzyme that acts as an effector protein in a receptor-effector system referred to as the cAMP signal transduction pathway. As such, it plays a key intermediate role in the conversion of extracellular signals, perceived by various receptors following binding of a particular ligand, into intracellular signals that, in turn, generate specific cellular responses.
A variety of hormones, neurotransmitters, and olfactants regulate the synthesis of cAMP by adenylate cyclases. In most tissues, regulation of cAMP synthesis is accomplished through three plasma membrane-associated components: G-protein-coupled receptors (GPCRs), which interact with regulatory hormones and neurotransmitters; heterotrimeric G proteins that either stimulate or inhibit the catalytic subunit of adenylate cyclase in response to interaction of ligands with appropriate GPCRs; and the catalytic entity, adenylate cyclase. Each G protein contains a guanine nucleotide-binding alpha subunit and a complex of tightly associated &bgr;- and &ggr;-subunits. When a G protein is activated following binding of a ligand to a GPCR, GDP is released from the &agr;-subunit in exchange for GTP. Binding of the GTP results in conformational changes that yield dissociation of the GTP-bound a-subunit from the &bgr;-&ggr;-subunit complex. The resulting macromolecular complexes regulate catalytic activity of adenylate cyclase. Where the receptor is a stimulatory receptor (R
s
), interaction with a stimulatory G-protein, termed G
s
, results in activation of the adenylate cyclase catalytic subunit by the GTP-bound form of the G
s
&agr;-subunit. In contrast, where the receptor is an inhibitory receptor (R
i
), interaction with an inhibitory G-protein (one of several known G
i
s) results in inhibition of the adenylate cyclase catalytic subunit by the GTP-bound form of the G
i
&agr;-subunit. In addition, the G-protein &bgr;-&ggr;-subunit complex may interact with and influence adenylate cyclase activity independent of or in parallel with the GTP-bound &agr;-subunit, depending upon the adenylate cyclase isoform involved. See Taussig and Gilman (1995)
J. Biol. Chem.
6:1-4; Hardman et al., eds. (1996)
Goodman and Gilman's Pharmacological Basis of Therapeutics
(McGraw-Hill Company, New York, N.Y.).
When activated, the catalytic subunit of adenylate cyclase converts intracellular ATP into cAMP. This second messenger then activates protein kinases, particularly protein kinase A. Activation of this protein kinase causes the phosphorylation of downstream target proteins involved in a number of metabolic pathways, thus initiating a signal transduction cascade.
The extent to which adenylate cyclase converts ATP to cAMP is highly dependent on the state of phosphorylation of the various components of the hormone-sensitive adenylate cyclase system. For example, stimulatory and inhibitory receptors are desensitized and down-regulated following phosphorylation by various kinases, particularly cAMP-dependent protein kinases, protein kinase C, and other receptor-specific kinases that preferentially use agonist-bound forms of receptors as substrates. In this manner, tight regulation of the cellular cAMP concentration, and hence regulation of the cAMP signal transduction pathway, is achieved (Taussig and Gilman (1995)
J. Biol. Chem.
270:1-4).
Adenylate cyclase activation may also occur through increased intracellular calcium concentration, especially in nervous system and cardiovascular tissues. After depolarization, the influx of calcium elicits the activation of calmodulin, an intracellular calcium-binding protein. In the cardiovascular system, this effect gives rise to the contraction of the blood vessels or cardiac myocytes. The activated calmodulin has been shown to bind and activate some isoforms of adenylate cyclase.
Several novel isoforms of mammalian adenylate cyclase have been identified through molecular cloning. Type I adenylate cyclase (CYA1) is primarily localized in brain tissues (see Krupinski et al (1989)
Science
244:1558-1564; Gilman (1987)
Ann. Rev. Biochem.
56:615-649, citing Salter et al. (1981)
J. Biol. Chem.
256:9830-9833; Andreasen et al. (1983)
Biochemistry
22:2757-2762; and Smigel et al (1986)
J. Biol Chem.
261:1976-1982 for bovine CYA1; and Villacres et al. (1993)
Genomics
16:473-478 for human CYA1). The type II adenylate cyclase (CYA2) is localized in brain and lung tissues (see Feinstein et al. (1991)
Proc. Natl. Acad. Sci. USA
88:10173-10177 for rat CYA2; and Stengel et al. (1992)
Hum. Genet.
90:126-130 for human CYA2). Type III adenylate cyclase (CYA3) is primarily localized in olfactory neuroepithelium and is thought to mediate olfactory receptor responses (Bakalyar and Reed (1990)
Science
250:1403-1406; Glatt and Snyder (1993)
Nature
361:536-538; and Xia (1992)
Neurosci. Lett.
144:169-173). Type IV adenylate cyclase (CYA4) most resembles type II, but is expressed in a variety of peripheral tissues and in the central nervous system (Gao and Gilman (1991)
Proc. Natl. Acad. Sci. USA
88:10178-10182, for rat CYA4). Type V adenylate cyclase (CYA5) (Ishikawa et al. (1992)
J. Biol. Chem.
267:13553-13557; Premont et al. (1992)
Proc. Natl. Acad. Sci. USA
89:9809-9813; and Glatt and Snyder (1993)
Nature
361:536-538; Krupinski et al. (1992)
J. Biol. Chem.
267:24858-24862) and type VI adenylate cyclase (CYA6) (Premont et al. (1992)
Proc. Natl. Acad. Sci. USA
89:9808-9813; Yoshimura and Cooper (1992)
Proc. Natl. Acad. Sci. USA
89:6716-6720; Katsushika et al. (1992)
Proc. Natl. Acad. Sci. USA
89:8774-8778; and Krupinski etal. (1992)
J. Biol. Chem.
267:24858-24862) both exhibit a widely distributed expression pattern, with type V having high expression in heart and striatum, and type VI having high expression in heart and brain. Type VII adenylate cyclase (CYA7) is widely distributed, though may be absent from brain tissues (Krupinski et al (1992)
J. Biol. Chem.
267:24858-24862). Type VIII adenylate cyclase (CYA8) is abundant in brain tissues (Krupinski et al. (1992)
J. Biol. Chem.
267:24858-24862; and Parma et al. (1991)
Biochem. Biophys. Res. Commun.
179:455-462). Type IX adenylate cyclase (CYA9) is widely expressed, at high levels in skeletal muscle and brain (Premont et al. (1996)
J. Biol. Chem.
271:13900-13907).
The different isoforms of adenylate cyclase exhibit unique patterns of regulatory responses (see Sunahara et al. (1996)
Annu. Tev. Pharmacol. Toxicol
36:461-480). For example, all of these isoforms are activated by the &agr;-subunit of a particular G protein, termed G
s
, which couples the stimulatory action of the ligand-bound receptor to activation of adenylate cyclase. The adenylate cyclases designated type I, III, and VIII are also stimulated by Ca
2+
/calmodulin in vitro, while type II, IV, V, VI, VII, and IX are not. Type I is inhibited by G protein &bgr;-&ggr;-subunit complex, independently of G
s
activation, while Type II is highly stimulated by G protein &bgr;-&ggr;-subunit complex when simultaneously activated by Gs alpha subunit. Type III, in contrast, is not affected by G protein &bgr;-&ggr;-subunit complex. Type V and type VI are both are inhibited by low levels of Ca
2+
, but appear to be unaffected by G protein &bgr;-&ggr;-subunit complex. Type IX is unique in that it is stimulated by Mg
2+
, but is not affected by G protein &bgr;-&ggr;-subunit complex.
The genes for these adenylate cyclases all encode proteins having molecular weights of approximately 120,000 and which range from 1064 to 1353 amino acid residues. These proteins are predicted to have a short cytoplasmic amino terminus followed by a first motif consisting of six transmembrane spans and a cytoplasmic (domain C
1
), and then a second motif, also consisting of six transmembrane spans and a second cyt

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