Publications

• Raf kinase activation of adenylyl cyclases: isoform-selective regulation.

  Ding Q., Gros R., Gray I.D., Taussig R., Ferguson S.S., Feldman R.D.

  Adenylyl cyclases (AC), a family of enzymes that catalyze the synthesis of cyclic AMP, are critical regulators of cellular functions. The activity of adenylyl cyclase is stimulated by a range of hormone receptors, primarily via interactions with G-proteins; however, recently we identified an alter ... >> More

  Adenylyl cyclases (AC), a family of enzymes that catalyze the synthesis of cyclic AMP, are critical regulators of cellular functions. The activity of adenylyl cyclase is stimulated by a range of hormone receptors, primarily via interactions with G-proteins; however, recently we identified an alternate mechanism by which growth factors sensitize adenylyl cyclase activation. We suggested that this mechanism might involve a Raf kinase-mediated serine phosphorylation of adenylyl cyclase. However, the direct involvement of a specific form of Raf kinase is yet to be demonstrated. Furthermore, whether this mechanism is generalized to other isoforms of adenylyl cyclase is unknown. In human embryonic kidney 293 cells, we now demonstrate that in reconstitution studies, c-Raf kinase can mediate phosphorylation of AC VI. Furthermore, AC VI coimmunoprecipitates with c-Raf. Raf kinase-dependent regulation of adenylyl cyclase VI is dependent on the integrity of Ser750 in the fourth intracellular loop of the enzyme and Ser603/Ser608 in the C1b region of the molecule. To examine how generalized this effect is, we studied representative isoforms of the major subfamilies of adenylyl cyclase viz., AC I, AC II, and AC V. Raf kinase-dependent sensitization/ phosphorylation of adenylyl cyclases is common to AC VI, AC V, and AC II isoforms but not AC I. In aggregate, these studies indicate that Raf kinase associates with adenylyl cyclases. Furthermore, Raf kinase regulation of adenylyl cyclase is isoform-selective. These functional interactions (as well as the physical association) between adenylyl cyclases and Raf kinases suggest an important but previously unrecognized interaction between these two key regulatory enzymes. << Less

  Mol. Pharmacol. 66:921-928(2004) [PubMed] [EuropePMC]

• Two-metal-ion catalysis in adenylyl cyclase.

  Tesmer J.J.G., Sunahara R.K., Johnson R.A., Gosselin G., Gilman A.G., Sprang S.R.

  Adenylyl cyclase (AC) converts adenosine triphosphate (ATP) to cyclic adenosine monophosphate, a ubiquitous second messenger that regulates many cellular functions. Recent structural studies have revealed much about the structure and function of mammalian AC but have not fully defined its active s ... >> More

  Adenylyl cyclase (AC) converts adenosine triphosphate (ATP) to cyclic adenosine monophosphate, a ubiquitous second messenger that regulates many cellular functions. Recent structural studies have revealed much about the structure and function of mammalian AC but have not fully defined its active site or catalytic mechanism. Four crystal structures were determined of the catalytic domains of AC in complex with two different ATP analogs and various divalent metal ions. These structures provide a model for the enzyme-substrate complex and conclusively demonstrate that two metal ions bind in the active site. The similarity of the active site of AC to those of DNA polymerases suggests that the enzymes catalyze phosphoryl transfer by the same two-metal-ion mechanism and likely have evolved from a common ancestor. << Less

  Science 285:756-760(1999) [PubMed] [EuropePMC]

• Molecular cloning and characterization of a Ca2+/calmodulin-insensitive adenylyl cyclase from rat brain.

  Feinstein P.G., Schrader K.A., Bakalyar H.A., Tang W.J., Krupinski J., Gilman A.G., Reed R.R.

  Biochemical, immunological, and molecular cloning studies have suggested the existence of multiple forms of adenylyl cyclase (EC 4.6.1.1). An adenylyl cyclase cDNA clone (type II) was isolated from a rat brain library and found to encode a protein of 1090 amino acids that was homologous to but dis ... >> More

  Biochemical, immunological, and molecular cloning studies have suggested the existence of multiple forms of adenylyl cyclase (EC 4.6.1.1). An adenylyl cyclase cDNA clone (type II) was isolated from a rat brain library and found to encode a protein of 1090 amino acids that was homologous to but distinct from the previously described Ca2+/calmodulin-stimulated adenylyl cyclase from bovine brain. Expression of the type II cDNA in an insect cell line resulted in an increased level of adenylyl cyclase activity that was insensitive to Ca2+/calmodulin. Addition of activated Gs alpha protein to type II-containing membranes increased enzyme activity. The mRNA encoding the type II protein was expressed at high levels in brain tissue and at low levels in olfactory epithelium and lung. The existence of multiple adenylyl cyclase enzymes may provide for complex and distinct modes of biochemical regulation of cAMP levels in the brain. << Less

  Proc. Natl. Acad. Sci. U.S.A. 88:10173-10177(1991) [PubMed] [EuropePMC]

• Cloning and expression of a widely distributed (type IV) adenylyl cyclase.

  Gao B., Gilman A.G.

  We have cloned and expressed a cDNA that encodes a widely distributed form of mammalian adenylyl cyclase (EC 4.6.1.1). Although those adenylyl cyclases described previously have a rather narrow tissue distribution, this enzyme (type IV) is apparently synthesized in a variety of peripheral tissues ... >> More

  We have cloned and expressed a cDNA that encodes a widely distributed form of mammalian adenylyl cyclase (EC 4.6.1.1). Although those adenylyl cyclases described previously have a rather narrow tissue distribution, this enzyme (type IV) is apparently synthesized in a variety of peripheral tissues and in the central nervous system. The protein resembles the other adenylyl cyclases in its proposed structure. It most resembles the type II adenylyl cyclase described in the preceding paper [Feinstein, P. G., Schrader, K. A., Bakalyar, H. A., Tang, W.-J., Krupinski, J., Gilman, A. G. & Reed, R. R. (1991) Proc. Natl. Acad. Sci. USA 88, 10173-10177] in its amino acid sequence, lack of response to calmodulin, and synergistic activation by a combination of the Gs alpha subunit and the G-protein beta gamma subunit complex. << Less

  Proc. Natl. Acad. Sci. U.S.A. 88:10178-10182(1991) [PubMed] [EuropePMC]

• Crystal structure of the catalytic domains of adenylyl cyclase in a complex with Gsalpha.GTPgammaS.

  Tesmer J.J.G., Sunahara R.K., Gilman A.G., Sprang S.R.

  The crystal structure of a soluble, catalytically active form of adenylyl cyclase in a complex with its stimulatory heterotrimeric G protein alpha subunit (Gsalpha) and forskolin was determined to a resolution of 2.3 angstroms. When P-site inhibitors were soaked into native crystals of the complex ... >> More

  The crystal structure of a soluble, catalytically active form of adenylyl cyclase in a complex with its stimulatory heterotrimeric G protein alpha subunit (Gsalpha) and forskolin was determined to a resolution of 2.3 angstroms. When P-site inhibitors were soaked into native crystals of the complex, the active site of adenylyl cyclase was located and structural elements important for substrate recognition and catalysis were identified. On the basis of these and other structures, a molecular mechanism is proposed for the activation of adenylyl cyclase by Gsalpha. << Less

  Science 278:1907-1916(1997) [PubMed] [EuropePMC]

• Adenylyl cyclase Rv1264 from Mycobacterium tuberculosis has an autoinhibitory N-terminal domain.

  Linder J.U., Schultz A., Schultz J.E.

  Mycobacterium tuberculosis contains 15 class III adenylyl cyclase genes. The gene Rv1264 is predicted to be composed of two distinct protein modules. The C terminus seems to code for a catalytic domain belonging to a subfamily of adenylyl cyclase isozymes mostly found in Gram-positive bacteria. Th ... >> More

  Mycobacterium tuberculosis contains 15 class III adenylyl cyclase genes. The gene Rv1264 is predicted to be composed of two distinct protein modules. The C terminus seems to code for a catalytic domain belonging to a subfamily of adenylyl cyclase isozymes mostly found in Gram-positive bacteria. The expressed protein was shown to function as a homodimeric adenylyl cyclase (1 micromol of cAMP x mg(-1) x min(-1)). In analogy to the structure of the mammalian adenylyl cyclase catalyst, six amino acids were targeted by point mutations and found to be essential for catalysis. The N-terminal region represents a novel protein domain, the occurrence of which is restricted to several adenylyl cyclases present in Gram-positive bacteria. The purified full-length enzyme was 300-fold less active than the catalytic domain alone. Thus, the N-terminal domain appeared to be autoinhibitory. The N-terminal domain contains three prominent polar amino acid residues (Asp(107), Arg(132), and Arg(191)) that are invariant in all seven sequences of this domain currently available. Mutation of Asp(107) to Ala relaxed the inhibition and resulted in a 6-fold increase in activity of the Rv1264 holoenzyme, thus supporting the role of this domain as a potential novel regulator of adenylyl cyclase activity. << Less

  J. Biol. Chem. 277:15271-15276(2002) [PubMed] [EuropePMC]