Novel short isoforms of adenylyl cyclase as negative regulators of cAMP production

Here, we cloned a new family of four adenylyl cyclase (AC) splice variants from interleukin-1β (IL-1β)-transdifferentiated vascular smooth muscle cells (VSMCs) encoding short forms of AC8 that we have named “AC8E-H”. Using biosensor imaging and biochemical approaches, we showed that AC8E-H isoforms have no cyclase activity and act as dominant-negative regulators by forming heterodimers with other full-length ACs, impeding the traffic of functional units towards the plasma membrane. The existence of these dominant-negative isoforms may account for an unsuspected additional degree of cAMP signaling regulation. It also reconciles the induction of an AC in transdifferentiated VSMCs with the vasoprotective influence of cAMP. The generation of alternative splice variants of ACs may constitute a generalized strategy of adaptation to the cell's environment whose scope had so far been ignored in physiological and/or pathological contexts.     

Molecular details of cAMP generation in mammalian cells: a tale of two systems

The second messenger cAMP has been extensively studied for half a century, but the plethora of regulatory mechanisms controlling cAMP synthesis in mammalian cells is just beginning to be revealed. In mammalian cells, cAMP is produced by two evolutionary related families of adenylyl cyclases, soluble adenylyl cyclases (sAC) and transmembrane adenylyl cyclases (tmAC). These two enzyme families serve distinct physiological functions. They share a conserved overall architecture in their catalytic domains and a common catalytic mechanism, but they differ in their sub-cellular localizations and responses to various regulators. The major regulators of tmACs are heterotrimeric G proteins, which transduce extracellular signals via G protein-coupled receptors. sAC enzymes, in contrast, are regulated by the intracellular signaling molecules bicarbonate and calcium. Here, we discuss and compare the biochemical, structural and regulatory characteristics of the two mammalian AC families. This comparison reveals the mechanisms underlying their different properties but also illustrates many unifying themes for these evolutionary related signaling enzymes.     

An isoform-specific interaction of the membrane anchors affects mammalian adenylyl cyclase type V activity.

The nine membrane-bound mammalian adenylyl cyclases (ACs) contain two highly diverged membrane anchors, M1 and M2, with six transmembrane spans each and two conserved cytosolic domains which coalesce into a pseudoheterodimeric catalytic unit. Previously, the catalytic segments, bacterially expressed as soluble proteins, were characterized extensively whereas the function of the membrane domains remained unexplored. Using the catalytic C1 and C2 domains of AC type V we employed the membrane anchors from type V and VII ACs for construction of enzymes with duplicated, inverted, fully swapped and chimeric membrane anchors. Further, in the M1 membrane domain individual transmembrane spans were removed or exchanged between type V and VII ACs. The constructs were expressed in HEK293 cells, the expression levels and membrane localization was assessed by Western blotting. Cell-free basal, forskolin-, GTP gamma S-and G(s alpha)/GTP gamma S-stimulated AC activities were determined. The results demonstrate that enzymatic activities were only maintained when the M1 and M2 membrane domains were derived from either AC V or VII. Constructs with chimeric membrane domains, i.e. M1 from type V and M2 from type VII AC or vice versa, were essentially inactive although the expression levels and membrane localization appeared to be normal. The data indicate a functionally important interaction of the membrane domains of ACs in that they seem to interact in a pair-like, isoform delimited manner. This interaction directly impinges on the formation of the catalytic interface. We propose that protein-protein interactions of the AC membrane domains may constitute another, yet unexplored level of AC regulation.     

Cellular Localisation of Adenylyl Cyclase: A Post-genome Perspective

The intracellular messenger cAMP is essential for vital processes ranging from ovulation to cognition. There are 10 genes for adenylyl cyclase (AC), the biosynthetic enzyme of cAMP. Nine of these encode membrane-bound proteins and one gives rise to soluble AC. The understanding of the biological significance of this molecular diversity is incomplete. Membrane-bound ACs conform to the same structural blueprint but have markedly different regulatory characteristics. AC mRNAs are differentially distributed in the body suggesting non-redundant physiological functions. The subcellular localisation of AC isoforms has not been examined in detail. Here we discuss the current knowledge on the intracellular targeting of AC isoforms, and highlight the technical problems of AC detection, some of which appear to be caused by the poor quality-control of commercially supplied antibodies. The principal message is that intracellular targeting of ACs may be isoform-specific and also dependent on the cellular context of expression.Invocation: This paper was written to honour one of the founders of chemical neuroanatomy—Professor Miklós Palkovits on his 70th birthday.     

Particulate and soluble adenylyl cyclases participate in the sperm acrosome reaction

cAMP is important in sea urchin sperm signaling, yet the molecular nature of the adenylyl cyclases (ACs) involved remained unknown. These cells were recently shown to contain an ortholog of the mammalian soluble adenylyl cyclase (sAC). Here, we show that sAC is present in the sperm head and as in mammals is stimulated by bicarbonate. The acrosome reaction (AR), a process essential for fertilization, is influenced by the bicarbonate concentration in seawater. By using functional assays and immunofluorescence techniques we document that sea urchin sperm also express orthologs of multiple isoforms of transmembrane ACs (tmACs). Our findings employing selective inhibitors for each class of AC indicate that both sAC and tmACs participate in the sperm acrosome reaction.     

Physiological roles for G protein-regulated adenylyl cyclase isoforms: insights from knockout and overexpression studies

Cyclic AMP is a universal second messenger, produced by a family of adenylyl cyclase (AC) enzymes. The last three decades have brought a wealth of new information about the regulation of cyclic AMP production by ACs. Nine hormone-sensitive, membrane-bound AC isoforms have been identified in addition to a tenth isoform that lacks membrane spans and more closely resembles the cyanobacterial AC enzymes. New model systems for purifying and characterizing the catalytic domains of AC have led to the crystal structure of these domains and the mapping of numerous interaction sites. However, big hurdles remain in unraveling the roles of individual AC isoforms and their regulation in physiological systems. In this review we explore the latest on AC knockout and overexpression studies to better understand the roles of G protein regulation of ACs in the brain, olfactory bulb, and heart.     

Adenylyl cyclases in the digestive system

Adenylyl cyclases (ACs) are a group of widely distributed enzymes whose functions are very diverse. There are nine known transmembrane AC isoforms activated by Gαs. Each has its own pattern of expression in the digestive system and differential regulation of function by Ca^2 + and other intracellular signals. In addition to the transmembrane isoforms, one AC is soluble and exhibits distinct regulation. In this review, the basic structure, regulation and physiological roles of ACs in the digestive system are discussed.     

Adenylyl cyclase 3 mediates prostaglandin E(2)-induced growth inhibition in arterial smooth muscle cells.

Arterial smooth muscle cell (SMC) proliferation contributes to a number of vascular pathologies. Prostaglandin E(2) (PGE(2)), produced by the endothelium and by SMCs themselves, acts as a potent SMC growth inhibitor. The growth-inhibitory effects of PGE(2) are mediated through activation of G-protein-coupled membrane receptors, activation of adenylyl cyclases (ACs), formation of cAMP, and subsequent inhibition of mitogenic signal transduction pathways in SMCs. Of the 10 different mammalian AC isoforms known today, seven isoforms (AC2-7 and AC9) are expressed in SMCs from various species. We show that, despite the presence of several different AC isoforms, the principal AC isoform activated by PGE(2) in human arterial SMCs is a calmodulin kinase II-inhibited AC with characteristics similar to those of AC3. AC3 is expressed in isolated human arterial SMCs and in intact aorta. We further show that arterial SMCs isolated from AC3-deficient mice are resistant to PGE(2)-induced growth inhibition. In summary, AC3 is the principal AC isoform activated by PGE(2) in arterial SMCs, and AC3 mediates the growth-inhibitory effects of PGE(2). Because AC3 activity is inhibited by intracellular calcium through calmodulin kinase II, AC3 may serve as an important integrator of growth-inhibitory signals that stimulate cAMP formation and growth factors that increase intracellular calcium.     

Localized Na+/H+ exchanger 1 expression protects Ca2+-regulated adenylyl cyclases from changes in intracellular pH

The Ca2+-sensitive adenylyl cyclases (ACs) are exclusively regulated by capacitative Ca2+ entry (CCE) in nonexcitable cells. The present study investigates whether this Ca2+-dependent modulation of AC activity is further regulated by local pH changes that can arise beneath the plasma membrane as a consequence of cellular activity. Ca2+ stimulation of AC8 expressed in HEK 293 cells and inhibition of endogenous AC6 in C6-2B glioma cells exhibited clear sensitivity to modest pH changes in vitro. Acid pH (pH 7.14) reduced the Ca2+ sensitivity of both ACs, whereas alkaline pH (pH 7.85) enhanced the responsiveness of the enzymes to Ca2+, compared with controls (pH 7.50). Surprisingly, in the intact cell, the response of AC8 and AC6 to CCE was largely unperturbed by similar changes in intracellular pH (pH(i)), imposed using a weak acid (propionate) or weak base (trimethylamine). A range of hypotheses were tested to identify the mechanism(s) that could underlie this lack of pH effect in the intact cell. The pH sensitivity of CCE in HEK 293 cells is likely to dampen the effects of pH(i) on Ca2+-regulated ACs and may partly explain the discrepancy between in vitro and in vivo data. However, we have found that the Na+/H+ exchanger (NHE), NHE1, is functionally active in these cells, and like AC8 (and AC6) it resides in lipid rafts or caveolae, which may create cellular microdomains where pH(i) is tightly regulated. An abundance of NHE1 in these cellular subdomains may generate a privileged environment that protects the Ca2+-sensitive ACs and other caveolar proteins from local acid shifts.     

Characterization of the human adenylyl cyclase gene family: cDNA,gene structure,and tissue distribution of the nine isoforms

The membrane-bound adenylyl cyclases (ACs) represent one of the major families of effector enzymes for G protein-coupled receptors. Eight human AC isoforms, encoded by separate genes, have been identified up to now. However, in several cases only partial cDNA sequences are available (ADCY1,2,5). A ninth expected isoform, the human ortholog of rat ADCY4, has not been described yet. Using the high inter-species homology of mammalian AC isoforms, we searched the human genome and we succeeded to identify full-length coding sequences for all enzymes. Where required, missing sequence information was provided experimentally. Analysis of genomic sequences from the Celera database also allowed us to determine the exon-intron boundaries for ADCY1-9 and to establish the gene structures. We found that human AC genes comprise 11 to 26 exons, which are distributed over 16 to 430kb. We further report expression profiles for the nine ACs in a panel of 16 human tissues and in human embryonic kidney (HEK) cells.     

Hydroxamate based inhibitors of adenylyl cyclase. Part 1: the effect of acyclic linkers on P-site binding

The adenylyl cyclases (ACs) are a family of enzymes that are key elements of signal transduction by virtue of their ability to convert ATP to cAMP. The catalytic mechanism of this transformation proceeds through initial binding of ATP to the purine binding site (P-site) followed by metal mediated cyclization with loss of pyrophosphate. Crystallographic analysis of ACs with known inhibitors reveals the presence of two metals in the active site. Presently, nine isoforms of adenylyl cyclase are known and unique isoform combinations are expressed in a tissue specific manner. The development of isoform specific inhibitors of adenylyl cyclase may prove to be a useful strategy toward the design of novel therapeutic agents. In order to develop novel AC inhibitors, we have chosen a design approach utilizing molecules with the adenine ring system joined to a metal-coordinating hydroxamic acid via flexible acyclic linkers. The designed inhibitors were assayed against type V AC with the size and heteroatom content of the linkers varied to probe the interaction of the nucleotide and metal binding sites within the enzyme.     

Regulation of adenylyl cyclase in the central nervous system

Adenylyl cyclases (ACs) are a family of enzymes that synthesize one of the major second messengers, cAMP, upon stimulation. Since the report of the first adenylyl cyclase (AC) gene in 1989, tremendous efforts have been devoted to identifying and characterizing more AC isozymes. In the past decade, significant knowledge regarding the basic structure, tissue distribution, and regulation of AC isozymes has been accumulated. Because members of the AC superfamily are tightly controlled by various signals, one of the most important impacts of these AC isozymes is their contribution to the complexity and fine-tuning of cellular signalling, especially in the central nervous system (CNS) where multiple signals constantly occur. This review focuses on recent progress toward understanding the physiological roles of ACs in the CNS.     

CHARACTERIZATION OF THE HUMAN ADENYLYL CYCLASE GENE FAMILY: cDNA,GENE STRUCTURE,AND TISSUE DISTRIBUTION OF THE NINE ISOFORMS

ABSTRACT

The membrane-bound adenylyl cyclases (ACs) represent one of the major families of effector enzymes for G protein-coupled receptors. Eight human AC isoforms, encoded by separate genes, have been identified up to now. However, in several cases only partial cDNA sequences are available (ADCY1,2,5). A ninth expected isoform, the human ortholog of rat ADCY4, has not been described yet. Using the high inter-species homology of mammalian AC isoforms, we searched the human genome and we succeeded to identify full-length coding sequences for all enzymes. Where required, missing sequence information was provided experimentally. Analysis of genomic sequences from the Celera database also allowed us to determine the exon–intron boundaries for ADCY1–9 and to establish the gene structures. We found that human AC genes comprise 11 to 26 exons, which are distributed over 16 to 430?kb. We further report expression profiles for the nine ACs in a panel of 16 human tissues and in human embryonic kidney (HEK) cells.     

Use of an in silico approach to define the gene structure of eukaryotic adenylyl cyclases.

The limited information available regarding the gene structure of adenylyl cyclases (AC), which catalyze the synthesis of cAMP, suggests a complex arrangement with many exons and large introns such that molecular techniques to define these gene structures are time- and labor-intensive. We report here the use of a computer-based approach involving the assembly of fragmented sequence data generated by the Human Genome Project and nucleic acid analysis software to decipher the gene structure of human and murine AC 6 and other human AC isoforms (ACs 3, 7, and 8). The results, which document 21 exons in human and murine AC 6, human AC 3, 18 exons in AC 8, and 24 exons in AC 7, show substantial conservation of exon organization in the AC family and in particular regions of the AC protein. Application of such in silico methods should prove useful to characterize genes for other ACs and protein families and data provided here should facilitate studies of polymorphisms in AC genes.     

Gsα stimulation of mammalian adenylate cyclases regulated by their hexahelical membrane anchors

Mammalian adenylate cyclases (ACs) are pseudoheterodimers with dissimilar hexahelical membrane-anchors, isoform-specifically conserved for more than half a billion years. We exchanged both membrane anchors of the AC isoform 2 by the quorum-sensing receptor from Vibrio harveyi, CqsS, which has a ligand, Cholera-Autoinducer-1 (CAI-1). In the chimera, AC activity was stimulated by Gsα, CAI-1 had no effect. Surprisingly, CAI-1 inhibited Gsα stimulation. We report that Gsα stimulation of human AC isoforms 2, 3, 5, and 9 expressed in Sf9 cells is inhibited by serum as is AC activity in membranes isolated from rat brain cortex. AC2 activation by forskolin or forskolin/Gsα was similarly inhibited. Obviously, serum contains as yet unidentified factors affecting AC activity. The data establish a linkage in ACs, in which the membrane anchors, as receptors, transduce extracellular signals to the cytosolic catalytic dimer. A mechanistic three state model of AC regulation is presented compatible with all known regulatory inputs into mammalian ACs. The data allow designating the membrane anchors of mammalian ACs as orphan receptors, and establish a new level of AC regulation.     

Insect Stage-Specific Receptor Adenylate Cyclases Are Localized to Distinct Subdomains of the Trypanosoma brucei Flagellar Membrane

Increasing evidence indicates that the Trypanosoma brucei flagellum (synonymous with cilium) plays important roles in host-parasite interactions. Several studies have identified virulence factors and signaling proteins in the flagellar membrane of bloodstream-stage T. brucei, but less is known about flagellar membrane proteins in procyclic, insect-stage parasites. Here we report on the identification of several receptor-type flagellar adenylate cyclases (ACs) that are specifically upregulated in procyclic T. brucei parasites. Identification of insect stage-specific ACs is novel, as previously studied ACs were constitutively expressed or confined to bloodstream-stage parasites. We show that procyclic stage-specific ACs are glycosylated, surface-exposed proteins that dimerize and possess catalytic activity. We used gene-specific tags to examine the distribution of individual AC isoforms. All ACs examined localized to the flagellum. Notably, however, while some ACs were distributed along the length of the flagellum, others specifically localized to the flagellum tip. These are the first transmembrane domain proteins to be localized specifically at the flagellum tip in T. brucei, emphasizing that the flagellum membrane is organized into specific subdomains. Deletion analysis reveals that C-terminal sequences are critical for targeting ACs to the flagellum, and sequence comparisons suggest that differential subflagellar localization might be specified by isoform-specific C termini. Our combined results suggest insect stage-specific roles for a subset of flagellar adenylate cyclases and support a microdomain model for flagellar cyclic AMP (cAMP) signaling in T. brucei. In this model, cAMP production is compartmentalized through differential localization of individual ACs, thereby allowing diverse cellular responses to be controlled by a common signaling molecule.     

Residence of adenylyl cyclase type 8 in caveolae is necessary but not sufficient for regulation by capacitative Ca(2+) entry.

Ca(2+)-sensitive adenylyl cyclases (ACs) depend on capacitative Ca(2+) entry (CCE) for their regulation. Residence of the endogenous Ca(2+)-inhibitable adenylyl cyclase of C6-2B glioma cells in cholesterol-enriched caveolae is essential for its regulation by CCE (Fagan, K. A., Smith, K. E., and Cooper, D. M. F. (2000) J. Biol. Chem. 275, 26530-26537). In the present study, we established that depletion of cellular cholesterol ablated the regulation by CCE of a Ca(2+)-stimulable adenylyl cyclase, AC8, heterologously expressed in HEK293 cells. We considered the possibility that a calmodulin-binding domain in the N terminus of AC8, which is not required for in vitro regulation by Ca(2+), might play a targeting role. Deletion and mutation of the N terminus did attenuate the enzyme's sensitivity to CCE without altering its in vitro responsiveness to Ca(2+)/calmodulin. Both N terminus-deleted AC8 and wild type AC8 were expressed at the plasma membrane, as shown by imaging analysis of green fluorescence protein-tagged constructs. However, not only wild type AC8 but also the CCE-insensitive mutants occurred in caveolar fractions of the plasma membranes, even though a Ca(2+)-insensitive adenylyl cyclase, AC7, was excluded from caveolae. Finally, the AC8 mutants were no more responsive to nonphysiological elevation of Ca(2+) than the wild type. We conclude that (i) not all adenylyl cyclases reside in caveolae, (ii) the calmodulin-binding domain in the N terminus of AC8 does not play a role in caveolar targeting, (iii) the N terminus does play a role in associating AC8 with factors that confer sensitivity to CCE, and (iv) residence of Ca(2+)-sensitive adenylyl cyclases in caveolae is essential but not sufficient for regulation by CCE.     

Transmembrane Signaling in Chimeras of the Escherichia coli Aspartate and Serine Chemotaxis Receptors and Bacterial Class III Adenylyl Cyclases

The Escherichia coli chemoreceptors for serine (Tsr) and aspartate (Tar) and several bacterial class III adenylyl cyclases (ACs) share a common molecular architecture; that is, a membrane anchor that is linked via a cytoplasmic HAMP domain to a C-terminal signal output unit. Functionality of both proteins requires homodimerization. The chemotaxis receptors are well characterized, whereas the typical hexahelical membrane anchor (6TM) of class III ACs, suggested to operate as a channel or transporter, has no known function beyond a membrane anchor. We joined the intramolecular networks of Tsr or Tar and two bacterial ACs, Rv3645 from Mycobacterium tuberculosis and CyaG from Arthrospira platensis, across their signal transmission sites, connecting the chemotaxis receptors via different HAMP domains to the catalytic AC domains. AC activity in the chimeras was inhibited by micromolar concentrations of l-serine or l-aspartate in vitro and in vivo. Single point mutations known to abolish ligand binding in Tar (R69E or T154I) or Tsr (R69E or T156K) abrogated AC regulation. Co-expression of mutant pairs, which functionally complement each other, restored regulation in vitro and in vivo. Taken together, these studies demonstrate chemotaxis receptor-mediated regulation of chimeric bacterial ACs and connect chemical sensing and AC regulation.