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.     

Functional characterization and expression analysis of members of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase family from Drosophila melanogaster

Here we report the cloning and functional characterization of eight members of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase gene family from Drosophila melanogaster (polypeptide GalNAc transferase = pgant1-8). Full-length cDNAs were isolated from a Drosophila embryonic library based on homology to known ppGaNTases. Alignments with characterized mammalian isoforms revealed strong sequence similarities between certain fly and mammalian isoforms, highlighting putative orthologues between the species. In vitro activity assays demonstrated biochemical transferase activity for each gene, with three isoforms requiring glycosylated substrates. Comparison of the activities of Drosophila and mammalian orthologues revealed conservation of substrate preferences against a panel of peptide and glycopeptide substrates. Furthermore, Edman degradation analysis demonstrated that preferred sites of GalNac addition were also conserved between certain fly and mammalian orthologues. Semi-quantitative PCR amplification of Drosophila cDNA revealed expression of most isoforms at each developmental stage, with some isoforms being less abundant at certain stages relative to others. In situ hybridization to Drosophila embryos revealed specific staining of pgant5 and pgant6 in the salivary glands and pgant5 in the developing hindgut. Additionally, pgant5 and pgant6 expression within the egg chamber was restricted to the follicle cells, cells known to be involved in egg formation and subsequent embryonic patterning. The characterization reported here provides additional insight into the use of this model system to dissect the biological role of this enzyme family in vivo during both fly and mammalian development.     

Dynamic expression of alternate splice forms of D-cbl during embryogenesis

The Cbl family of proteins act as E3 ubiquitin-protein ligases and have been associated with the down regulation of a variety of receptor tyrosine kinases. Cbl proteins associate with many different cell signalling molecules suggesting that they may have functions outside of the RING finger-mediated ubiquitin ligase activity. The Drosophila melanogaster cbl gene (D-cbl) encodes two splice forms (Oncogene 19 (2000) 3299). Here we report on the differential expression of these isoforms during Drosophila embryogenesis. Both isoforms are maternally expressed but the long isoform of D-cbl is also transiently expressed in the invaginating mesoderm and later is specifically expressed in neurons of the central nervous system (CNS). Cbl protein is shown to be localised to axons of the longitudinal connectives and commissures in the central nervous system.     

Cloning and expression of sprint, a Drosophila homologue of RIN1

The small GTPase Ras is critical for regulation of growth and differentiation during development. The mammalian protein RIN1 is a potential Ras effector protein, which can also interact with the Abelson tyrosine kinase. However, its biological function is unknown. We have identified the Drosophila homologue of RIN1, called sprint, for SH2, poly-proline containing Ras interactor. The sprint locus is very large and contains at least two differentially expressed isoforms (sprint-a and sprint-b). Both isoforms are expressed in the ovary and maternal mRNA is deposited into embryos. In addition, sprint is zygotically expressed in the developing midgut, amnioserosa and in a specific subset of CNS neurons. The expression patterns of the two sprint isoforms are temporally distinct suggesting that the isoforms may have unique functions.     

Characterization of two novel lipocalins expressed in the Drosophila embryonic nervous system

We have found two novel lipocalins in the fruit fly Drosophila melanogaster that are homologous to the grasshopper Lazarillo, a singular lipocalin within this protein family which functions in axon guidance during nervous system development. Sequence analysis suggests that the two Drosophila proteins are secreted and possess peptide regions unique in the lipocalin family. The mRNAs of DNLaz (for Drosophila neural Lazarillo) and DGLaz (for Drosophila glial Lazarillo) are expressed with different temporal patterns during embryogenesis. They show low levels of larval expression and are highly expressed in pupa and adult flies. DNLaz mRNA is transcribed in a subset of neurons and neuronal precursors in the embryonic CNS. DGLaz mRNA is found in a subset of glial cells of the CNS: the longitudinal glia and the medial cell body glia. Both lipocalins are also expressed outside the nervous system in the developing gut, fat body and amnioserosa. The DNLaz protein is detected in a subset of axons in the developing CNS. Treatment with a secretion blocker enhances the antibody labeling, indicating the DNLaz secreted nature. These findings make the embryonic nervous system expression of lipocalins a feature more widespread than previously thought. We propose that DNLaz and DGLaz may have a role in axonal outgrowth and pathfinding, although other putative functions are also discussed.     

Compartmentalization of Distinct cAMP Signaling Pathways in Mammalian Sperm

Fertilization competence is acquired in the female tract in a process known as capacitation. Capacitation is needed for the activation of motility (e.g. hyperactivation) and to prepare the sperm for an exocytotic process known as acrosome reaction. Although the HCO₃⁻-dependent soluble adenylyl cyclase Adcy10 plays a role in motility, less is known about the source of cAMP in the sperm head. Transmembrane adenylyl cyclases (tmACs) are another possible source of cAMP. These enzymes are regulated by stimulatory heterotrimeric G_s proteins; however, the presence of G_s or tmACs in mammalian sperm has been controversial. In this study, we used Western blotting and cholera toxin-dependent ADP-ribosylation to show the G_s presence in the sperm head. Also, we showed that forskolin, a tmAC-specific activator, induces cAMP accumulation in sperm from both WT and Adcy10-null mice. This increase is blocked by the tmAC inhibitor SQ22536 but not by the Adcy10 inhibitor KH7. Although G_s immunoreactivity and tmAC activity are detected in the sperm head, PKA is only found in the tail, where Adcy10 was previously shown to reside. Consistent with an acrosomal localization, G_s reactivity is lost in acrosome-reacted sperm, and forskolin is able to increase intracellular Ca²⁺ and induce the acrosome reaction. Altogether, these data suggest that cAMP pathways are compartmentalized in sperm, with G_s and tmAC in the head and Adcy10 and PKA in the flagellum.     

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.     

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.     

Two domains are critical for the nuclear localization of soluble adenylyl cyclase

Soluble adenylyl cyclase (sAC) is a newly identified source of cyclic adenosine 3',5'-monophosphate (cAMP). Unlike the well-known transmembrane adenylyl cyclases (tmACs), sAC locates to the nucleus, mitochondria and microtubules. For most cAMP-signaling microdomains, there is always an AC nearby, for example tmAC. But it was until the discovery of sAC that there was not known cAMP resource in the nucleus. sAC associates with nuclear cAMP-signaling microdomains, which were once considered to depend on the diffusion of cAMP produced by tmAC. In this report, we focus on the truncated soluble adenylyl cyclase (tsAC), the most common existence form of sAC in tissues. Two domains (145-200 aa and 257-318 aa) related with sAC nuclear localization were present here. The findings provide evidence that these two domains are critical for the nuclear localization of sAC and they collocated with the catalytic domains.     

Function of RNA-binding protein Musashi-1 in stem cells

Musashi is an evolutionarily conserved family of RNA-binding proteins that is preferentially expressed in the nervous system. The first member of the Musashi family was identified in Drosophila. This protein plays an essential role in regulating the asymmetric cell division of ectodermal precursor cells known as sensory organ precursor cells through the translational regulation of target mRNA. In the CNS of Drosophila larvae, however, Musashi is expressed in proliferating neuroblasts and likely has a different function. Its probable mammalian homologue, Musashi-1, is a neural RNA-binding protein that is strongly expressed in fetal and adult neural stem cells (NSCs). Mammalian Musashi-1 augments Notch signaling through the translational repression of its target mRNA, m-Numb, thereby contributing to the self-renewal of NSCs. In addition to its functions in NSCs, the role of mammalian Musashi-1 protein in epithelial stem cells, including intestinal and mammary gland stem cells, is attracting increasing interest.     

NKD, a developmentally regulated tachykinin receptor in Drosophila.

A number of neuropeptides have been described which are present in the insect nervous system. The physiological role of these neuropeptides has not yet been clarified. We have characterized a Drosophila melanogaster cDNA coding for a protein, NKD, whose sequence resembles that of mammalian G protein-coupled neuropeptide receptors. This protein shows 38% homology with the mammalian tachykinin NK3 receptor within the transmembrane domain region. Stable cell lines expressing this cDNA are responsive to Locusta migratoria tachykinin but not to other peptides of the tachykinin family. The expression of this gene is detected principally in adult fly heads, but also in the adult body and in embryos. Interestingly, NKD mRNA is detected at very early stages of Drosophila embryonic development (3 h) and reaches the highest level of expression at 12-16 h, a time which correlates with the period of major neuronal development. In situ hybridization experiments demonstrate that NKD is expressed in the central nervous system, as well as in subsets of neurons in each segment of the developing ventral ganglia. The cytological localization of this gene is at position 86C on the Drosophila third chromosome.