rutabaga
A novel isoform of adenylyl cyclase, DAC78C, has been identified in Drosophila that encodes two structurally distinct proteins in a developmentally
restricted manner. The protein corresponding to
one transcript is potently activated by G protein and protein
kinase C and is expressed ubiquitously. The protein
corresponding to the second transcript is expressed in a
dynamic pattern in gastrulation stage embryos; it is restricted
to the cephalic furrow and dorsal transverse
folds, active regions of cell movement of unknown function
in the Drosophila embryo. It is proposed that
DAC78C and the cAMP pathway play an important role
in directing these morphogenetic movements:
this gene may provide clues to the functional significance
of these structures in gastrulation (Cann, 2000a).
The hydrophobicity profile of the ORF has revealed a proposed structure
resembling the known transmembrane adenylyl cyclase (tmAC) isoforms, consisting of
12 putative transmembrane domains; homology
searches have identified two presumptive catalytic domains. This novel Drosophila tmAC displays additional structural characteristics distinct from other
known tmACs. The ORF has a large, approximately
100 amino acid extracellular loop between transmembrane
domains 9 and 10 containing four potential N-linked
glycosylation sites. The ORF also has a 300 amino acid long intracellular N terminal domain of unknown function. Both regions are considerably longer than the analogous domains in previously identified mammalian and Drosophila tmACs (Cann, 2000a).
To determine whether DAC78C is the ortholog of a
previously identified mammalian AC gene
extensive phylogenetic analysis was performed. CAC79C
is not a clear ortholog of any previously identified
mammalian AC. DAC78C is the sole member of a distinct evolutionary
clade. Other Drosophila tmAC isoforms have clear mammalian
orthologs based on similar analysis; Rutabaga is orthologous to bovine AC1 (38.3% similar), DAC39E is orthologous
to rat AC3 (33.4% similar), and DAC9 is orthologous to mouse AC9 (28.7% similar). In each case the mammalian tmAC is more similar to its Drosophila ortholog than it is to the other mammalian isoforms. Among the known mammalian
tmACs, DAC78C is most similar to rat AC8 (30.4%
similar) and rat AC5 (29.1% similar); however, rat AC8
and rat AC5 are more similar to other mammalian tmACs
than to DAC78C, making relationships based purely on
primary sequence data problematic. Another Drosophila tmAC has been identified,
DAC76E, corresponding to mammalian tmAC types 2, 4, and 7. Drosophila therefore possesses a single tmAC isoform corresponding to each clade of related
mammalian tmACs: Rutabaga with AC1, AC5, and AC6; DAC39E with AC3; DAC9 with AC9, and
DAC76E with AC2, AC4, and AC7. In contrast DAC78C is the sole member of a distinct clade among the higher eukaryotic tmACs (Cann, 2000a).
The larger, approximately 6.5 kb transcript is
ubiquitously expressed in embryos, larvae, and adults,
and the smaller, approximately 4.5 kb transcript is present,
seemingly, only in embryos. Using developmentally
staged embryonic total RNA, the 6.5 kb transcript has been
shown to be ubiquitous from hour 8 of embryogenesis
while the 4.5 kb transcript is expressed specifically in
2-4 h embryos. To characterize the molecular difference between the 6.5 and 4.5 kb transcripts, the genomic structure of the entire DAC78C locus was
solved. The 4.5 kb transcript encodes
an N terminal truncation of the full-length
DAC78C protein, missing transmembrane domains 1-6. The 6.5 kb transcript was thereafter denoted DAC78C L, and the 4.5 kb transcript was denoted
DAC78C S. Transmembrane domains 1-6 are intact in the
DAC78C L transcript. Despite possessing two catalytic domains, which have
been shown to be both necessary and sufficient for catalytic
activity, AC activity could not be demonstrated for DAC78C S using a variety of
heterologous expression systems (Cann, 2000a).
The DAC78C L transcript is
expressed ubiquitously in late embryos.
DAC78C S, which is temporally restricted to the earliest
(2-4) hours of embryogenesis, is first detected at stage
4 just prior to cellularization, throughout the embryo. Stage 4 corresponds approximately to a 2-h-old
embryo, and its initial appearance at this stage implies
that DAC78C S is zygotically transcribed rather then maternally
deposited in the embryo. As the embryo cellularizes,
DAC78C S mRNA levels rise and become excluded from the anterior and posterior poles.
As germ band extension commences, expression decreases
along the entire ventral surface of the embryo and is restricted to the CF and DTFs, active regions of cell involution of unknown function. By late
stage 7, strong expression of DAC78C S is present only in
the CF and DTFs on the dorsal and lateral surfaces of the
embryo. A view of the dorsal surface of a late
stage 7 embryo demonstrates the restriction of mRNA
expression to the cephalic furrow (CF) and dorsal transverse folds (DTFs). Expression in the CF persists longer dorsally than ventrally. This may
reflect distinct mechanisms that control normal CF formation
and DAC78C S expression dorsally and laterally.
At stage 8, corresponding to an approximately 4-h-old
embryo, DAC78C S mRNA is no longer detectable. Thus
DAC78C S represents the first example of an AC specifically
expressed during development. The function of these gastrulating regions and the
mechanisms that direct these morphogenetic movements are unknown (Cann, 2000a).
The cloning and characterization
of a new gene family of adenylyl cyclase related genes in
Drosophila is described. The five adenylyl cyclase-like genes that define
this family are clearly distinct from previously
known adenylyl cyclases. One member forms a unique
locus on chromosome 3 whereas the other four members
form a tightly clustered, tandemly repeated array on chromosome
2. The genes on chromosome 2 are transcribed
in the male germline in a doublesex dependent manner
and are expressed in postmitotic, meiotic, and early differentiating
sperm. These genes therefore provide the first
evidence for a role for the cAMP signaling pathway in
Drosophila spermatogenesis. Expression from this locus
is under the control of the always early, cannonball, meiosis arrest, and spermatocyte arrest genes that are required
for the G2/M transition of meiosis I during spermatogenesis,
implying a mechanism for the coordination
of differentiation and proliferation. Evidence is also provided
for positive selection at the locus on chromosome 2,
which suggests this gene family is actively evolving and
may play a novel role in spermatogenesis (Cann, 2000b).
cDNAs have been isolated that correspond to two previously identified
transmembrane adenylyl cyclase (tmAC) related genomic DNA fragments, AC34A and
AC62D. Their predicted protein products
are highly related to each other, and closely related
to known mammalian and Drosophila tmACs. PCR with
degenerate primers designed to selectively amplify similar
genes has identified two additional highly related sequences
that were subsequently cloned. The four genes have been renamed
Drosophila AC 'X' (DACX)A (formerly called
AC62D); DACXB (formerly called AC34A); DACXC,
and DACXD, to reflect the fact that they are a related
family of genes distinct from the known tmACs. The reconstructed
full-length cDNA clones each possess a single
open reading frame (ORF) homologous to previously
characterized mammalian and Drosophila tmACs. The
hydrophobicity profiles of each ORF reveal a proposed
structure resembling the known tmAC isoforms consisting
of 12 putative transmembrane domains; homology
searches have identified two presumptive catalytic domains. To determine whether DACXA-D are orthologs
of any previously identified mammalian AC gene
phylogenetic analysis was performed using the complete amino
acid sequences. The phylogenetic tree reveals
that the DACX ORFs represent a novel gene family distinct
from any previously identified tmACs (70% similar
to one another compared to 25%-35% similarity to all other
tmACs in the catalytic regions). The proteins are only distantly related to Drosophila Rutabaga (E-value = e-42). Examination of the
Caenorhabditis elegans genome sequence and the GenBank
Expressed Sequence Tag database does not reveal any
likely orthologous sequences. Comparison of the amino
acid sequences of DACXA-D with the known tmACs reveals
that all residues shown by the tmAC crystal structure
to be essential for catalytic activity are intact; however, contact residues for Gsalpha.GTPgammaS and
forskolin are not conserved (Cann, 2000b).
Since DACXA and DACXB had been previously cytologically
localized to polytene bands 62D and 34A, respectively, P1 clones were obtained
that encompassed these loci: 62D (DS00542) and 34A
(DS03829). Surprisingly, DACXC also resides
on P1 DS03829 (34A) and DACXD on P1
DS00542 (62D). Further analysis reveals that DACXA
is also found on P1 DS03829 (34A) and not at 62D, as
previously described. This false cytology is thought to reflect
the extensive cross-hybridization observed between
the DACX family members. The intron/exon
structures of the loci at 34A and 62D were mapped by a
combination of subcloning, PCR, and DNA sequence
analysis. These analyses have revealed a fifth member
of the gene family, DACXE, which resides on P1
DS03829 (34A). A putative ORF was reconstructed for
DACXE that demonstrates that it is highly related to
the other DACX family members. This gene
was not analyzed further. DACXD is found as a single
gene at 62D whereas DACXA, DACXB, DACXC, and
DACXE form a tandem array of directly repeated genes
at 34A with 39 bp (DACXC to DACXB), 84 bp (DA-CXB
to DACXA), and 166 bp (DACXA to DACXE) between
transcription units. All 11 intron/exon boundaries
are conserved between the DACX family members at
34A although there is some variation in intron size. Five
of these intron/exon boundaries are also conserved with
DACXD (Cann, 2000b).
The expression profiles of DACXA-D were examined
by Northern blotting of developmentally staged RNA. DACXD is expressed predominantly during late
embryogenesis, in late larvae, and in male and female
adults. In contrast, DACXA, DACXB, and DACXC,
which reside in the tandem array at 34A, are expressed at
low levels in late larvae, increase during the pupal stage,
and exclusively in adult males. Presumably the expression
in late larvae and pupae is restricted to males of
these mixed populations of males and females.
Spatial expression of DACXA, DACXB, and DACXC
mRNA was examined by in situ hybridization to
whole Drosophila testes with antisense riboprobes.
The expression profile is similar for all three genes.
Expression is absent in the stem cells at the distal tip of
the testis and in the mitotic cells that undergo four
rounds of division within somatically derived cyst cells. Expression is first evident in prophase pre-meiotic
spermatocytes that undergo considerable growth
before meiosis. mRNA is detectable throughout
meiosis and in early haploid postmeiotic
spermatids (Cann, 2000b).
Although cAMP has been
known to play an essential role in fruit fly oogenesis,
these AC-like genes provide the first evidence for a potential
role of cAMP signaling in Drosophila spermatogenesis.
Developing sperm undergo a number of predefined
developmental processes that are tightly controlled.
Perhaps the best described of these processes is the
checkpoint that controls the transition through the G2/M
phase of meiosis I. aly is the master gene essential for
the normal function of can, sa, mia, and the transcriptional
activation of the cdc25 phosphatase ortholog,
twine. The expression of DACXA, DACXB, and
DACXC is downregulated in adult males mutant
for aly, can, mia, or sa. The control of the DACX
genes by the G2/M checkpoint is likely to provide a powerful
mechanism for coordinating the precise requirements for
cell cycle progression with postmeiotic differentiation.
Mammals
also possess a distinct AC-related gene, sAC, which is
specifically expressed in the male germline. Previous studies have demonstrated that dsx in
Drosophila and mab-3, a dsx homolog in C. elegans, direct
the transcription of yolk proteins that are entirely
distinct between species. The
DACX family and sAC may also represent convergent
evolution for a specific and novel role for cAMP in Drosophila
and mammalian spermatogenesis (Cann, 2000b).
The mechanism of P-site inhibition of adenylyl cyclase has been probed by equilibrium binding
measurements using 2'-[3H]deoxyadenosine, a P-site inhibitor, and by kinetic analysis of both the
forward and reverse reactions (i.e. cyclic AMP and ATP synthesis, respectively). There is one binding
site for 2'-deoxyadenosine per C1/C2 heterodimer. Binding is
observed only in the presence of one of the products of the adenylyl cyclase reaction, pyrophosphate
(PPi). A substrate analog, Ap(CH2)pp (alpha,beta-methylene adenosine 5'-triphosphate), and cyclic
AMP compete for the P-site in the presence of PPi, but P-site analogs do not compete for substrate
binding (in the absence of PPi). Kinetic analysis indicates that the release of products from the enzyme is
random. These facts permit formulation of a model for the adenylyl cyclase reaction, for which kinetic support is provided. It is proposed that P-site analogs act as dead-end inhibitors of
product release, stabilizing an enzyme-product (E-PPi) complex by binding at the active site. Although
product release is random, cyclic AMP dissociates from the enzyme preferentially. Release of PPi is
slow and partially rate-limiting (Dessauer, 1997).
Mammalian adenylyl cyclases contain two conserved regions, C1 and C2, which are responsible for forskolin- and G-protein-stimulated catalysis. The structure of the C2 catalytic region of type II rat adenylyl cyclase has an alpha/beta class fold in a
wreath-like dimer, which has a central cleft. Two forskolin molecules bind in hydrophobic pockets at the ends of cleft. The central part of the cleft is lined by charged residues implicated in ATP binding. Forskolin appears to activate adenylyl cyclase by promoting the assembly of the active dimer and by direct interaction within the catalytic cleft. Other adenylyl cyclase regulators act at the dimer
interface or on a flexible C-terminal region (Zhang, 1997).
Adenylyl cyclases possess complex structures like those of the ATP binding cassette (ABC) transporter family, which includes the cystic fibrosis transmembrane regulator, the P-glycoprotein, and ATP-sensitive K+ channels. These structures comprise a cytosolic N terminus followed by two tandem six-transmembrane cassettes, each associated with a highly homologous (ATP binding) cytosolic loop. The catalytic domains, which are located in the two large cytoplasmic loops, are highly conserved and well studied. Nothing is known of the function or organization of the 12 transmembrane segments. In the present study a range of strategies is adopted to analyze the trafficking and activity of this molecule. When expressed as individual peptides, the two transmembrane domains -- largely independent of any cytosolic region -- formed a tight complex that is delivered to the plasma membrane. This cooperation between the two intact transmembrane domains is essential and sufficient to target the enzyme to the plasma membrane of the cell. The extracellular loop between the ninth and tenth transmembrane segments, which contains an N-glycosylation site, is also necessary. Furthermore, the interaction between the two transmembrane clusters played a critical role in bringing together the cytosolic catalytic domains to express functional adenylyl cyclase activity in the intact cell (Gu, 2001).
Dictyostelium development is induced by starvation. The adenylyl cyclase gene ACA is one of the first
genes expressed upon starvation. ACA produces extracellular cAMP that induces chemotaxis,
aggregation, and differentiation in neighboring cells. Using insertional mutagenesis, a
mutant has been isolated that does not aggregate upon starvation but is rescued by adding extracellular cAMP.
Sequencing of the mutated locus reveals a new gene, DdMYB2, whose product contains three Myb
repeats, the DNA-binding motif of Myb-related transcription factors. Ddmyb2-null cells show
undetectable levels of ACA transcript and no cAMP production. Ectopic expression of ACA from a
constitutive promotor rescues the differentiation and morphogenesis of Ddmyb2-null mutants. The results
suggest that development in Dictyostelium starts by starvation-mediated DdMyb2 activation, which
induces adenylyl cyclase activity producing the differentiation-inducing signal cAMP (Otsuka, 1998).
The role of the adenylyl cyclase ACA in Dictyostelium discoideum chemotaxis and streaming was studied. In this process, cells orient themselves in a head to tail fashion as they are migrating to form aggregates. Cells lacking ACA are capable of moving up a chemoattractant gradient, but are unable to stream. Imaging of ACA-YFP reveals plasma membrane labeling highly enriched at the uropod (a slender posterior appendage) of polarized cells. This localization requires the actin cytoskeleton but is independent of the regulator CRAC and the effector PKA. A constitutively active mutant of ACA shows dramatically reduced uropod enrichment and has severe streaming defects. It is proposed that the asymmetric distribution of ACA provides a compartment from which cAMP is secreted to locally act as a chemoattractant, thereby providing a unique mechanism to amplify chemical gradients. This could represent a general mechanism that cells use to amplify chemotactic responses (Kriebel, 2003).
A variety of extracellular signals leads to the accumulation
of cAMP, which can act as a second message within cells by
activating protein kinase A (PKA). Expression of many
of the essential developmental genes in Dictyostelium
discoideum are known to depend on PKA activity. Cells in
which the receptor-coupled adenylyl cyclase gene, acaA, is
genetically inactivated grow well but are unable to develop.
Surprisingly, acaA- mutant cells can be rescued by
developing them in mixtures with wild-type cells,
suggesting that another adenylyl cyclase is present in
developing cells that can provide the internal cAMP
necessary to activate PKA. However, the only other known
adenylyl cyclase gene in Dictyostelium, acgA, is only
expressed during germination of spores and plays no role
in the formation of fruiting bodies. By screening
morphological mutants generated by Restriction Enzyme
Mediated Integration (REMI), a novel
adenylyl cyclase gene, acrA, was discovered. acrA is expressed at low levels
in growing cells and at more than 25-fold higher levels
during development. Growth and development up to the
slug stage are unaffected in acrA- mutant strains but the
cells make almost no viable spores and produce
unnaturally long stalks. Adenylyl cyclase activity increases
during aggregation; plateaus during the slug stage, and then
increases considerably during terminal differentiation. The
increase in activity following aggregation fails to occur in
acrA- cells. As long as ACA is fully active, ACR is not
required until culmination but then plays a critical role in
sporulation and construction of the stalk (Söderbom, 1999).
Activation of cAMP-dependent protein kinase (PKA) triggers terminal differentiation in Dictyostelium, without an obvious requirement for the G-protein-coupled
adenylyl cyclase, ACA, or the osmosensory adenylyl cyclase, ACG. A third adenylyl cyclase, ACB, was recently detected in rapidly developing mutants. The
specific characteristics of ACA, ACG, and ACB were used to determine their respective activities during development of wild-type cells. ACA is highly active
during aggregation, with negligible activity in the slug stage. ACG activity is not present at significant levels until mature spores have formed. ACB activity increases
strongly after slugs have formed with optimal activity at early fruiting body formation. The same high activity is observed in slugs of ACG null mutants and ACA null
mutants that overexpress PKA (acaA/PKA), indicating that it is not due to either ACA or ACG. The detection of high adenylyl cyclase activity in acaA/PKA null
mutants contradicts earlier conclusions that these mutants can develop into fruiting bodies in the complete
absence of cAMP. In contrast to slugs of null mutants for the intracellular cAMP-phosphodiesterase REGA, where both intact cells and lysates show ACB activity,
wild-type slugs only show activity in lysates. This indicates that cAMP accumulation by ACB in living cells is controlled by REGA. Both REGA inhibition and PKA
overexpression cause precocious terminal differentiation. The developmental regulation of ACB and its relationship to REGA suggest that ACB activates PKA and
induces terminal differentiation (Meima, 1999).
There are at least six mammalian adenylcyclases. Type I, to which rutabaga is homologous, is a Calmodulin-sensitive form found in the brain and other tissues. Calmodulin is a ubiquitious receptor of Ca++ that senses the intracellular Ca++ concentration: based on these levels it will bind to and activate other proteins. Types II, III and IV are found in brain and lung, olfactory tissue and testis respectively. All these are Calmodulin insensitive. Each of these proteins share a similar predicted structure. Each has two clusters of six transmembrane segments separated by two homologous cytoplasmic domains. Sequences within each cytoplasmic domain display significant homology with the catalytic portion of guanylyl cyclases, suggesting their involvement in cyclase activity. Two large cyclase domains have been found in Rutabaga with homology to bovine type I cyclase (Levin, 1992).
The distribution of type I calmodulin-sensitive adenylyl cyclase in bovine and rat tissues was examined
by northern blot analysis and in situ hybridization. Messenger RNA for type I adenylyl cyclase is found only in
brain, retina, and adrenal medulla, suggesting that this enzyme is neural specific. In situ hybridization
studies using bovine, rabbit, and rat retina indicate that mRNA for type I adenylyl cyclase is found in
all three nuclear layers of the neural retina and is particularly abundant in the inner segment of the
photoreceptor cells. The neural-specific distribution of type I adenylyl cyclase mRNA and its restricted
expression in areas of brain implicated in neuroplasticity are consistent with the proposal that this
enzyme plays an important role in various neuronal functions, including learning and memory (Xia, 1993).
The distribution of mRNA encoding the Calmodulin-sensitive Adenylate cyclase in rat
brain have been examined by in situ hybridization. mRNA for this enzyme is expressed in specific areas of
brain that have been implicated in learning and memory, including the neocortex, the hippocampus,
and the olfactory system. The presence of mRNA for this enzyme in the pyramidal and granule cells
of the hippocampal formation provides evidence that it is found in neurons. These data are
consistent with the proposal that the Calmodulin-sensitive Adenylate cyclase plays an important role
in learning and memory (Xia, 1991).
Spatiotemporal organization of cAMP signaling begins with the tight control of second messenger synthesis. In response to agonist stimulation of G protein-coupled receptors, membrane-associated adenylyl cyclases (ACs) generate cAMP that diffuses throughout the cell. The availability of cAMP activates various intracellular effectors, including protein kinase A (PKA). Specificity in PKA action is achieved by the localization of the enzyme near its substrates through association with A-kinase anchoring proteins (AKAPs). Evidence is provided for interactions between AKAP79/150 and AC isoenzymes ACV and ACVI. PKA anchoring facilitates the preferential phosphorylation of AC to inhibit cAMP synthesis. Real-time cellular imaging experiments show that PKA anchoring with the cAMP synthesis machinery ensures rapid termination of cAMP signaling upon activation of the kinase. This protein configuration permits the formation of a negative feedback loop that temporally regulates cAMP production (Bauman, 2006).
It has been suggested that all intracellular signaling by cAMP during development of Dictyostelium is mediated by the cAMP-dependent protein kinase, PKA, since cells carrying null mutations in the acaA gene that encodes adenylyl cyclase can develop so as to form fruiting bodies under some conditions if PKA is made constitutive by overexpressing the catalytic subunit. However, a second adenylyl cyclase encoded by acrA has recently been found
that functions in a cell autonomous fashion during late development. Expression of a modified acaA gene rescues acrA- mutant cells, indicating that the only role played by ACR is to produce cAMP. To determine whether cells lacking both adenylyl cyclase genes can develop when PKA is constitutive, acrA was disrupted in a acaA- PKA-Cover strain. When developed at high cell densities, acrA- acaA- PKA-Cover cells form mounds, express cell type-specific genes at reduced levels and secrete cellulose coats but do not form fruiting bodies or significant numbers of viable spores. Thus, it appears that synthesis of cAMP is required for spore differentiation in Dictyostelium even if PKA activity is high (Anjard, 2001).
Mutant mice in which type I Ca(2+)-sensitive adenylyl cyclase has been inactivated by targeted mutagenesis show
deficient spatial memory and altered long term potentiation. Long term potentiation in the CA1 region of the rat hippocampus develops
during the first 2 weeks after birth and reaches maximal expression at postnatal day 15 with a gradual
decline at later stages of development. Ca(2+)-stimulated adenylyl cyclase activity
in rat hippocampus, cerebellum, and cortex increases significantly between postnatal days 1-16. This
increase appears to be due to enhanced expression of type I adenylyl cyclase rather than type VIII
adenylyl cyclase, the other adenylyl cyclase that is directly stimulated by Ca2+ and calmodulin. Type I
adenylyl cyclase mRNA in the hippocampus increases 7-fold during this developmental period. The
developmental expression of Ca(2+)-stimulated adenylyl cyclase activity in mouse brain is attenuated
in mutant mice lacking type I adenylyl cyclase. Changes in expression of the type I adenylyl cyclase
during the period of long term potentiation development are consistent with the hypothesis that this
enzyme is important for neuroplasticity and spatial memory in vertebrates (Villacres, 1995).
The somatosensory (SI) cortex of mice displays a patterned, nonuniform distribution of neurons in layer IV termed the 'barrelfield' because of the barrel shapes formed by cylindrical arrays of neurons within the region. Thalamocortical afferents (TCAs) that terminate in layer IV are segregated such that each 'barrel' (the readily visible cylindrical array of neurons surrounding a cell-sparse center) represents a distinct receptive field. TCA arbors are confined to the hollow part of the so-called barrel; the arbors synapse on barrel-wall neurons whose dendrites are oriented toward the center of the barrel. Mice homozygous for the barrelless (brl) mutation, which occurred spontaneously in ICR stock at Universite de Lausanne (Switzerland), fail to develop this patterned distribution of neurons, but still display normal topological organization of the SI cortex. Despite the absence of barrels in these mutants, and the overlapping zones of TCA arborization, the size of individual whisker representations, as judged by 2-deoxyglucose uptake, is similar to that of wild-type mice. Adenylyl cyclase type I (Adcy1) has been identified as the gene disrupted in brl mutant mice, by using fine mapping of proximal chromosome 11, enzyme assay, mutation analysis and by examining mice homozygous for a targeted disruption of Adcy1. These results provide the first evidence for involvement of cAMP signaling pathways in brain pattern formation, and they suggest that an activity dependent process is involved in the wiring of the barrel cortex (Abdel-Majid, 1998).
Adenylyl cyclase types 1 (AC1) and 8 (AC8), the two major calmodulin-stimulated adenylyl cyclases in the brain, couple NMDA receptor activation to cAMP signaling pathways. Cyclic AMP signaling pathways are important for many brain functions, such as learning and memory, drug addiction, and development. Wild-type, AC1, AC8, or AC1&8 double knockout (DKO) mice are indistinguishable in tests of acute pain, whereas behavioral responses to peripheral injection of two inflammatory stimuli, formalin and complete Freund's adjuvant, are reduced or abolished in AC1&8 DKO mice. AC1 and AC8 are highly expressed in the anterior cingulate cortex (ACC), and contribute to inflammation-induced activation of CREB. Allodynia is the inflammation-related behavioral sensitization to a non-noxious stimulus. Intra-ACC administration of forskolin rescues behavioral allodynia defective in the AC1&8 DKO mice. These studies suggest that AC1 and AC8 in the ACC selectively contribute to behavioral allodynia (Wei, 2002).
The ACC plays important roles in the cognitive, motor, and emotional functions of the brain. It has been suggested to contribute to the perception of pain, to the learning processes associated with the prediction and avoidance of noxious sensory stimuli, as well as to pathological phantom pain. Recent studies from animals and humans demonstrate that ACC neurons play key roles in behavioral nociceptive responses to injury in animals and pain perception or unpleasantness in humans. In humans, results from electrophysiological recordings from the ACC and functional imaging studies show that the ACC neurons respond to noxious stimuli. In animals, ACC neurons respond to peripheral noxious stimuli or electrical shocks at high intensities. It is proposed that activity in the ACC may underlie the unpleasantness or discomfort associated with some somatosensory stimuli. Consistently, lesions in the ACC can reduce chronic pain in patients. In animal models of acute pain and persistent pain, lesions of the ACC produce antinociceptive effects. In freely moving animals, local administration of various opioid receptor agonists in the ACC produces powerful antinociceptive effects. In the present studies, it was found that AC1 and AC8 both are highly expressed in the ACC neurons and that genetic deletion of AC1 and AC8 leads to a complete abolishment of the behavioral allodynia caused by tissue injury and inflammation. Consistent with these findings, behavioral nociceptive responses in the formalin test were also significantly reduced. These results support a role for the ACC in the processing of pain-related information. Behavioral responses to acute noxious stimuli are normal in these mutant mice. This strongly suggests that AC1 and AC8 are selectively involved in mediating the behavioral responses to injury. These results are consistent with findings from in vitro brain slices that the activity of adenylyl cyclases is primarily required for plastic changes, while basal synaptic transmission is unaffected by deletion of AC1 and AC8. The pharmacological rescue of behavioral allodynia by local forskolin microinjection into the ACC provides further evidence for an important role of the ACC in persistent pain (Wei, 2002).
What is the possible synaptic mechanism for the action of AC1 and AC8 within the ACC? It is thought that activation of adenylyl cyclases in the ACC may lead to long-lasting changes in synaptic transmission. Glutamate is a major fast excitatory transmitter within the ACC. Theta burst stimulation causes long-term potentiation of synaptic responses in ACC slices from adult mice. The potentiation is completely absent in mice lacking both AC1 and AC8, suggesting that Ca2+-CaM sensitive adenylyl cyclases are important for synaptic potentiation. cAMP clearly contributes to the synaptic potentiation observed 5-40 min after the induction. These results suggest that the enhancement of synaptic responses within the ACC may serve as a synaptic mechanism contributing to injury-related behavioral sensitization. The possible presynaptic effects of forskolin within the ACC cannot be ruled out. It is quite possible that both pre- and postsynaptic changes occur within the ACC during forskolin treatment. Future studies are clearly needed to dissect the detailed synaptic mechanisms within the ACC (Wei, 2002).
Evidence is presented that AC1 and AC8 are important for CREB activation following tissue injury and inflammation in the ACC and insular cortex. Deletion of AC1 or AC8 causes significant reduction of CREB activated by inflammation. Interestingly, no further reduction was found in the AC1&8 DKO mice. Furthermore, injury-triggered CREB activation is not completely blocked in any mice, suggesting that other signaling pathways also contribute to CREB activation in the forebrain. These findings are slightly different from those in the spinal cord. In spinal cord dorsal horn, injury-induced activation of CREB is completely blocked in AC1&8 DKO mice. Future studies are needed to identify other signaling molecules for injury-related CREB activation in the ACC and insular cortex. In both the spinal cord and ACC, signaling molecules downstream of activated CREB remain to be identified (Wei, 2002).
Mammalian fertilization is dependent upon a series of bicarbonate-induced,
cAMP-dependent processes sperm undergo as they 'capacitate,' i.e., acquire the
ability to fertilize eggs. Male mice lacking the bicarbonate- and
calcium-responsive soluble adenylyl cyclase (sAC), the predominant source of
cAMP in male germ cells, are infertile, since the sperm are immotile.
Membrane-permeable cAMP analogs are reported to rescue the motility defect, but these 'rescued' null sperm are not hyperactive, display
flagellar angulation, and remain unable to fertilize eggs in vitro. These
deficits uncover a requirement for sAC during spermatogenesis and/or epididymal
maturation and reveal limitations inherent in studying sAC function using
knockout mice. To circumvent this restriction, a specific sAC inhibitor was identified that allows temporal control over sAC activity. This inhibitor
revealed that capacitation is defined by separable events: induction of protein
tyrosine phosphorylation and motility are sAC dependent while acrosomal
exocytosis is not dependent on sAC (Hess, 2005).
The vertebrate olfactory bulb is a remarkably organized neuronal structure, in which hundreds of functionally different sensory inputs are organized into a highly stereotyped topographical map. How this wiring is achieved is not yet understood. This study shows that the olfactory bulb topographical map is modified in adenylyl cyclase 3 (adenylate cyclase 3)-deficient mice. In these mutants, axonal projection targets corresponding to specific odorant receptors are disorganized, are no longer exclusively innervated by functionally identical axonal projections and shift dramatically along the anteroposterior axis of the olfactory bulb. Moreover, the cyclase depletion leads to the prevention of neuropilin 1 (Nrp1) expression in olfactory sensory neuron axonal projections. Taken together, these data point to a major role played by a crucial element of the odorant-induced transduction cascade, adenylyl cyclase 3, in the targeting of olfactory sensory neuron axons towards the brain. This mechanism probably involves the regulation of receptor genes known to be crucial in axonal guidance processes (Col, 2007).
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