DO THE CALMODULIN STIMULATED ADENYLYL CYCLASES PLAY A ROLE IN NEUROPLASTICITY?
Zhengui Xia (1),
We propose that long term changes in neurons and at synapses may require synergism between the cAMP and Ca2+ signal transduction systems which regulates transcription and synthesis of specific proteins required for long term synaptic changes. During LTP, protein kinase C is activated and intracellular Ca2+ increases. We hypothesize that the CaM regulated adenylyl cyclases may be activated during LTP because of increases in intracellular Ca2+, release of free CaM from neuromodulin, activation by protein kinase C, release of neurotransmitters, or a combination of these events. Synergistic activation of CaM sensitive adenylyl cyclases may produce a robust or prolonged cAMP signal required for transcriptional control. Furthermore, the coupling of the Ca2+ and cAMP systems may provide positive feedback regulation of Ca2+ channels by cAMP dependent protein kinase.
One area of intense interest in neurobiology is the molecular mechanism underlying short-term and long-term adaptive changes in synaptic function. Several regulatory systems have been implicated in neuroplasticity including the cAMP and the Ca2+ signal transduction systems. Ca2+, calmodulin (CaM), CaM-sensitive protein kinases, and adenylyl cyclases have all been implicated in long term adaptive responses in neurons and synaptic plasticity. The purpose of this target article is to present a hypothesis that the calmodulin (CaM) sensitive adenylyl cyclases may play a crucial role in long term adaptive responses in brain and to present molecular models to explain the role of these enzymes in these processes. Evidence supporting this hypothesis is derived from studies of the invertebrate and mammalian CaM sensitive adenylyl cyclases, as well as data concerning the role of the cAMP signal transduction system for LTP in brain.
In mammalian brain, adenylyl cyclase activity is regulated by neurotransmitter and hormone receptors coupled to the enzyme through the G regulatory proteins, Gs and Gi (reviewed by Ross and Gilman,1980), as well as by intracellular free calcium (reviewed by Cheung and Storm, 1982). Protein phosphorylations catalyzed by the cAMP dependent protein kinase regulate several important aspects of neuronal function including ion channel activity, gene expression and neurotransmitter synthesis (reviewed by Krebs and Beavo,1979; Nestler and Greengard, 1983; Nairn et al., 1985). Furthermore, cAMP has been implicated in the regulation of synaptic plasticity and may play an important role in mechanisms underlying learning and memory (reviewed by Kandel and Schwartz, 1982; Dudai, 1988).
Elucidation of molecular mechanisms for neuroplasticity requires a multidisciplinary approach with electrophysiology as the bridging discipline between signal transduction molecular biology and the behavioral sciences. One useful electrophysiological model for neuroplasticity is LTP which occurs in many areas of brain including the hippocampus. The relationship between LTP and behavior is still controversial. However, both spatial learning in rats and LTP in the hippocampus are blocked by NMDA antagonists (Davis et al., 1992) demonstrating a possible relationship between these two phenonema . Other evidence suggesting a correlation between LTP and learning comes from gene disruption studies which have demonstrated that mice lacking the alpha form of CaM kinase II (Silva et al., 1992a, 1992b) or the fyn-tyrosine kinase (Grant et al., 1992) are deficient in LTP and spatial learning. On the other hand, PKC-gamma mutant mice showed impaired LTP in the hippocampus but can perform the hidden platform test of the Morris water task indicating that LTP induced by conventional tetanic stimulation is not essential for this specific type of learning (Abellovich et al., 1993a,b). These mice were, however, deficient in the transfer test of the Morris water task which may be a more reliable measure of spatial learning.
Although many forms of LTP decay over several hours, long-lasting LTP (L-LTP) which is produced by multiple trains of high frequency stimulation can last hours or even days depending upon the stability of the preparation (reviewed in Matties et al., 1990) and is sensitive to inhibitors of transcription and translation (Squire and Davis, 1981; Krug et al., 1984; Frey et al., 1988; Grecksch, and Matthies,1980). Various forms of LTP in the hippocampus require entry of Ca2+ through NMDA receptors or voltage gated Ca2+ channels (reviewed in Nicoll et al., 1988; Kauer et al., 1988) and an increase in intracellular Ca2+ can induce LTP (Malenka et al., 1988). Consequently, Ca2+ sensitive adenylyl cyclases may be stimulated when Ca+ channels are activated during LTP.
The Aplysia Adenylyl Cyclase System and the Gill Withdrawal Reflex
One of the most interesting systems demonstrating the possible importance of Ca2+ sensitive adenylyl cyclases for learning and memory is the gill withdrawal reflex of the invertebrate Aplysia (Castellucci and Kandel, 1976; Byrne, 1987). In this system, the conditioned stimulus, a weak touch to the tail, illicits a minimal defensive response by withdrawing the siphon. In contrast, the unconditioned stimulus which is a shock to the tail, produces a strong withdrawal reflex. If the conditioned stimulus is paired with the unconditioned stimulus the animal learns to associate touch with the shock and shows a strong withdrawal response to touch alone. The length of the memory for this response depends upon the number of stimuli applied (Frost et al., 1985) and can last for days when 4 or 5 noxious signals are administered (Castellucci et al., 1989). An extensive analysis of this system has lead to the conclusion that the conditioned stimulus results in an increase in intracellular Ca2+ within the sensory neuron, and that the unconditioned stimulus results in the release of serotonin unto the same cell (Abrams et al., 1991). It has been proposed that paired activation of the Aplysia adenylyl cyclase by Ca2+ /CaM and serotonin results in a synergistic activation of adenylyl cyclase which may be required for long term memory. Since long term facilitation can be induced by the injection of cAMP into the presynaptic sensory neuron (Schacher et al., 1988; Scholz and Byrne, 1988) and blocked by inhibitors of protein kinase A (Ghirardi et al., 1992), it is very likely that cAMP protein kinase plays a central role in long term facilitation in Aplysia. Furthermore, long term facilitation in this system is sensitive to inhibitors of RNA and protein synthesis (Castellucci et al., 1989; Montarolo et al., 1986) suggesting that cAMP mediated transcription may be required for long term facilitation in Aplysia (Kaang et al., 1993).
The gene for the Aplysia adenylyl cyclase(s) has not been cloned and its relationship to the cloned mammalian adenylyl cyclases has not been determined. However, Ca2+/CaM sensitive adenylyl cyclase activity is present in Aplysia membranes and Yovell and Abrams, (1992) have demonstrated synergism for activation of Aplysia adenylyl cyclase by Ca2+ and serotonin when brief pulses of Ca2+ followed by serotonin were paired. Synergistic activation of this adenylyl cyclase by Ca2+ and Gs is quite novel in that it was only observed with sequential applications of Ca2+ and serotonin and is not observable in steady-state assays. Although this synergism was relatively weak and its physiological significance remains to be established, the small increment in cAMP signal may be functionally important because of the signal amplification that is inherent in the cAMP signal transduction pathway. Synergistic activation of the type I adenylyl cyclases by Ca2+ and Gs in vivo is observable in steady state assays and does not require ordered administration of Ca2+ and activators of Gs (Wayman et al., 1994). Consequently, the Aplysia adenylyl cyclase may be quite distinct from the mammalian type I and type III CaM sensitive adenylyl cyclases.
If cAMP regulated transcription plays a major role in long term facilitation in Aplysia neurons, then cAMP signals generated at synapses must either diffuse to the cell body in order to affect transcription in the nucleus or a secondary signal arising from the initial synaptic cAMP signal must reach the nucleus. This is of particular concern in neurons because of substantial distances required for transport or diffusion of cAMP along neurites. Recently, this question has been directly addressed in Aplysia neurons by monitoring gradients of cAMP produced in response to serotonin stimulation of adenylyl cyclase (Bacskai et al., 1993). Bath application of serotonin produced an extensive cAMP gradient between the processes and the cells body which was consistent with the diffusion of cAMP from neurite tips to the cell body of the neuron. Nuclear translocation of the catalytic subunit of cAMP dependent protein kinase was relatively slow and probably only occurs after prolonged or enhanced cAMP signals. In contrast to other cAMP regulated phenonema such as regulation of metabolism and ion channel function, which are rapid short term responses, cAMP regulation of transcription in neurons may require strong or persistent activation of adenylyl cyclase activities.
The Drosophilia Rutabaga Learning Mutant
Other evidence that Ca 2+ sensitive adenylyl cyclases may be important for synaptic plasticity in invertebrates has come from studies of the Drosophilia learning mutant, rutabaga. Rutabaga is an X-linked recessive mutant that is deficient in associative learning (Livingston et al., 1984; Dudai and Zvi, 1984 and 1985; Livingston, 1985). In contrast to wild type Drosophilia, the rutabaga fly lacks Ca2+/CaM sensitive adenylyl cyclase activity. The gene for an adenylyl cyclase similar to the type I adenylyl cyclase maps within a region on the X chromosome that includes the rut locus and a single point mutation in this gene is sufficient to destroy all enzyme activity (Levin et al., 1992). Feany (1990) has proposed that calcium responsiveness, rather than the overall cAMP synthesis may be the crucial component of adenylyl cyclase activity required for associative learning in Drosophila. In rutabaga larvae, voltage clamp analysis of neuromuscular transmission indicated deficient synaptic facilitation and post-tetanic potentiation (Zhong and Wu, 1991). The rutabaga mutant has provided convincing evidence that the type I adenylyl cyclase is important for associative learning in invertebrates and synaptic facilitation.
The Family of Mammalian Adenylyl Cyclases
The existence of distinct CaM stimulated and CaM insensitive adenylyl cyclases in brain was first demonstrated by the separation of these two forms of the enzyme from bovine brain using CaM-Sepharose affinity chromatography (Westcott et al., 1979). The CaM sensitive adenylyl cyclase activity absorbed to CaM-Sepharose in the presence of Ca2+ and was stimulated by Ca2+ when reconstituted with CaM. Half-maximal stimulation of the enzyme occurred at 80 nM free Ca2+. The CaM insensitive forms of adenylyl cyclase present in brain did not absorb to CaM-Sepharose in the presence or absence of Ca2+, and were not stimulated by Ca2+. Furthermore, polyclonal and monoclonal antibodies have been isolated that distinguish between the CaM -sensitive and CaM-insensitive adenylyl cyclases in brain providing further evidence for separate adenylyl cyclases (Rosenberg et al., 1987a; Mollner et al., 1988; Mollner and Pfeuffer, 1991). The catalytic subunit of a CaM-sensitive adenylyl cyclase was purified to homogeneity from bovine brain using CaM-Sepharose and Forskolin-Sepharose affinity chromatography (Yeager et al., 1985; Smigel et al., 1986; Minocherhomjee et al., 1987). Characterization of the purified CaM-sensitive adenylyl cyclase from brain indicated that it is a glycoprotein which interacts directly with CaM (Minocherhomjee et al., 1987). Furthermore this enzyme can couple to Gs and beta-adrenergic receptors (May et al., 1985; Rosenberg et al., 1987b) as well as Gi and muscarinic receptors (Tota et al., 1990; Dittman et al., 1994). The expression of the type I adenylyl cyclases in the insect/Bacculovirus system and the development of new strategies for its purification from these cells offers great promise for direct characterization of the protein (Taussig et al., 1993).
cDNA clones for the type I adenylyl cyclase have been isolated from bovine brain (Krupinski et al., 1989) and human brain cDNA libraries (Villacres et al., 1992). To date, cDNA clones for eight distinct ACs have been published (Krupinski et al., 1989; Feinstein et al., 1991; Bakalyar & Reed, 1990; Gao & Gilman, 1991; Ishikawa et al., 1992; Yoshimura & Cooper, 1992; Cali et al., 1994). Although these enzymes share sequence homology, they contain hypervariable regions and exhibit different regulatory properties. I-AC (Tang et al ., 1991; Choi et al., 1992a), III-AC (Choi et al., 1992b), and VIII-AC (Cali et al., 1994) are stimulated by Ca2+ and CaM in vitro whereas II-AC, IV-AC, V-AC and VI-AC are not. Although these enzymes share considerable sequence homology, they contain hypervariable regions and exhibit different regulatory properties. The diversity of this enzyme system undoubtedly reflects different mechanisms for regulation of cAMP levels in animal cells, and the variety of physiological processes that are regulated by intracellular cAMP.
Regulation of the Type I and III Adenylyl Cyclases by Calcium and CaM
Type III adenylyl cyclase- Modulation of adenylyl cyclase activity by Ca2+ has been demonstrated in several tissues including brain and retina and it has been proposed that cAMP levels may be controlled by fluctuations in intracellular free Ca2+. Most mammalian tissues contain mixture of adenylyl cyclases, and the Ca2+ sensitivity of specific forms of adenylyl cyclase has only recently been addressed. The type III adenylyl cyclase was expressed in human kidney 293 cells to determine if it is stimulated by Ca2+ and CaM (Choi et al., 1992b). In isolated membranes, the type III enzyme was not stimulated by Ca2+and CaM in the absence of other effectors. It was, however, stimulated by Ca2+ through CaM when the enzyme was concomitantly activated by forskolin (Figure 1b). The concentrations of free Ca2+ for half-maximal stimulation of type III adenylyl cyclases was 5.0 5M Ca2+ (Figure 2b). The sensitivity of the type III adenylyl cyclase to Ca2+ in vivo has not been reported.
Toscano et al. (1979) isolated a partially purified adenylyl cyclase from bovine brain that was not stimulated by GppNHp, NaF, or Ca2+/ CaM. Sensitivity to these effectors were restored by incubation of the adenylyl cyclase preparation with detergent solubilized membranes from cerebral cortex. Reconstitution of Ca2+/CaM sensitivity required the presence of guanyl nucleotides. The properties of this adenylyl cyclase are similar to the type III adenylyl cyclase and these early studies showed synergistic activation of an adenylyl cyclase by Ca2+ and Gs.
Is Ca2+ regulation of the type III adenylyl cyclase at concentrations of 5 5M Ca2+ physiologically significant, and what role could it play in signal transduction pathways in olfactory sensory neurons, retina or brain? Free Ca2+ in many animal cells generally varies from less than 0.1 to 10 5M. Local Ca2+ concentrations at the membrane surface in neurons may increase to 100 5M or even higher during action potentials (Smith and Augustine, 1988). Since the type III adenylyl cyclase is only activated at the higher end of the free Ca2+ range, and when the enzyme is activated by other effectors, the enzyme may allow Ca2+ amplification of cAMP signals. For example, the existence of cyclic AMP gated ion channels in neurons suggests that initial cyclic AMP signals, generated through receptors coupled to adenylyl cyclase, may be further amplified by increases in intracellular Ca2+. Thus the type III adenylyl cyclase is able to integrate multiple signals and may function as a "coincidence detector" (Bourne and Nicoll, 1993).
Type I adenylyl cyclase- CaM stimulates the type I adenylyl cyclase activity in membranes from CDM8(I-AC)-transfected 293 cells at a half-maximal concentration of approximately 20 nM (Figure 1). Similar CaM sensitivities have been reported for the CaM sensitive adenylyl cyclase purified from bovine brain (Minocherhomjee et al., 1987) and the type I adenylyl cyclase expressed in insect SF9 cells (Tang et al., 1991). Half-maximal stimulation of type I adenylyl cyclase occurred at approximately 50 nM free Ca2+ (Figure 2), which is consistent with the Ca2+ sensitivity of the CaM stimulated adenylyl cyclase isolated from bovine brain calculated from the data of Westcott et al., 1979 . These Ca2+ dependencies were determined using EGTA/Ca2+ buffers. Because of the uncertainties associated with calculating free Ca2+ by this method, these data are only estimates of the Ca2+ dependence of the enzyme (Yovell, 1992).
It has been generally assumed that CaM sensitive adenylyl cyclases can function to couple increases in intracellular free Ca2+ to elevations in cAMP in vivo. The purified type I adenylyl cyclase, and the enzyme in membrane preparations, is stimulated by Ca2+ and CaM . However, it was important to demonstrate that increases in intracellular free Ca2+ can actually stimulate the type I adenylyl cyclase in intact cells. Therefore, we expressed the enzyme in human 293 cells and examined the effect of a Ca2+ ionophore, A23187, and extracellular Ca2+ on intracellular cAMP (Choi et al., 1992a) . Human 293 cells were selected for these studies because their endogenous adenylyl cyclase activity is quite low and insensitive to extracellular Ca2+ and the Ca2+ ionophore A23187 (Figure 3a). In the presence of 2 mM extracellular Ca2+ , the intracellular cAMP levels of control cells, transfected with the CDM8 vector alone, were unaffected by addition of A23187. In contrast, intracellular cAMP in 293 cells stably expressing the type I adenylyl cyclase increased approximately 16 fold with addition of 10 5M A23187 (Figure 3a). Under these conditions, the intracellular free Ca2+ increased to 1.0 5M. The increase in intracellular cAMP stimulated by A23187 depended upon the concentration of Ca2+ applied (Figure 3B). Elevated cAMP was detectable within a few minutes after addition of 10 5M A23187 and 2 mM Ca2+ (Figure 3C).
The data described above indicated that intracellular Ca2+ can stimulate the type I adenylyl cyclase activity in vivo. Ca2+ stimulation of the enzyme in vivo may be due to direct interactions of the enzyme with Ca2+ and CaM, or indirect mechanism involving stimulation of the enzyme by Ca2+ activated protein kinases. We have made several point mutations within the calmodulin binding domain to determine if the Ca2+ sensitivity of the enzyme can be modified by mutagenesis (Wu et al., 1993). The catalytic activities of the mutant enzymes were comparable to wild type type I adenylyl cyclase. Ca2+ and CaM stimulation was abolished by substitution of Phe-503 with Arg-503 (FR-I-AC). Stimulation of type I adenylyl cyclase activity in vivo by intracellular Ca2+ was also greatly diminished with the Arg-503 mutant indicating that Ca2+ stimulation of the enzyme in vivo is due primarily to direct interactions with CaM and Ca2+. These data demonstrated that the Ca2+ sensitivity of this enzyme can be modulated by point mutagenesis within the putative calmodulin binding domain, and indicate that the enzyme can be directly regulated by Ca2+ and CaM, in vivo.
Kidney 293 cells contain various receptors that can couple directly or indirectly to adenylyl cyclases, including muscarinic receptors. Therefore, we examined the influence of carbachol, a muscarinic agonist, on the intracellular cAMP levels of 293 cells expressing the type I adenylyl cyclase (Choi et al., 1992a). Carbachol stimulated intracellular cAMP levels approximately 3 fold in 293 cells stably expressing type I adenylyl cyclase, but was without significant effect on cAMP in control cells (Figure 4A). Maximal stimulation by carbachol occurred at approximately 100 5M, and this increase in cAMP was inhibited by the muscarinic antagonist, atropine. No carbachol stimulation of intracellular cAMP was seen without forskolin, even when IBMX was present. The requirement for forskolin reflects the low sensitivity of this assay for cAMP and more sensitive assays using a CRE-beta-galactosidase reporter construct can detect Ca2+ stimulated cAMP signals in 293 cells without the presence of forskolin or phosphodiesterase inhibitors (Impey and Storm, unpublished observations).
These data illustrated that the the type I adenylyl cyclase can be regulated by muscarinic receptors in vivo either by direct coupling through a G regulatory protein or indirectly by mobilization of intracellular Ca2+. The latter explanation seems most likely since carbachol had no effect on the basal, CaM stimulated, or forskolin stimulated activities of type I adenylyl cyclase activity in isolated membranes . Furthermore, we analyzed the influence of carbachol on intracellular free Ca2+ in 293 cells using fura-2. One mM carbachol increased free Ca2+ from a baseline of 40 nM to 140 nM and this increase was blocked by 100 5M BAPTA/ AM, the intracellular Ca2+ chelator. Furthermore, 100 5M BAPTA/AM completely inhibited carbachol stimulated increases in intracellular cAMP (Figure 4B) . We concluded that the type I adenylyl cyclase can function to couple increases in intracellular Ca2+ to cAMP production in whole cells, and that the enzyme can also be indirectly stimulated through muscarinic receptors by mobilization of intracellular free Ca2+.
Synergistic Regulation of Type I Adenylyl Cyclase by Ca2+ and Isoproterenol In vivo-Evidence from several studies has indicated that adenylyl cyclase activity from various areas of mammalian brain may be synergistically activated by CaM/ Ca2+ and Gs, or Gs coupled receptors (Toscano et al., 1979; Gnegy and Treisman, 1981; Natsukari et al., 1990; Choi et al., 1992a). However, other investigators have found that CaM and G protein stimulation of AC activities are additive and not synergistic (Piascik et al., 1981; Salter et al., 1981; Sano, 1985). This apparent discrepancy reflects the regulatory diversity of the ACs, their distribution within brain, and the different preparations used in these studies.
Although the type I adenylyl cyclase is stimulated by Ca2+ in vivo, we have discovered that it is not stimulated by Gs coupled receptors in vivo unless it is also activated by Ca2+/ CaM (Wayman and Storm, 1994). We examined the sensitivity of I-AC expressed in HEK-293 cells to isoproterenol or glucagon when intracellular Ca2+ was elevated . The cells used in this study were stable transfectants expressing type I adenylyl cyclase and glucagon receptors. The Ca2+ ionophore A23187 stimulated the enzyme approximately three fold, isoproterenol did not stimulate the enzyme, but the combination of the two stimulated adenylyl cyclase activity 13 fold in vivo. Similarly, glucagon did not stimulate the enzyme but the combination of A23187 and glucagon activated the enzyme 90 fold. This phenonema was not observed with a mutant enzyme (FR-I-AC) that is insensitive to Ca2+ and CaM. Therefore, I-AC may couple Ca2+ and neurotransmitter signals to generate optimal cAMP levels, a property of the enzyme which may be important for learning and memory in mammals. We have also demonstrated that the expression of type I adenylyl cyclase in HEK-293 cells allows Ca2+ to stimulate reporter gene activity mediated through the CRE response element (Impey and Storm, unpublished observations). Simultaneous activation by Ca2+ and isoproterenol caused synergistic stimulation of CRE-mediated transcription in HEK-293 cells and cultured neurons.
Regulation of the CaM Regulated Adenylyl Cyclases by Protein Kinase C
Activation of the type I and type III adenylyl cyclases by activators of protein kinase C- Protein kinase C (PKC) is activated during many forms of LTP and phorbol esters and other activators of PKC can also affect intracellular cAMP levels in various tissues and cultured cells. Therefore, the effect of phorbol esters on the activity of the type I and type III adenylyl cyclases in whole cells has been examined using stably transfected 293 cells expressing either enzyme (Choi et al., 1993). TPA markedly enhanced the forskolin responsiveness of the type I and type III adenylyl cyclases expressed in kidney 293 cells. The effect of TPA on the activity of the CaM sensitive adenylyl cyclases was not mediated through increases in intracellular free calcium. Jacobowitz et al., (1993) have also examined the sensitivities of various adenylyl cyclases to phorbol esters by transient expression of the enzymes in 293 cells. Their data indicated that the type II adenylyl cyclase was particularly sensitive to phorbol esters and that types IV, V and VI showed modest stimulations upon PMA treatment. Since PKC is activated during some forms of LTP, cAMP levels may be regulated during LTP.
Does protein kinase C regulate adenylyl cyclase activities in the brain by controlling the levels of free CaM? - The activity of the CaM sensitive adenylyl cyclases depends on the concentrations of free intracellular Ca2+ and free CaM in neurons. We have described the biochemical properties of a neurospecific CaM binding protein, neuromodulin (GAP-43), that may regulate the levels of free CaM present in neurons and consequently may play a key role in CaM regulated processes in neurons . The neurobiology of neuromodulin has recently been reviewed (Benowitz and Routtenberg, 1987; Skene,1989; Liu and Storm, 1990).
Neuromodulin is neurospecific and bovine brain contains approximately 60 pmol neuromodulin/mg of membrane protein making it the most abundant CaM binding protein in brain with a concentration comparable to CaM itself (Cimler et al., 1985). Neuromodulin is a prominent constituent of neuronal growth cone membranes, comprising up to 1% of the total growth cone membrane protein and it is transported by rapid axoplasmic transport (Pfenninger et al., 1983; Skene et al.,1986).
Neuromodulin has been implicated in several neuromodulatory roles including axon growth and synaptic plasticity and it has been studied extensively as a potential mediator of synapse formation and modification. Phosphorylation of neuromodulin by PKC has been correlated with synaptic long-term potentiation (Nelson and Routtenberg, 1985).
On the basis of the affinity of neuromodulin for CaM under physiologically relevant ionic strength (Alexander et al., 1987) and the concentrations of these two abundant proteins in brain, we predict that the majority of CaM will be complexed to neuromodulin in vivo. Neuromodulin is phosphorylated by PKC with a phosphate: protein molar ratio of 1:1, CaM decreases the rate of phosphorylation of neuromodulin by PKC, and phosphorylation prevents neuromodulin binding to CaM ((Alexander et al., 1987). Phosphorylation of neuromodulin by PKC inhibits CaM binding because the phosphorylation site is within the CaM binding domain of the protein (Apel et al., 1990). In addition, phosphoneuromodulin is an excellent substrate for calcineurin, the CaM stimulated phosphatase which is particularly abundant in brain (Liu and Storm, 1989). On the basis of these observations, we have proposed that neuromodulin may bind and localize CaM at specific sites within neurons and that PKC may regulate the levels of free CaM available in neurons for stimulation of various enzymes including adenylyl cyclases and protein kinases (Andreasen et al., 1993; Alexander et al.,1987; Liu and Storm, 1990).
Evidence that protein kinase C may regulate the levels of free CaM in neurons or neuronal-like cells in vivo comes from several different studies. For example, activation of protein kinase C in PC 12 cells increased the levels of free CaM, presumably because of the phosphorylation of neuromodulin or related proteins (MacNicol and Schulman, 1992). Mangels and Gnegy, 1990 demonstrated that the ratio of cytosolic to membrane associated CaM increases when neuroblastoma cells when PKC is activated through muscarinic receptors or by phorbol esters. Since these cells contain membrane associated neuromodulin, it was concluded that the redistribution of CaM may be due to PKC phosphorylation of neuromodulin. If this hypothesis is true, then CaM may be liberated during LTP by activation of PKC and phosphorylation of neuromodulin.
Tissue Distribution of the Type I and Type III Adenylyl Cyclases
Tissue distribution of the enzymes- Although the type III adenylyl cyclase was originally cloned from an olfactory cDNA library and is greatly enriched in this tissue, type III adenylyl cyclase mRNA is also expressed in brain, spinal cord, adrenal medulla, adrenal cortex, heart atrium, aorta, lung, retina, 293 cells and PC 12 cells (Xia et al., 1992). In contrast, the type I adenylyl cyclase is neurospecific (Xia et al., 1993). The only bovine tissues showing a positive signal for type I mRNA were brain, retina and adrenal medulla (Figure 5). The weak signal seen with whole adrenal was most likely due to adrenal medulla since the cortex gave a negative signal. In addition to the expected transcript seen at approximately 11.7 kb, retina also contained a second transcript at 6.5 kb. Several cultured cell lines including neuroblastoma cell N1E-115, neuroglio hybridoma cell NG-108, rat glioma 36B-10 cell, and PC-12 cells were also analyzed and found not to express mRNA for the type I adenylyl cyclase. The restricted expression of type I adenylyl cyclase mRNA in neural tissues contrasts sharply with most of the other mammalian enzymes which show fairly broad distribution in both neural and non-neuronal tissues.
Distribution of type I adenylyl cyclase mRNA in rat retina examined by in situ hybridization - The presence of type I adenylyl cyclase mRNA in retina is consistent with the Ca2+ and CaM sensitivities of adenylyl cyclase activities reported for retina (Gnegy et al. (1984). The distribution of type I adenylyl cyclase mRNA in retina was examined in more detail by in situ hybridization. Retina cross sections from rat, rabbit, and bovine were analyzed with a 35S-UTP labeled bovine riboprobe specific for the type I adenylyl cyclase (Xia et al., 1991, 1993). mRNA for the type I adenylyl cyclase was detected in the inner segment layer of the photoreceptors (IS), and in all three nuclear layers of the neural retina (Figure 6). The outer nuclear layer (ONL) which contains rods and cones, the inner nuclear layer (INL) which contains horizontal cells, bipolar cells and amacrine cells, and the ganglion cell layer (GCL) all contained mRNA for type I adenylyl cyclase. The intensity of the labeling was strongest in the IS (the cytoplasm of photoreceptors) and the ONL.
Distribution of type I adenylyl cyclase mRNA in rat brain examined by in situ hybridization - The distribution of mRNA encoding the type I adenylyl cyclase in rat brain was also examined by in situ hybridization (Xia et al., 1991). In situ hybridizations in adult rat brain revealed high levels of type I adenylyl cyclase mRNA in specific areas of brain including the hippocampal formation, the neocortex, entorhinal cortex, cerebellum cortex, and the olfactory system (Figures 7, 8). The dentate gyrus in the hippocampal formation showed very intense labeling which appeared to be associated with the granule cell layer. Moderately strong labeling was also evident in association with the pyramidal cells in CA1, CA2, and CA3 layers of the hippocampus. The data reported in Figure 7 shows expression of mRNA for the CaM-sensitive adenylyl cyclase in granule cells of the dentate gyrus and in pyramidal cells of the hippocampus, providing the first evidence that the enzyme is expressed in neurons.
These data illustrate that mRNA for the type I adenylyl cyclase is not generally distributed throughout the brain, suggesting that it does not play a general regulatory role (e.g., in regulation of cell metabolism), and that it may be important for specific neuronal functions. Messenger RNA for the type I adenylyl cyclase is highly localized to specific regions of brain, including those areas that show long-term potentiation and have been implicated in learning and memory. Although these data do not define the function of the type I CaM-sensitive adenylyl cyclase, its mRNA distribution is consistent with the proposal that this enzyme may be important for learning and memory.
Disruption of the Gene for the Type I Adenylyl Cyclase Leads to Deficiencies in Spatial Learning- Recently, we evaluated the role of the type I adenylyl cyclase for learning and memory by disruption of the gene for the enzyme in mice (Wu and Storm, unpublished observations) . Brain coronal sections showed no detectable anatomical differences in the hippocampus, neocortex or cerebellum between wild type and mutant mice. There were also no differences in the arrangement of cell body layers of the hippocampus or cerebellum. The mutant mice had normal motor coordination, suckling behavior, weight gain and reproduced with litter sizes comparable to wild type mice. An analysis of Ca2+ sensitive adenylyl cyclase activity in membranes from the cerebellum, neocortex, hippocampus, and brain stem revealed decreases of 62%, 38%, 46%, and 6%, respectively.
Mutant and wild type mice were analyzed for spatial learning by the Morris water task, a set of assays that has been used to exam spatial learning in other mutant mice deficient in specific genes. Both sets of animals showed decreased escape latencies with training, and there were no statistically significant differences in the ability of the mutant and wild type mice to find the visible or hidden platform. However, escape latencies in the hidden platform task are a poor indicator of spatial learning and even rodents with hippocampal lesions that affect other forms of spatial learning can learn to find the hidden platform in the Morris water task (Morris et al., 1982, 1986, 1990; Davis et al.,1992). A better indicator of spatial learning is the transfer test in which the animal is trained to find the hidden platform at a specific site in the pool. The platform is then removed, and the number of times that a mouse swims across the target area or the time in the target quadrant is quantitated . There were significant and reproducible differences in transfer test behavior between the mutant and wild type mice. Wild type mice crossed the target area 6 1 0.4 times during a 60 sec. trial whereas the mutant mice crossed only 4 1 0.4 times (p < .002). The difference in transfer ability was also evident when the time in various quadrants was analyzed. Only wild type mice showed a bias for quadrant A. They spent 42% 1 3.0 of their time in quadrant A searching for the platform. The mutant mice showed no significant preference for quadrant A (27% 1 2.0) indicating an impaired ability in this specific task (p <.001). These data illustrate that the mutant mice have a significant and lasting place navigational impairment that was dissociated from visual, motivational, or motor requirements of the test. We conclude that I-AC may play an important role for signal transduction pathways underlying some forms of learning and memory.
Role of Adenylyl Cyclases and cAMP in LTP
The general hypothesis under consideration in this paper suggests that the Ca2+ sensitive adenylyl cyclases may play an important role in neuroplasticity and participate in signal transduction pathways underlying long term adaptive responses in neurons such as LTP. Since LTP results in postsynaptic increases in Ca2+ and various areas of the hippocampus including CA1, CA3 and the dentate gyrus contain type I adenylyl cyclase (Xia et al., 1991), one might expect that activation of NMDA receptors or other Ca2+ channels during LTP may elevate cAMP in these regions. Indeed, activation of NMDA receptors gives increased cAMP in area CA1 of the hippocampus (Chetkovich et al., 1991). Furthermore, LTP in the dentate gyrus (Stanton and Sarvey, 1985a) and the CA1 (Chetkovich and Sweatt, 1993) have both been reported to produce increases in cAMP. Although the cAMP increase caused by LTP in the CA1 was very small (20 to 25%), it may be difficult to detect these increases against a background of cells in the preparation which are not potentiated.
There is also evidence that adenylyl cyclases, cAMP, and cAMP dependent protein kinases may play an important role in some forms of LTP, particularly in the hippocampus. For example, stimulation of adenylyl cyclase activity in the dentate gyrus by norepinephrine produces LTP (Stanton and Sarvey, 1985b; Hopkins and Johnston, 1988). The early phase of LTP in the CA1 persists only 1 to 2 hours and is initiated by a single train of high-frequency stimulation whereas the late phase requires three of more trains of high-frequency stimulation and lasts up to 10 hours. Since D1 dopamine antagonists block L-LTP and D1 receptors are coupled to stimulation of adenylyl cyclase (Frey et al., 1991) it has been proposed that cAMP stimulated protein kinase may play a pivotal role in L-LTP. In fact, dibutyryl cAMP induces increases in synaptic efficacy in the CA1 region of the hippocampus (Slack and Pockett, 1991) and L-LTP in the CA1 is blocked by Rp-cAMPS, an inhibitor of cAMP dependent protein kinase (Frey et al., 1993). Furthermore, Sp-cAMPS, which activates cAMP dependent kinase, produces L-LTP in the CA1. Biochemical Model for the Role of the CaM Regulated Adenylyl Cyclases in Neuroplasticity
The type I adenylyl cyclase is a neural specific adenylyl cyclase (Xia et al., 1993) with a highly restricted expression in mammalian brain which includes the dentate gyrus, CA1, CA2, and CA3 regions of the hippocampus (Xia et al., 1991). Furthermore, the type III and VIII adenylyl cyclases are also expressed in the hippocampus (Glatt and Snyder, 1993; Cali et al., 1994). What is the function of these enzymes in neurons, and what possible role(s) might they have in signal transduction systems important for neuroplasticity?
During LTP, PKC is activated (reviewed by Linden and Routtenberg, 1989) intracellular Ca2+ increases and neuromodulin is phosphorylated by PKC (Routtenberg, 1985). We hypothesize that phosphorylation of neuromodulin at specific sites in neurons may regulate the concentrations of free CaM available to activate the CaM regulated adenylyl cyclases and other enzymes including CaM kinases and NO synthetase (Figure 9).
Long term changes in synaptic function may be due, at least in part, to cAMP control of transcription through cAMP responsive DNA elements such as CRE (reviewed by Mitchell and Tjian,1989). We hypothesize that synergistic stimulation of adenylyl cyclases by Ca2+ and neurotransmitters or PKC may produce exceptionally strong or prolonged cAMP signals required for stimulation of transcription . Stimulation of transcription by cAMP, which requires the nuclear translocation of PKA (Nigg et al., 1985; Hagiwara et al., 1993), requires higher or more persistent cAMP signals than other cAMP regulated events, particularly in neurons. For example, stimulation of PKA nuclear translocation in Aplysia neurons (Bacskai et al., 1993) and serotonin stimulation of transcription through CRE (Kaang et al., 1993) are relatively slow processes that require multiple doses of serotonin for AC activation. Robust cAMP signals may be required for transcriptional control in neurons because a significant cAMP gradient must be established from the synapse to the cell body. Elevated cAMP signals arising from synergistic activation of the type I adenylyl cyclase by Ca2+ and neurotransmitters, or other signals, may therefore play an important role in synaptic plasticity. Alternatively, the coupling of the Ca2+ and cAMP systems may result in simultaneous or sequentially ordered activation of the Ca2+ and cAMP stimulated protein kinases, or provide positive feedback regulation of Ca2+ channels by cAMP dependent protein kinase. All of these mechanisms are dependent upon the unique property of the CaM sensitive adenylyl cyclases to integrate multiple signals for modification of synaptic function.
Abeliovich, A., Chen, Chong, Goda, Y., Silva, A. J., Stevens, C. F., and Tonegawa, S. (1993a) Modified hippocampal long term potentiation in PKC-gamma mutant mice. Cell 75: 1253-1262.
Abeliovich, A. Paylor, R. Chen, C. Kim. J. J. Wehner, J.M. and Tonegawa, S. (1993b) PKC gamma mutant mice exhibit mild deficits in spatial and contextual learning. Cell 75:1263-1271.
Abrams, T.W., Karl. K.A. and Kandel, E. R. (1991) Biochemical studies of stimulus convergence during classical conditioning in Aplysia: dual regulation of adenylate cyclase by Ca2+/calmodulin and transmitter. J. Neurosci. 11: 2655-2665.
Alexander, K. A., Cimler, B. M., Meier, K. E. & Storm, D. R. (1987) Regulation of calmodulin binding to P-57. J. Biol. Chem. 262:6108-6113.
Alexander, K. A., Wakim, B. T., Doyle, G. S., Walsh, K. A. & Storm, D. R. (1988) Identification and characterization of the calmodulin binding domain of neuromodulin, a neurospecific calmodulin binding protein. J. Biol. Chem. 263:544-7549.
Andreasen, T. J., Keller, C. H., LaPorte, D. C., Edelman, A. M. & Storm, D. R. (1981) Preparation of Azido-calmodulin: A Photoaffinity Label for Calmodulin-Binding Proteins. Proc. Natl. Acad. Sci. U. S. A . 78:2782-2785
Andreasen, T. J., Luetje, C. W., Heideman, W. & Storm, D. R. (1983) Purification of a novel calmodulin binding protein from bovine cerebral cortex membranes. Biochemistry 22:4615-4618.
Apel, E. D., Byford, M. F., Au, D., Walsh, K. A. and Storm, D.R. (1990) Identification of the protein kinase C phosphorylation site in neuromodulin. Biochemistry 29: 2330-2335.
Bacskai, B. J., Hochner, B., Mahaut-Smith, M., Adams, S.R., Kaang, B. K., Kandel, E. R., Tsien, R. Y. (1993) Spatially resolved dynamics of cAMP and protein kinase A subunits in Aplysia sensory neurons. Science 260: 222-226.
Bakalyar, H. A. & Reed, R. R. (1990) Identification of a specialized adenylyl cyclase that may mediate odorant detection. Science 250:1403-1406.
Benowitz, L. I. and Routtenberg, A. (1987) A membrane phosphoprotein associated with neural development axonal regeneration, phospholipid metabolism, and synaptic plasticity. Trends in Neurosci. 10: 527-532.
Bourne, H. R. and Nicoll, R. (1993) Molecular machines integrate coincident synaptic signals. Cell 72:65-75.
Byrne, J. H. (1987) Presynaptic facilitation as a mechanism for behavioral sensitization in Aplysia. Physiol. Rev. 67:329-439.
Cali, J. F., Zwaagstra, J. C., Mons, N., Cooper, D. M. F., and Krupinski, J. (1994) Type VIII Adenylyl Cyclase. J. Biol. Chem. 269:12190-12195.
Castellucci, V. F. and Kandel, E. R.(1976) Presynaptic facilitation as a mechanism for behavioral sensitization in Aplysia. Science 194:1176-1178.
Castellucci, V.F., Blumenfeld, H., Goelet, P., and Kandel, E.R. (1989) Inhibitor of protein synthesis blocks long-term behavioral sensitization in the isolated gill-withdrawal reflex of Aplysia. J. Neurobiol. 20:1-9.
Chetkovich, D. M., Gray, R., Johnston, D. and Sweatt, J. D. (1991) N-methyl-D-aspartate receptor activation increases cAMP levels and voltage gated Ca2+ channel activity in area CA1 of hippocampus. Proc. Natl. Acad. Sci. U.S.A. 88:6467-6471.
Cheung, W.Y. and Storm D.R. (1982) Calmodulin regulation of cAMP metabolism. Handbook of Exper. Pharmacol. 58:301-317.
Choi, E. J., Wong, S. T., Hinds, T. J. and Storm, D. R. (1992a) Calcium and muscarinic agonist stimulation of type I adenylyl cyclase in whole cells. J. Biol. Chem. 267: 12440-12442.
Choi, E. J., Xia Z., and Storm D.R. (1992b) Stimulation of the type III olfactory adenylyl cyclase by calcium and calmodulin. Biochemistry 31:6492-6498.
Choi, E. J., Wong, S. T., Dittman, A. H., and Storm, D.R. (1993). Phorbol Ester Stimulation of the Type I & Type III Adenylyl Cyclases In Whole Cells. Biochemistry 32:1891-1894.
Cimler, B. M., Andreasen, T. J., Andreasen, K. I. & Storm, D. R. (1985) P-57 is a neural specific calmodulin-binding protein. J. Biol. Chem. 260:10784-10788.
Davis, S., Butcher, S. P. Morris, R. G. (1992) The NMDA receptor antagonist D-2-amino-5-phosphonopentanoate (D-AP5) impairs spatial learning and LTP in vivo at intracerebral concentrations comparable to those that block LTP in vitro. J. Neuroscience 12: 21-34.
Dittman, A. H., Weber, J. P., Hinds, T. R., Choi, E. J., Migeon, J. C., and Nathanson, N. M. and Daniel R. Storm (1994) A Novel Mechanism for Coupling of m4 Muscarinic Acetylcholine Receptors to Calmodulin Sensitive Adenylyl Cyclases: Cross-Over from G Protein Coupled Inhibition to Stimulation. Biochemistry (in press).
Dudai, Y. and Zvi, S. (1984) Adenylate cyclase in the Drosophila memory mutant rutabaga displays an altered Ca2+ sensitivity. Neurosci. Lett.. 47:19-24.
Dudai, Y., and Zvi, S. (1985) Multiple defects in the activity of adenylyl cyclase from the Drosophila memory mutant rutabaga. J. Neurochem. 45:355-364.
Dudai, Y. (1988) Neurogenetic dissection of learning and short-term memory in Drosophila. Annu. Rev. Neurosci. 11:537-563.
Feany, M. B. (1990) Rescue of the learning defect in dunce, a Drosophila learning mutant, by an allele of rutabaga, a second learning mutant. Proc. Natl. Acad. Sci. USA 87:2795-2799.
Feinstein, P.G., Schrader, A., Bakalyar, H. A., Tang,W. J., Krupinski, J., Gilman A. G., & Reed R. R. (1991) Molecular cloning and characterization of a calcium calmodulin insensitive adenylyl cyclase (typeII) from rat brain. Proc. Natl . Acad. Sci. USA 88:10173-10177.
Frey, U., Krug, M., Reymann, K.G., and Matthies, H. (1988) Anisomycin, an inhibitor of protein synthesis, blocks late phases of LTP phenomena in the hippocampal CA1 region in vitro. Brain Res. 452:57-65.
Frey, U., Matthies, H., Reymann, K. G., and Matthies, H (1991). The effect of dopaminergic D1 receptor blockade during tetanization on the expression of long-term potentiation in the rat CA1 region in vitro. Neurosci. Lett. 29:111-4.
Frey, U., Huang. Y.Y. and Kandel, E. R. (1993) Effects of cAMP simulate a late stage of LTP in hippocampal CA1 neurons. Science 260: 1661-1664.
Frost, W. N., Castellucci, V. F., Hawkins, R. D., and Kandel, E. R. (1985) Monosynaptic connections from the sensory neurons of the gill- and siphon-withdrawal reflex in Aplysia participate in the storage of long-term memory for sensitization. Proc. Natl . Acad. Sci. USA 82:8266-8269.
Gao B. & Gilman A. (1991) Cloning and expression of a widely distributed (type IV) adenylyl cyclase. Proc. Natl. Acad. Sci. USA 88:10178-10182
Ghirardi, M., Braha, O., Hochner, B., Montarolo, P.G., Kandel, E. R. (1992) Roles of PKA and PKC in facilitation of evoked and spontaneous transmitter release at depressed and nondepressed synapses in Aplysia sensory neurons. Neuron 9:479-89.
Glatt, C. E. and Snyder, S. H. (1993) Cloning and expression of an adenylyl cyclase localized to the corpus striatum. Nature 363:679-680.
Gnegy, M. and Treisman, G. (1981) Effect of calmodulin on dopamine-sensitive adenylate cyclase activity in rat striatal membranes. Mol. Pharmacol. 19:256-63.
Gnegy M., Muirhead N., Roberts-Lewis J. M., and Treisman, G. (1984) Calmodulin stimulates adenylyl cyclase activity and increases dopamine activation in bovine retina. J. Neurosci. 4:2712-2717.
Grant, S. G. N., O'Dell, T. J., Karl, K. A., Stein, P. L., Soriano, P., Kandel, E. R. (1992) Impaired long term potentiation, spatial learning and hippocampal development in fyn mutant mice. Science 258:1903-1992.
Grecksch, G. and Matthies, H. (1980) Two sensitive periods for the amnesic effect of anisomycin. Pharmacol. Biochem. Behav. 12:663-5.
Greenberg, S. M., Castellucci, V. F., Bayley, H., and Schwartz, J. H. (1987) A molecular mechanism for long-term sensitization in Aplysia. Neuron 329: 62-65.
Hagiwara, M., Brindle, P., Harootunian, A., Armstrong, R., Rivier, J., Vale, J., Tsien, R. and Montminy, M. R. (1993) Coupling of hormonal stimulation and transcription via the cAMP responsive factor CREB is rate limited by nuclear entry of protein kinase A. Mol. and Cell Biology 13, 4852-4859.
Hopkins, W. F., and Johnston, D. (1988) Noradrenergic enhancement of long-term potentiation at mossy fiber synapses in the hippocampus. J. Neurophysiol. 59:667-687.
Ishikawa, Y., Katsushika, S., Chen, L., Halnon, N. J., Kawabe, J. & Homcy, C. J. (1992) Isolation and characterization of a novel cardiac adenylyl cyclase cDNA. J. Biol. Chem. 267:13553-13557.
Jacobowitz, O. Chen, J., Premont, R. T., and Iyengar, R (1993) Stimulation of specific types of Gs-stimulated adenylyl cyclases by phorbol ester treatment. J. Biol. Chem. 268:3829-3822.
Kaang, B.K., Kandel, E. R., and Grant, S.G. (1993) Activation of cAMP-responsive genes by stimuli that produce long-term facilitation in Aplysia sensory neurons. Neuron 10: 427-435.
Kandel, E. R. and Schwartz, J. H. (1982) Molecular biology of learning : modulation of transmitter release. Science 218:433-443.
Kauer, J.A., Malenka, R. C., and Nicoll, R. A. (1988) NMDA application potentiates synaptic transmission in the hippocampus. Nature 334:250-252.
Klein, M., Hochner, B., and Kandel, E. R. (1986) Facilitatory transmitters and cAMP can modulate accommodation as well as transmitter release in Aplysia sensory neurons:Evidence for parallel processing in a single cell. Proc. Natl. Acad. Sci. USA 83:7994-7998.
Krebs, E. G., and Beavo, J. A (1979) Phosphorylation-dephosphorylation of enzymes. Annu. Rev. Biochem. 48:923-959.
Krupinski, J., Coussen, F., Bakalyar, H. A., Tang, W. J., Feinstein, P.G., Orth, K., Slaughter, C., Reed, R. R., and Gilman, A. G. (1989) Adenylyl cyclase amino acid sequence: possible channel- or transporter-like structure. Science 244:1558-1564.
Krug, M. and Lossner, B. and Ott, T.. (1984) Anisomycin blocks the late phase of long-term potentiation in the dentate gyrus of freely moving rats. Brain Res. Bull. 13:39-42.
Levin L.R., Han P.L., Hwang P.M., Feinstein P.G., Davis R.L., Reed, R., R. (1992) The drosophila learning and memory gene rutabaga encodes a Ca2+/CaM-responsive adenylyl cyclase. Cell 68: 479-489.
Liu, Yuechueng and Storm, D. R. (1989) Dephosphorylation of neuromodulin by calcineurin. J. Biol. Chem. 264:12800-12804.
Liu, Y. L., & Storm, D. R. (1990) Regulation of Free Calmodulin Levels in Neurons by Neuromodulin: Relationship to Neuron Growth and Regeneration. Trend in Pharmacological Sciences 11:107-111.
Livingston, M. S., Sziber, P. P., and Quinn, W. G. (1984). Loss of calcium calmodulin responsiveness in adenylyl cyclase of rutabaga, a Drosophila learning mutant. Cell 37: 205-215.
Livingston, M. S. (1985) Genetic dissection of Drosophila adenylyl cyclase. Proc. Natl. Acad. Sci. USA 82:5992-5996.
Malenka, R. C., Kauer, J. A., Zucker, R. S. and Nicoll, R.A. (1988) Postsynaptic calcium is sufficient for potentiation of hippocampal synaptic transmission. Science 242:81-84.
Matthies, H. Frey, U., Reymann, K., Krug, M., Jork, R., Schroeder, H. (1990) Different mechanisms and multiple stages of LTP. Adv. Exp. Med. Biol. 268:359-68.
May, D., Ross, E., Gilman, A., and Smigel, M. (1985). Reconstitution of Catecholamine-stimulated Adenylate cyclase Activity Using Three Purified Proteins. J. Biol. Chem. 260:15829-15833.
MacNicol, M., and Schulman, H. (1992) Cross-talk between protein kinase C and multifunctional Ca2+/calmodulin-dependent protein kinase. J. Biol. Chem. 267:12197-201.
Mitchell, P. J. and Tjian, R. (1989) Transcriptional regulation in mammalian cells by sequence specific DNA binding proteins.Science 245:371-378.
Mollner, S. and Pfeuffer, T. (1988) Two different adenylyl cyclases in brain distinguished by monoclonal antibodies. Eur. J. Biochem. 171:265-271.
Mollner, S., Simmoteit, R., Palm, D., Pfeuffer, T. (1991) Monoclonal antibodies against various forms of the adenylyl cyclase catalytic subunit and associated proteins. European. J. Biochem. 195:281-286.
Montarolo, P.G., Goelet, P., Castellucci, V. F., Morgan, J., Kandel, E. R. and Schacher, S.(1986) A critical period for macromolecular synthesis in long-term heterosynaptic facilitation in Aplysia. Science 234: 1249-1254.
Morris, R.G., Garrud, P., Rawlins, J.N. and O'Keefe, J. (1982) Place navigation impaired in rats with hippocampal lesions. Nature 297:681-683.
Morris, R. G., Anderson, E., Lynch, G. S., and Baudry, M. (1986) Selective impairment of learning and blockade of long-term potentiation by an N-methyl-D-aspartate receptor antagonist, AP5. Nature 319:774-776.
Morris, R. G. (1900) Toward a representational hypothesis of the role of hippocampal synaptic plasticity in spatial and other forms of learning. Cold Spring Harbor Symp. Quant. Biol. 50:161-173.
Nairn, A. C., Hemmings, H. C., and Greengard, P. (1985) Protein kinases in the brain.Annu. Rev. Biochem. 54:931-976.
Natsukari, N., Hanai, H., Matsunaga, T., and Fujita, M. (1990) Synergistic activation of brain adenylate cyclase by calmodulin, and either GTP or catecholamines including dopamine. Brain Res. 534:170-176.
Nelson, R. B. and Routtenberg, A. (1985) Characterization of protein F1: a kinase C substrate directly related to neural plasticity. Exp. Neurol. 89:213-224
Nestler, E. J., and Greengard, P. (1983) Protein phosphorylation in the Brain. Nature 305:583-588.
Nicoll, R. A., Kauer, J. A., and Malenka, R. C. (1988) The current excitement in long-term potentiation. Neuron 1: 97-103.
Nigg, E.A., Hilz, H., Eppenberger, H.M., and Dutly, F. (1985) Rapid and reversible translocation of the catalytic subunit of cAMP-dependent protein kinase type II from the Golgi complex to the nucleus. EMBO J. 4:2801-2806.
Pfenninger, K. H., Ellis, L., Johnson, M. P., Friedman, L. B. & Somlo, S. (1983) Nerve growth cones isolated from fetal rat brain: subcellular fractionation and characterization. Cell 35: 573-584
Piascik, M. T., Piascik, M. F., Hitzemann, R. J., and Potter, J. D. (1981)
Rosenberg, G. B., and Storm, D. R. (1987a) Immunological distinction between calmodulin-sensitive and calmodulin-insensitive adenylyl cyclases. J. Biol. Chem. 262:7623-7628 .
Rosenberg, G.B., Minocherhomjee, A., Storm, D.R. (1987b) Reconstitution of calmodulin sensitive adenylate cyclase. Methods in Enzymology 139:776-79.
Ross, E. M. and Gilman A.G. (1980) Biochemical properties of hormone sensitive adenylyl cyclase. Ann. Rev. Biochem. 49:533-564.
Salter, R. S., Krinks, M. H., Klee, C. B., and Neer, E. J. (1981) Calmodulin activates the isolated catalytic unit of brain adenylate cyclase. J. Biol. Chem. 256:9830-9833.
Sano, M. (1985) Calmodulin-dependent adenylate cyclase in rat retina and the response to dopamine. Brain Research 345:337-340
Schacher, S., Castellucci, V. F., and Kandel, E.R. (1988) cAMP evokes long-term facilitation in Aplysia sensory neurons that requires new protein synthesis. Science 240: 1667-1669.
Scholz, K.P. and Byrne, J. H. (1988) Intracellular injection of cAMP induces a long-term reduction of neuronal K+ currents. Science 240: 1664-1666.
Silva, A.J., Stevens, C.F., Tonegawa, S. and Wang, Y. (1992a) Deficient hippocampal long-term potentiation in alpha-calmodulin kinase II mutant mice.Science 257:201-206.
Silva, A. J., Paylor, R., Wehner, J. M., and Tonegawa, S. (1992) Impaired spatial learning in alpha calcium calmodulin kinase II mutant mice. Science 257:206-210.
Skene, J. H. P. and Willard, M., J. (1981a) Changes in axonally transported proteins during axon regeneration in toad retinal ganglion cells. J. Cell Biol. 89:86-95.
Skene, J. H. P., Jacobson, R. D., Snipes, G. J., McGuire, C. B., Norden, J. J. & Freeman, J. A. (1986) A protein induced during nerve growth (GAP-43) is a major component of growth-cone membranes. Science 233:783-786
Skene, J.H.P. (1989) Axonal growth-associated proteins. Ann. Rev. Neurosci. 12:127-156.
Slack, J. R. and Pockett, S. (1991) Cyclic AMP induces long-term increase in synaptic efficacy in CA1 region of rat hippocampus. Neurosci. Lett. 130: 69-72.
Smigel, M.D. (1986) Purification of brain adenylyl cyclase using forskolin affinity chromatography and WGA-Sepharose. J. Biol. Chem. 261:1976-1982.
Smith, S. J. and Augustine, G. J. (1988) Calcium ions, active zones and synaptic transmitter release. Trends Neurosci. 11:458-464.
Squire, L.R. and Davis, H.P. (1981) The pharmacology of memory: a neurobiological perspective. Annu. Rev. Pharmacol. Toxicol. 21: 323-56.
Stanton, P. K., and Sarvey, J. M. (1985a) The effect of high-frequency electrical stimulation and norepinephrine on cyclic AMP levels in normal versus norepinephrine-depleted rat hippocampal slices. Brain Res. 358: 343-348.
Stanton, P. K., and Sarvey, J. M. (1985b) Depletion of norepinephrine, but not serotonin, reduces long-term potentiation in the dentate gyrus of rat hippocampal slices. J. Neurosci. 5:2169-2176.
Tang W. J., Krupinski J. Gilman A. G. (1991) Expression and characterization of calmodulin activated (type I) adenylyl cyclase. J. Biol. Chem. 266:8595-8603.
Taussig, R.T., Quarmby, L. R., and Gilman, A. G. (1993a) Regulation of purified type I & type III adenylyl cyclases by G protein beta/gamma subunits. J. Biol. Chem. 268: 9-12
Toscano, W.A., Jr., Westcott, K.R., LaPorte, D.C., and Storm, D. R. (1979) Evidence for a dissociable protein subunit required for calmodulin stimulation of brain adenylate cyclase. Proc. Natl. Acad. Sci. U.S.A. 76:P 5582-6
Tota, M. R., Xia, Z., Storm, D. R., and Schimerlik, M. I. (1990). Reconstitution of Muscarinic Receptor Mediated Inhibition of Adenylyl Cyclase. Mol. Pharm. 37:950-957.
Villacres, E. C., Xia, Z., Bookbinder, L. H., Edelhoff, S., Adler, D. A., Disteche, C. M., and Storm, D. R. (1993) Cloning, Chromosomal Mapping, and Expression of Human Fetal Brain Type I Adenylyl Cyclase. Genomics 16: 473-478.
Wayman, G. A., Impey, S., Wu, Z., Kinsvogel, W., and Prichard. L. and Storm, D. R. (1994) Synergistic Activation of the Type I Adenylyl Cyclase by Ca2+ and Gs Coupled Receptors In Vivo. J. Biol. Chem. (in press).
Walters, E. T., and Byrne J. H. (1983) Associative conditioning of single sensory neurons suggests a cellular mechanism for learning. Science 219:405-408.
Westcott, K. R., LaPorte D.C., and Storm D.R. (1979) Resolution of adenylyl cyclase sensitive and insensitive to Ca2+ and CDR by CDR-Sepharose affinity chromatography. Proc. Nat. Acad. Sci., USA 76:204-228.
Wu, Z., Wong, S. T., and Storm, D. R. (1993) Modification of the Calcium and Calmodulin Sensitivity of the Type I Adenylyl Cyclase by Mutagenesis of its Calmodulin Binding Domain. J. Biol. Chem. 268: 23766-23768.
Xia, Z., Refsdal, C. D., Merchant, K. M., Dorsa, D. M., & Storm, D.R. (1991) Distinct Patterns for the Distribution of mRNA for the Calmodulin Sensitive adenylyl Cyclase in Rat Brain: Expression in Areas Associated with Learning and Memory. Neuron 6:431-443.
Xia, Z., Choi, E. J., Wang, F., and Storm, D. R. (1992) The Type III Calcium/Calmodulin Sensitive Adenylyl Cyclase is not Specific to Olfactory Sensory Neurons. Neurosci. Letters 144: 169-173.
Xia, Z., Choi, E. J., Wang, F., Blazynski, C., & Storm, D. R. (1993) The Type I Calmodulin Sensitive Adenylyl Cyclase Is Neural Specific. J. Neurochem. 60:305-11.
Yovell, Y. Kandel, E.R., Dudai, Y., and Abrams, T.W. (1992) A quantitative study of the Ca2+/calmodulin sensitivity of adenylyl cyclase in Aplysia, Drosophila, and rat.J. Neurochem. 59:1736-1744.
Yoshimura, M. & Cooper, D. M. (1992) Cloning and expression of a Ca2+ inhibitable adenylyl cyclase from NCB-20 cells. Proc Natl. Acad. Sci. USA 89:6716-6720.
Zhong, Y. and Wu, C. F. (1991) Altered synaptic plasticity in Drosophila memory mutants with a defective cyclic AMP cascade. Science 251:198-201.
FIGURE LEGENDS: [figures available only in hard copy]
Figure 1. The effect of CaM on either type I (A) or type III (B) adenylyl cyclase activities transiently expressed in human 293 cells. Human 293 cells were transfected with control CDM8 vector, CDM8(I-AC) to express the type I adenylyl cyclase or CDM8(III-AC) to express the type III adenylyl cyclase. Cell membranes prepared from transfected cells were washed with buffer A containing 1 mM EGTA to remove endogenous CaM, and then assayed for adenylyl cyclase activity as a function of CaM concentration. A, the effect of CaM on control (O) or type I adenylyl cyclase activity (O). The assay was performed in the presence of 1.9 5M free Ca2+ and varying concentrations of CaM. Control activity was the adenylyl cyclase activity in cell membranes from CDM8-transfected 293 cells. B, the effect of CaM on type III adenylyl cyclase activity in the absence (O) or in the presence of 10 5M forskolin (O). The enzyme assay was performed in the presence of 30 5M free Ca2+ and varying concentrations of CaM. Adenylyl cyclase activities due to the type III enzyme were calculated from data shown in panel C by subtracting adenylyl cyclase activities in control cells from those of cells transfected with CDM8(III-AC). In panel C, cell membranes from control 293 cells (x, F) or cells transfected with CDM8(III-AC) (O, O) were assayed for adenylyl cyclase activity with varying concentrations of CaM and 30 5M Ca2+ in the absence (x, O) or presence of 10 5M forskolin (F, O). Data is from Choi et al., 1992a.
Figure 2. The effect of Ca2+ on type I (A) or type III (B) adenylyl cyclase activities expressed in 293 cells. Cell membranes from human 293 cells transfected with control CDM8 vector, CDM8(I-AC) to express the type I adenylyl cyclase, or CDM8(III-AC) to express the type III adenylyl cyclase were examined for adenylyl cyclase activity as a function of free Ca2+ concentration by varying concentrations of CaCl2 in the presence of 0.2 mM EGTA in the assay. A, the effect of Ca2+ on control (O) or type I adenylyl cyclase activity (O). The enzyme assay was performed in the presence of 2.4 5M CaM and various concentrations (0 - 3.7 5M) of free Ca2+. B, the effect of Ca2+ on type III adenylyl cyclase activity in the absence (O) or in the presence of 100 5M GppNHp (O). The enzyme assay was performed in the presence of 2.4 5M CaM and various concentrations (0 - 74 5M) of free Ca2+. Type III adenylyl cyclase activity was calculated from data shown in panel C by subtracting adenylyl cyclase activities in control cells from those of cells transfected with CDM8(III-AC). In panel C, cell membranes from control cells (F, F) or cells transfected with CDM8(III-AC) (O, O) were assayed for adenylyl cyclase activity with varying concentrations of free Ca and 2.4 5M CaM in the absence (F, O) or presence of 100 5M GppNHp (F, O). Data is from Choi et al., 1992b.
Figure 3. Ca2+ and A23187 stimulation of intracellular cAMP levels in 293 cells expressing the type I adenylyl cyclase. A, cultured 293 cells expressing the type I adenylyl cyclase (O) or control cells transfected with the CDM8 vector (O) were treated with varying concentrations of A23187 in the presence of 2 mM CaCl2 for 30 minutes. B, 293 cells expressing the type I adenylyl cyclase (O) or control cells (O) were treated with varying concentrations of CaCl2 for 30 minutes in the presence of 10 5M A23187. C, 293 cells expressing the type I adenylyl cyclase (O, F) or control cells (O, X) were treated for various periods of time with 2 mM CaCl2 and 10 5M A23187 in the presence (O, O) or absence (F, X) of one mM IBMX. Intracellular cAMP was assayed as described in Experimental Procedures. Data is from Choi et al., 1992a.
Figure 4. Carbachol stimulation of intracellular cAMP levels in 293 cells expressing the type I adenylyl cyclase. A, Control cells ([ ], [ ] ) or 293 cells expressing the type I adenylyl cyclase ([ ], [ ]) were treated with varying concentrations of carbachol for 30 minutes in the absence ([ ], [ ]) or presence ([ ], [ ]) of 1.0 5M atropine. One mM IBMX was present in all assays. B, pretreatment of 293 cells for one hour with BAPTA/AM at 100 5M completely blocked the increase in intracellular cAMP caused by carbachol. Data is from Choi et al., 1992a.
Figure 5. Northern analysis of the type I sensitive adenylyl cyclase using mRNA from various bovine tissues. Two micrograms of poly (A)+ selected RNA samples were electrophoresed to a 1.2% agarose/formaldehyde gel. The blots were hybridized with an alpha [32P]dCTP labeled cDNA probe 3C that is specific for the bovine type I adenylyl cyclase. The 0.24-9.5 kb RNA ladder from BRL was used as the molecular weight standard. Panel A, poly (A)+ selected RNA was isolated from various bovine tissues. Data is from Xia et al., 1993.
Figure 6. Distribution of the type I Ca2+/CaM sensitive adenylyl cyclase mRNA within various layers of bovine neural retina examined by in situ hybridizations. Cross sections of bovine eyecups hybridized with 35S-UTP labeled bovine type I adenylyl cyclase specific riboprobe 3C were treated with NTB2 emulsion for two weeks, and counterstained with cresyl violet acetate. Panel A, light microscope photomicrograph; panel B, phase contract photomicrograph; panel C, dark field photomicrograph. Abbreviations: IS, inner segment layer of the photoreceptor cells; ONL, the outer nuclear layer which contains nuclei of rods and cones; INL, the inner nuclear layer which contains cell bodies of horizontal cells, bipolar cells and amacrine cells; GCL, the ganglion cell layer. Data is from Xia et al., 1993
Figure 7. In situ hybridizations for type I adenylyl cyclase in middle rat brain. Rat brain sections were hybridized with either 35S-labeled antisense riboprobe 3C (A and C) or oligonucleotide probe ZX4 (E). The specificity of hybridization was demonstrated by incubation of the respective 35S-labeled probe with a 1000-fold molar excess of the unlabeled probe (B, D, F). Exposure time: 3 days (A-D), and 7 days (E-F). Abbreviations: Cx, neocortex; DG, dentate gyrus; Hi, hippocampus; IG, indusium griseum; Pir, piriform cortex; SHi, septohippocampal nucleus; Tu, olfactory tubercle. Data is from Xia et al., 1991.
Figure 8. in situ hybridizations for type I adenylyl cyclase in cerebellum. Rat brain cerebellum sections were hybridized with 35S-labeled antisense riboprobe 3C (A and B). Specificity of hybridization was demonstrated by incubation of the respective 35S-labeled probe with a 1000-fold molar excess of unlabeled probe (C). Exposure time: 3 days (A, B) and 7 days (C). Abbreviations: Cb, cerebellum; BS, brain stem. Data is from Xia et al., 1991.
Figure 9. Hypothetical model for the role of neuromodulin and the type I adenylyl cyclase in neuroplasticity. It is hypothesized that neuromodulin binds and concentrates CaM at specific sites in neurons and that the levels of free CaM are regulated by phosphorylation of neuromodulin by protein kinase C. During LTP protein kinase C is activated and Ca2+ is mobilized. We propose that the CaM sensitive adenylyl cyclases may be activated during LTP by Ca2+/CaM, neurotransmitters, and/or activation of protein kinase C. Activation of the CaM sensitive adenylyl cyclase(s) results in coupling of the Ca2+ and cAMP regulatory systems during LTP. Simultaneous or ordered activation of the Ca2+ and cAMP regulatory systems may be important for amplified cAMP signals required for transcription, synergism between Ca2+ and cAMP activated kinases, and/ or positive feedback regulation of Ca2+ channels by cAMP dependent kinase. Abbreviations: CaM, calmodulin; NM, neuromodulin (GAP-43), PKC, protein kinase C; NM-P, neuromodulin phosphorylated on Ser-41; PKA, cAMP dependent protein kinase.