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Siegel GJ, Agranoff BW, Albers RW, et al., editors. Basic Neurochemistry: Molecular, Cellular and Medical Aspects. 6th edition. Philadelphia: Lippincott-Raven; 1999.

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Basic Neurochemistry: Molecular, Cellular and Medical Aspects. 6th edition.

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Adenylyl Cyclases

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Regulation of cAMP formation by neurotransmitter receptors and intracellular messenger pathways is determined by the activity of the synthetic enzyme adenylyl cyclase, also termed adenylate cyclase. Adenylyl cyclase can be activated directly by forskolin, a plant diterpene which has been useful in studies of enzyme regulation and purification. The substrate for adenylyl cyclase is a complex of Mg2+ and ATP. In addition, free divalent cation, such as free Mg2+, in excess of ATP is a requisite cofactor for enzyme activity. As shown in Figure 22-2, adenylyl cyclase forms cAMP by creating a cyclic phosphodiester bond with the α-phosphate group of ATP, with the concomitant release of pyrophosphate, which provides energy for the reaction.

Figure 22-2. Chemical pathways for the synthesis and degradation of cAMP.

Figure 22-2

Chemical pathways for the synthesis and degradation of cAMP. cAMP is synthesized from ATP by the enzyme adenylyl cyclase with the release of pyrophosphate and hydrolyzed into 5′-AMP by the enzyme phosphodiesterase. Both reactions require Mg2+ (more...)

Biochemical and molecular cloning studies indicate the existence of several forms of adenylyl cyclase which comprise a distinct enzyme family [1,2]. To date, nine forms of the enzyme, referred to as types I through IX, have been identified, although type IX has not been fully characterized. Additional forms may be found when discrete brain regions and tissue types are examined. All of the enzyme forms that have been identified are membrane-bound and stimulated by Gαs and forskolin. However, the different forms of adenylyl cyclase exhibit distinct patterns of expression in brain and peripheral tissues and are regulated differentially by Ca2+/calmodulin, by the G protein subunits Gαi and Gβγ and by phosphorylation.

Multiple forms of adenylyl cyclase exist in the nervous system

As mentioned earlier, these enzymes show considerable variability in the levels and region-specific patterns of expression in the brain. Types I and VIII appear to be expressed exclusively in the brain, adrenal gland and retina [35]. In the brain, these enzymes are expressed at highest levels in the hippocampus and neocortex. The catalytic activities of types I and VIII are stimulated by Ca2+/calmodulin (see below) and thereby could be regulated by activation of N-methyl-d-aspartate (NMDA) glutamate receptors that flux Ca2+ into neurons and have been implicated in several forms of neural plasticity. Further evidence for a role of type I adenylyl cyclase in synaptic plasticity is the finding that mice lacking this form of the enzyme have deficits in spatial memory as well as long-term potentiation [6], a cellular model of learning and memory seen in hippocampus and other brain regions (see Chap. 50).

Type II adenylyl cyclase is expressed at high concentrations in many brain regions but is also found at lower concentrations in lung and olfactory tissue [3,5]. The highest concentrations are found in hippocampus, hypothalamus and cerebellum, with moderate concentrations in neocortex, piriform cortex and amygdala. Type III adenylyl cyclase is highly enriched in olfactory epithelium and is expressed at lower concentrations in other brain regions, as well as in lung and heart [7]. Type IV adenylyl cyclase is widely distributed and has been found in all tissues and cell types tested to date [8].

Type V adenylyl cyclase is highly enriched in brain and is localized largely to the striatum and related structures, such as the nucleus accumbens and olfactory tubercle, which are innervated by dopamine. As a result, it often is referred to as “striatal adenylyl cyclase” [9,10]. The type V enzyme is also expressed in heart and kidney, where it is associated with blood vessels; the anterior lobe of the pituitary; and the retina. All of these tissues share the common feature of having dopamine innervation, and it has been suggested that type V adenylyl cyclase is associated uniquely with dopamine actions [9]. Type VI adenylyl cyclase is structurally similar to the type V enzyme, and both are inhibited by free Ca2+ at physiological concentrations (0.1 to 1.0 μM) [11]. Type VI adenylyl cyclase is enriched in brain and heart, with low expression in other tissues, such as testes, muscle, kidney and lung.

Type VII adenylyl cyclase is found in several tissues; concentrations are highest in lung and spleen, moderate in heart and low in brain, kidney and skeletal muscle [12,13]. In the brain, concentrations are highest in cerebellar granule cells and lower in hippocampus, neocortex and striatum. Less is known about the type IX enzyme, which to date has not been characterized extensively.

The different forms of adenylyl cyclase are similar in structure

Hydropathicity profiles of amino acid sequences indicate that adenylyl cyclases contain two regions (M1 and M2), each of which consists of six putative membrane-spanning hydrophobic domains [1,2] (Fig. 22-3). This is preceded by a short, variable amino terminus. In addition, there are two large cytoplasmic domains (C1 and C2), one between the two hydrophobic regions and the other at the carboxy terminus of the protein. The predicted molecular weight of type I adenylyl cyclase is 124,000 [14], although other types of analysis indicate a native molecular mass of over 200 kDa. This suggests that adenylyl cyclases may exist as a dimer or as a complex with other regulatory proteins.

Figure 22-3. Schematic illustration of the proposed topographical structure of adenylyl cyclases.

Figure 22-3

Schematic illustration of the proposed topographical structure of adenylyl cyclases. Hydropathicity profiles predict that adenylyl cyclases contain two hydrophobic regions (M1 and M2), each of which contains six membrane-spanning regions and two relatively (more...)

The two cytoplasmic domains are the most highly conserved portions of the known forms of adenylyl cyclase and are similar to each other within a given enzyme molecule. C1 is larger, with 360 to 390 amino acids, than C2, with 255 to 330 amino acids. These cytoplasmic regions have been subdivided into a and b domains (Fig. 22-3). C1a and C2a are similar to each other and to certain sequences of both membrane-bound and soluble guanylyl cyclases, which catalyze the synthesis of cGMP (see below). These are thought to be the catalytic domains of the enzyme and to contain nucleotide-binding sites. Both C1 and C2 appear to be necessary for catalytic activity: there is no enzymatic activity when only one of the domains is expressed, but activity is reinstated when the two halves are coexpressed [8]. It is still unknown whether C1a and C2a act in concert as catalytic domains or if one is catalytic and the other regulatory. The smaller C1b and C2b regions appear to be involved in regulation of catalytic activity (see below). Adenylyl cyclases also are glycosylated and show several potential sites for phosphorylation, as will be discussed below. All known forms of adenylyl cyclase are inhibited by P-site inhibitors, which are adenosine analogues that probably act at the catalytic site of the enzyme.

The topographical structure of adenylyl cyclases is similar to that of membrane transporters and ion channels. However, there is currently no convincing evidence of a transporter or channel function for mammalian adenylyl cyclases. The structural similarity may indicate that these functionally divergent protein families are derived in an evolutionary sense from related proteins.

Adenylyl cyclases are regulated by Gαs and Gαi

Each of the different forms of adenylyl cyclase is stimulated by activated Gαs, that is, Gαs bound to guanosine triphosphate (GTP). As described in Chapter 20, activation by G protein-coupled receptors occurs when ligand binds to the receptor and catalyzes the exchange of guanosine diphosphate (GDP) for GTP at the α subunit. This promotes the dissociation of the α and βγ subunits, which allows for activated Gαs to interact with and stimulate adenylyl cyclase. GTPase activity contained within the α subunit hydrolyzes GTP to GDP and thereby leads to reassociation with the βγ complex.

It is also notable that adenylyl cyclase types I and VIII are activated synergistically by Gαs and Ca2+/calmodulin. This finding suggests that these forms of the enzyme are capable of responding to receptors coupled to Gαs as well as to those that increase intracellular Ca2+ levels. This may be of particular importance to the function of these proteins in integrating the consequences of multiple extracellular stimuli as well as in synaptic plasticity.

In contrast to Gαs, several forms of Gαi can inhibit the catalytic activity of adenylyl cyclase when the enzyme is activated by either Gαs or forskolin. This includes the Gαi1, Gαi2, Gαi3 and Gαz subtypes. Gαi-mediated inhibition of adenylyl cyclase is most dramatic for the type V and type VI enzymes. Type I adenylyl cyclase also can be inhibited by these Gαi subtypes, as well as by Gαo, but this inhibition is observed more readily when the type I enzyme is activated by Ca2+/calmodulin than by Gαs. Type VIII adenylyl cyclase differs from the type I enzyme in this respect since it does not appear to be inhibited appreciably by Gαi [15].

Adenylyl cyclase subtypes also are regulated by βγ subunits

The type of regulation seen varies dramatically with the different forms of adenylyl cyclase [1,2,16]. In the presence of Gαs, addition of βγ complexes inhibits type I but stimulates types II and IV adenylyl cyclases. The type III, V and VI enzymes do not appear to be influenced by βγ subunits. The concentration of βγ required for its inhibitory effect on type I adenylyl cyclase is much higher than that of free Gαs required to activate this enzyme. This implies that dissociation of βγ from other, more abundant G proteins, such as Gαi and Gαo, would be the primary source of βγ for this type of inhibition to occur in vivo. βγ also inhibits Ca2+/calmodulin-stimulated type I adenylyl cyclase and acts as a better inhibitor of this enzyme than either Gαi or Gαo.

Activation of types II and IV adenylyl cyclase by βγ complexes not only is dependent on the presence of Gαs but the enzymes are activated synergistically by these G protein subunits. As is the case for inhibition of type I adenylyl cyclase, the concentration of βγ for activation of the types II and IV enzymes is higher than that for Gαs. This implies that activation of receptors coupled to Gαs and Gαi/Gαo may be able to activate types II and IV adenylyl cyclase, and these responses could be synergistic under certain conditions. Studies on chimeras of the different enzymes indicate that the stimulatory effect of βγ resides in the carboxy half of the adenylyl cyclase molecule [8]. These studies involved “non-covalent” chimeras, where the amino half of the type I enzyme, containing the first membrane-spanning and cytoplasmic domains, is co-expressed in cultured cells with the carboxy half of either the type I or type II enzyme, containing the second membrane-spanning and cytoplasmic domains. Depending on whether the carboxy portion is from type I or II, the coexpressed chimera is either inhibited or stimulated, respectively, by βγ complexes.

In addition to the selective responses of adenylyl cyclases to βγ subunits, it is likely that different forms of β and γ subunits influence the various forms of adenylyl cyclase in different ways [17]. There are five known forms of β and seven known forms of γ. Differential expression and regulation of these subunits could provide additional mechanisms for selectively controlling adenylyl cyclase catalytic activity in specific neuronal cell types.

Adenylyl cyclases show differential regulation by Ca2+

This regulation [1,2] suggests an important series of mechanisms by which various forms of adenylyl cyclase may be regulated in vivo by receptors that influence ligand- or voltage-gated Ca2+ channels or receptors that influence intracellular Ca2+ levels via coupling to Gαo and release of inositol trisphosphate (IP3) (see Chap. 21). Types I, III and VIII adenylyl cyclase are activated by Ca2+/calmodulin, and this effect is synergistic with Gαs. In contrast, types II, IV, V and VI adenylyl cyclase are insensitive to Ca2+/calmodulin. The C1b domain of adenylyl cyclase appears to mediate activation of the enzyme by Ca2+/calmodulin (Fig. 22-3). This is established best for the type I enzyme. Activation of this form of adenylyl cyclase by Ca2+/calmodulin can be blocked by a peptide fragment of the C1b portion. This peptide is also capable of binding Ca2+/calmodulin itself. Current mutagenesis studies are defining the precise amino acid sequence required for Ca2+/calmodulin binding and activation of catalytic activity.

Although types V and VI adenylyl cyclase are not influenced by Ca2+/calmodulin, these two forms are inhibited by free Ca2+. The concentrations of Ca2+ required for this inhibition are in the physiological range (0.1 to 1.0 μM). It has been suggested that types V and VI adenylyl cyclase may be inhibited by entry of Ca2+ into neurons via voltage-gated channels [1].

Adenylyl cyclases are regulated upon phosphorylation

In addition to direct regulation by binding G protein subunits and Ca2+, certain forms of adenylyl cyclase are regulated upon their phosphorylation by second messenger-dependent protein kinases (see Chap. 21). Activation of cAMP-dependent protein kinase is reported to inhibit types V and VI adenylyl cyclase, and both of these enzymes contain consensus sequences for this kinase [18]. In contrast, types II and V adenylyl cyclase are phosphorylated and activated by protein kinase C (PKC). Although these initial observations are exciting, much further work is needed to define the physiological role played in adenylyl cyclase phosphorylation by second messenger-dependent protein kinases and perhaps by other protein kinases.

Three general categories of adenylyl cyclase can be delineated based on their regulatory properties

These include the following: (i) adenylyl cyclases (types I, III and VIII) that are activated synergistically by Gαs and Ca2+/calmodulin and inhibited, in at least some cases, by βγ subunits; (ii) adenylyl cyclases (types II, IV and perhaps VII) that are activated synergistically by Gαs and βγ; and (iii) adenylyl cyclases (types V and VI) that are inhibited by Gαi and free Ca2+ [1,2]. The differential sensitivity of adenylyl cyclase types I through VIII to G protein α and βγ subunits and to Ca2+ provides potentially complex mechanisms for the regulation of cAMP formation.

These pathways are illustrated schematically in Figure 22-4. While many features of the schemes remain hypothetical, they do suggest different patterns of regulation of cAMP formation in cells that contain various forms of adenylyl cyclase. For example, in cells that contain types I, III or VIII adenylyl cyclase (Fig. 22-4A), cAMP formation would be stimulated by extracellular signals that activate receptors coupled to Gαs as well as by those signals that increase Ca2+ entry into the cells. Neurons expressing type I adenylyl cyclase may have inherently high rates of cAMP formation due to basal stimulation by Ca2+/calmodulin. In fact, the high adenylyl cyclase enzyme activity known to be present in the brain may be partly explained in this way. This high rate of enzyme activity could underlie the requirement for additional mechanisms by which this type of adenylyl cyclase is inhibited: cAMP formation would be inhibited in these cells not only by signals that activate receptors coupled to Gαi but also by additional signals that activate receptors coupled to other G proteins, such as Gαo and Gαq, via the release of βγ subunits. This could provide a mechanism for keeping “in check” the high cAMP synthetic capacity present in the brain, as well as multiple mechanisms for the regulation of cAMP formation by a variety of extracellular signals.

Figure 22-4. Schematic illustration of the mechanisms by which the activity of adenylyl cyclases (AC) may be regulated.

Figure 22-4

Schematic illustration of the mechanisms by which the activity of adenylyl cyclases (AC) may be regulated. Whereas all forms of adenylyl cyclase are activated by Gαs(αs) and forskolin, different types of the enzyme can be distinguished (more...)

A very different situation would exist in cells that express types II, IV and perhaps VII adenylyl cyclase (Fig. 22-4B). In these cells, enzyme activity would not be stimulated by Ca2+/calmodulin but would be increased by signals that activate receptors coupled to Gαs, as well as by additional signals that activate receptors coupled to other G proteins through the generation of free βγ subunit complexes. This would provide a mechanism by which cAMP formation is regulated in an integrated manner by multiple extracellular stimuli. In fact, there are several examples in brain of interactions between receptors coupled to Gαs and those coupled to other G proteins. For example, stimulation of adenylyl cyclase activity in cerebral cortex by activation of β-adrenergic receptors, which are coupled to Gαs, can be potentiated by activation of α1-adrenergic or GABAB receptors, which are coupled to other G proteins. In these same cells, stimulation of α1-adrenergic or GABAB receptors alone has little or no effect on cAMP formation [19]. This potentiation could result from the release of free βγ complexes from the G proteins coupled to the α1-adrenergic or GABAB receptors, such as Gαo and/or Gαq. However, these potentiating effects are dependent on extracellular Ca2+ and, therefore, could be mediated by activation of Ca2+-dependent intracellular pathways.

The third situation would be cells that express types V and VI adenylyl cyclase. In these cells, these adenylyl cyclases would be activated potently by Gαs (Fig. 22-4C). However, this activation would be tightly controlled since the same signals would lead to activation of cAMP-dependent protein kinase, causing feedback inhibition through phosphorylation and inhibition of the adenylyl cyclases. Activation of the adenylyl cyclases would be controlled further by receptors coupled to Gαi and those that increase levels of free Ca2+ since both Gαi and free Ca2+ exert an inhibitory effect on these enzymes. In addition, receptors coupled to Gαq, which lead to activation of PKC, also would be expected to inhibit types V and VI adenylyl cyclase. These types of mechanisms may be of particular relevance to psychotropic drugs that act on dopamine-rich brain regions, such as neostriatum and nucleus accumbens, including drugs of abuse and antipsychotic drugs, since type V adenylyl cyclase is highly enriched in these brain areas.

Adenylyl cyclase is subject to long-term regulation in the nervous system

Prolonged exposure of cells to a receptor agonist typically leads to desensitization of receptor function, whereas prolonged exposure to antagonist can lead to sensitization of receptor function. Increasing evidence suggests that such desensitization and sensitization can be achieved, depending on the receptor and cell type involved, through alterations in the receptors themselves as well as through alterations in the series of proteins downstream of the receptor that mediate receptor function, including adenylyl cyclase. Investigation of the possible mechanisms underlying adaptations in adenylyl cyclase that are involved in desensitization and sensitization of receptor function have only begun; one such mechanism could be phosphorylation of the enzyme or alterations in its expression at transcriptional, translational and post-translational levels.

Processes of receptor desensitization and sensitization are understood best for receptors coupled positively or negatively to the cAMP system. Agonist- and antagonist-dependent changes in these receptors are discussed elsewhere (see Chaps. 10 and 12). However, research has indicated that adaptations in postreceptor signaling proteins also play an important role in controlling receptor sensitivity under a variety of physiological and pathological conditions. One example where adaptations in adenylyl cyclase have been related to long-term adaptations in receptor function is opiate tolerance and dependence. In this case, chronic exposure to opiates leads to coordinate upregulation in the activity of the cAMP cascade in specific opiate-responsive brain regions, including higher levels of adenylyl cyclase and cAMP-dependent protein kinase and, in some cases, lower levels of inhibitory G proteins [20]. Upregulation of the cAMP cascade contributes to opiate tolerance and dependence by opposing the acute actions of opiates, which stimulate receptors coupled to Gαi. Increased expression of adenylyl cyclase, which in some brain regions is seen selectively for the type I and VIII enzymes, may be achieved at the level of gene expression since higher levels of enzyme mRNA are observed as well [21,22] (see Box 53-1).

In addition to drug regulation of adenylyl cyclase mediated through agonist or antagonist effects on receptors, some drugs produce direct effects on the adenylyl cyclase protein. One example is forskolin, which directly stimulates the catalytic activity of all known forms of the enzyme, as mentioned earlier. Another example is lithium, which has been the drug of choice in the treatment of bipolar disorder. Lithium acutely inhibits adenylyl cyclase, apparently by interfering with the Mg2+-binding sites of the enzyme. This accounts for some of the side effects of lithium, such as inhibition of thyroid hormone production, which is dependent on the cAMP cascade. However, chronic lithium administration, which is required for its beneficial clinical effects, leads to increases in adenylyl cyclase expression in the brain [23], which may represent a compensatory response to acute inhibition of the enzyme. Whether this upregulation of adenylyl cyclase contributes to the clinical effects of lithium remains unknown.

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Copyright © 1999, American Society for Neurochemistry.
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