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New paradigms in cAMP signalling
Molecular and Cellular Endocrinology 353 (2012) 3–9
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Review
New paradigms in cAMP signalling Ferenc A. Antoni ⇑ Division of Preclinical Research, EGIS PLC, Bökényföldi út 116, 1165 Budapest, Hungary
a r t i c l e
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Article history: Available online 9 November 2011 Invocation: This paper was written in honour of the 70th birthday of Prof. András Spät. Long live the spirit of collective intellectual adventure! Keywords: Adenylyl cyclase Biosensor cAMP Phosphodiesterase Plasticity Signal transduction
a b s t r a c t Signalling through adenosine 30 50 monophosphate (cAMP) is known to be important in virtually every cell. The mapping of the human genome over the past two decades has revealed an unexpected complexity of cAMP signalling, which is shared from insects to mammals. A more recent technical advance is the ability to monitor intracellular cAMP levels at subcellular spatial resolution within the time-domains of fast biochemical reactions. Thus, new light has been shed on old paradigms, some of which turn out to be multiple new ones. The novel aspects of cAMP signalling are highlighted here: (1) agonist induced plasticity – showing how the repertory of cAMP signalling genes supports homeostatic adaptation; (2) sustained cAMP signalling after endocytosis; (3) pre-assembled receptor-Gs–adenylyl cyclase complexes. Finally, a hypothetical model of propagating neuronal cAMP signals travelling form dendrites to the cell body is presented. Ó 2011 Elsevier Ireland Ltd. All rights reserved.
Contents 1.
2. 3.
4. 5. 6.
Brief synopsis of the main components of intracellular cAMP signalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Adenylyl cyclases (ACs) and cyclic nucleotide phosphodiesterases (PDEs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Downstream targets of cAMP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Methodological break-through: molecular biosensors provide direct proof of cAMP compartments and intracellular oscillations. . . . . . . . . . . . . A selection of novel paradigms in cAMP signalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Agonist induced plasticity at the level of AC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Plasticity at the level of cyclic nucleotide phosphodiesterases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Translational aspects: relevance of the plasticity of intracellular cAMP signalling at the pituitary gland for the adaptive response during stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Persistence of activated adenylyl cyclase after receptor internalization into endosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evidence for preassembled receptor-Gs–adenylyl cyclase complexes and its translational application. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The intracellular traffic of tmAC and its unexplored consequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3 3 4 4 4 4 5 5 6 6 6 7
1. Brief synopsis of the main components of intracellular cAMP signalling
1.1. Adenylyl cyclases (ACs) and cyclic nucleotide phosphodiesterases (PDEs)
The molecular toolbox of cAMP signalling has been reviewed extensively (Houslay and Milligan, 1997; Antoni, 2000; Conti and Beavo, 2007; Houslay et al., 2007), hence only a cursory treatise necessary for understanding the subsequent sections is provided.
There are 10 mammalian AC genes each of which encodes a different protein. Transmembrane domain ACs (tmACs) are differentially controlled by heterotrimeric G proteins, intracellular Ca2+ and protein phosphorylation (Taussig and Gilman, 1995; Antoni, 2000; Willoughby and Cooper, 2007). In addition, the soluble enzyme AC10 is uniquely controlled by bicarbonate ions (Kamenetsky et al., 2006). More recent results also show selective targeting of tmAC isoforms to membrane microdomains such as
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lipid rafts (Ostrom and Insel, 2004; Crossthwaite et al., 2005) and differential association with scaffolding proteins (Dodge et al., 2001), particularly A-kinase anchoring proteins (AKAPs), all contributing to the formation of intracellular cAMP compartments (Lynch et al., 2006; Dessauer, 2009). Several signalling pathways converge on ACs, which act as molecular signal integrators. The AC isoforms have distinct tissue distributions indicating nonredundant physiological roles (Sadana and Dessauer, 2009). At the single cell level, expression of multiple AC-s isotypes is the rule rather than the exception. The situation with PDEs is similar to that of ACs, an even greater genetic variability is evident for these enzymes displaying unique regulation of cAMP hydrolysis, subcellular targeting and tissue topography (Houslay and Milligan, 1997; Conti and Beavo, 2007).
1.2. Downstream targets of cAMP The classical downstream target of cAMP is the protein kinase A (PKA) family of proteins which has been extensively characterized (Taylor et al., 2004). The important advance in the field is the discovery of the multiplicity of AKAPs, that have emerged as scaffolding proteins for a bewildering variety of signallosomes (Logue and Scott, 2010). Relatively novel targets of cAMP include ion channels directly influenced by cAMP. These come in two flavours – the hyperpolarization-activated and cyclic nucleotide-modulated channels (HCN) and channels directly modulated by cyclic nucleotides (CNG channels). Both of these channel types increase the electrical activity of cells in response to cAMP. Various types of PDE are modulated by cAMP usually via the N-terminal domains (Keravis and Lugnier, 2010). Last, but not least, exchange proteins activated by cAMP (EPACs) are the most recently recognized family of proteins the functional activity of which is regulated by cAMP– EPACs modulate the activity of the small GTPase Rap1 and have important functions regulating cell shape and secretory granule dynamics (Gloerich and Bos, 2010). Importantly, the cAMP sensitivity of this panoply of targets spans a 30-fold dynamic range. The PKA isoforms are the most responsive and are saturated at 1 lM, cyclic nucleotide gated channels are close to maximally active at 10 lM whilst EPAC may require up to 40 lM cAMP to be fully active.
2. Methodological break-through: molecular biosensors provide direct proof of cAMP compartments and intracellular oscillations Arguably the most significant recent achievement in furthering our understanding of cAMP signalling has been the introduction of methodologies for monitoring intracellular cAMP levels with spatial and temporal resolutions classical biochemical approaches could not achieve. The evolution of intracellular cAMP sensors from fluorescently tagged PKA subunits delivered into cells by microinjection (Bacskai et al., 1993) to genetically engineered mice expressing EPAC-based cAMP sensor protein (Calebiro et al., 2009) has been reviewed comprehensively (Nikolaev and Lohse, 2006; Willoughby and Cooper, 2008; Calebiro et al., 2010a,b). The chief achievement of these technologies is that unique and localized patterns of cAMP concentrations have been reported that can be connected to specified elements of the cAMP signalling toolbox thus providing important clues for the biological relevance of the genetic diversity of cAMP-signalling proteins. This is not to say that the classical biochemical approaches are no longer useful, as in pharmacological screens the relatively narrow dynamic range of the biosensors restricts their applicability (Hill et al., 2010). However, where practicable, the deployment of sensor-based methods is
important for discovery research and for the full validation of the cAMP response measured. 3. A selection of novel paradigms in cAMP signalling The accounts provided below are by no means a comprehensive treatise of all the novel aspects of cAMP signalling that are beginning to emerge on the back of the biosensor revolution. Notably, cells of the immune system and the role of soluble ACs are not covered, the reader is referred to excellent recent reviews (Gloerich and Bos, 2010; Buck and Levin, 2011). There are also detailed accounts of the multiplicity of cAMP signals involved in the control of insulin release by pancreatic beta cells (Ramos et al., 2008; Leech et al., 2010). The examples highlighted here are as yet not in the mainstream of work in the field, but appear to be highly significant for physiological control. Moreover, these paradigms are amenable to further study, for example, by making use of new biosensor and RNA interference technologies. 3.1. Agonist induced plasticity at the level of AC The example cited here is the adenohypophyseal corticotrope cell. These cells produce adrenocorticotropin (ACTH) to stimulate adrenocortical steroid production and are thus the main endocrine mediators of the CNS control of the stress-response. Briefly, corticotropes are stimulated by the neuropeptides corticotropin releasing factor-41 (CRF) and arginine vasopressin (AVP) via the neurohaemal interface of the median eminence and are under feedback inhibition by adrenal corticosteroids (Antoni, 1986). With respect to the underlying signalling mechanisms, it is well established that CRF activates adenylyl cyclase (AC) and that this effect is amplified by AVP through activation of protein kinase C (PKC) (Carvallo and Aguilera, 1989; Antoni, 1993). Whilst the exact mechanism of AVP potentiation remains to be established, work over the years clearly points to the involvement of a PKC activated AC such as AC2 or AC7 (Antoni, 1993, 2000). Inhibition by adrenal corticosteroids is largely mediated by the Type 2 nuclear glucocorticoid receptor, and is characterized by distinct time domains (Dallman et al., 1992; Antoni, 1996). The steroid effect that largely determines the size of the stress response in the short and medium term (i.e. from 20 min to 4 h after the induction of stress) is called early delayed inhibition and requires the synthesis of new protein(s) (Dallman et al., 1992; Antoni, 1996). Work in the mouse pituitary cell line AtT20 showed conclusively that the effect of glucocorticoids is mediated by an alteration of the balance of kinase/phosphatase control of STREX-variant, large-conductance (BK) potassium channels (Shipston et al., 1996, 1999). These channels are inhibited by PKA activated by CRF, whilst protein phosphatase 2A recruited by glucocorticoids can override the effect of PKA and thus suppress the stimulation of ACTH release (Tian et al., 1998, 2001). Significantly, in AtT-20 cells clamping of the CRF-activation of tmAC by Ca2+ derived through voltage-operated L-channels appeared to be important for maintaining the efficacy of inhibition by glucocorticoids (Shipston et al., 1994; Antoni et al., 1995). Whilst glucocorticoid feedback inhibition of pituitary ACTH release in non-tumoral rat pituitary corticotrope cells shares the pivotal features of that observed in AtT20 cells, there was no indication for the involvement of BK channels (Lim et al., 1998). Functionally relevant BK- and SK-channels are present in rat adenohypophyseal corticotrope cells, as the ACTH release inhibiting action of atrial natriuretic peptides as well as cyclic GMP analogues is fully opposed upon blockade of these channels (Antoni and Dayanithi, 1990). However, two independent studies concur, that in rat corticotrope cells K+ channels inhibited by PKA and insensitive to
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tetraethylammonium as well as apamin, such as HERG or TREK-1 are likely to underlie glucocorticoid feedback inhibition in Lim et al. (2002) and Yamashita et al. (2009). As HERG and TREK-1 channels are thought to be largely Ca2+independent, an important question that arose from these findings was whether or not the Ca2+-clamp of cAMP synthesis is operational and functionally relevant in normal pituitary cells? Correlated molecular and pharmacological analysis showed that in rat adenohypophyseal corticotrope cells Ca2+ mobilized from ryanodine-sensitive intracellular pools suppressed cAMP synthesis induced by physiological concentrations of CRF and that the plausible target of Ca2+ is AC9 (Antoni et al., 2003). In contrast, supraphysiological concentrations of CRF (>0.3 nM) produced a dramatic escape from inhibition by intracellular free Ca2+, indicating the presence of substantial Ca2+-independent synthesizing capacity (Antoni et al., 2003). Further experiments showed that Ca2+-independent cAMP synthesis in anterior pituitary corticotropes is attributable to AC2 or AC7 and is invoked by AVP, the physiological co-agonist of CRF. Concentrations of CRF and AVP observed in the pituitary portal circulation during intensive physical stress such as endurance exercise training, haemorrhage, autoimmune inflammation (Plotsky, 1991; Antoni, 1993; Chowdrey et al., 1995) induced dramatic increases of cAMP production that were largely resistant to inhibition by Ca2+. Recent work with genetically modified mice suggests that AC7 is indeed an important molecular component of the hypothalamic–pituitary–adrenocortical system, and that overexpression of this enzyme in the CNS and the pituitary gland enhances plasma ACTH and corticosterone levels (Pronko et al., 2010). In summary, these data show that cAMP production induced by physiological concentrations of CRF is largely controlled by intracellular Ca2+ feedback impinging on AC9. Under conditions of stress that mobilize high levels of AVP, a protein kinase C activated AC, most likely AC7 becomes active, and thus the Ca2+ clamp on cAMP synthesis is removed. 3.2. Plasticity at the level of cyclic nucleotide phosphodiesterases The analysis of cAMP synthesis induced by CRF and activators of protein kinase C indicated intracellular cAMP levels far above the Km-s of PDE1C or PDE4, the high-affinity low capacity cAMP PDEs expressed in rat adenohypophyseal tissue (Carvallo and Aguilera, 1989; Ang and Antoni, 2002; Antoni et al., 2003). As upon PKC activation Ca2+ inhibition of cAMP levels was only evident in the absence of PDE blockers, the involvement of Ca2+-dependent PDEs could be inferred. Enter PDE1A, the Km of which for cAMP is around 10 lM and is prominently expressed in rat adenohypophysis (Ang and Antoni, 2002). Indeed, application of various classes of PDE inhibitor to cells exposed to combinations of CRF and AVP
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indicated plasticity of PDE usage: at low levels of stimulation, as seen with CRF (0.3 nM<), rolipram, a selective inhibitor of PDE4 (Conti and Beavo, 2007; Houslay et al., 2007), had a prominent effect to enhance cAMP levels, whilst vinpocetine, a prototype PDE1 blocker (Conti and Beavo, 2007) had no significant impact. In contrast, vinpocetine effectively enhanced cAMP levels when the cells were exposed to CRF and AVP concentrations characteristic of intensive stress (Ang and Antoni, 2002). These data suggest than in parallel to the emergence of a PKC stimulated tmAC as the mediator of CNS stimulatory input, cAMP hydrolysis also shifts from PDE4 to PDE1A, reflecting the high levels of intracellular cAMP induced by CRF and AVP – notably, still within the concentration range that occurs during the stress response (Fig. 1). 3.3. Translational aspects: relevance of the plasticity of intracellular cAMP signalling at the pituitary gland for the adaptive response during stress The anterior pituitary corticotrope cell is an important site of integrating CNS and peripheral signals into a hormonal secretory response (Antoni, 1993, 1996). The inhibitory feedback effects of adrenal corticosteroids are clearly apparent at the pituitary level. Moreover, dexamethasone a synthetic glucocorticoid still used in clinical diagnosis, acts largely at the pituitary gland (Meijer et al., 1998). An important aspect of the dual control of corticotrope cells by CRF and AVP is the relative insensitivity of AVP-induced changes to prolonged glucocorticoid exposure (Bilezikjian et al., 1987). When the effects of glucocorticoids on ACTH release induced by CRF are examined in vitro, it transpires that high stimulus strength does not lead to proportional increases of ACTH release but instead produces resistance to early delayed inhibition by corticosterone (‘‘glucocorticoid escape’’) (Lim et al., 2002). Importantly, the amounts of CRF (3 nM or higher) required to demonstrate this exceed the highest concentrations of the neuropeptide measured in the pituitary portal circulation by 10-fold or higher (Plotsky, 1991). Augmentation of the cAMP response to physiological concentrations of CRF by rolipram also produced glucocorticoid escape of ACTH secretion and last, but not least, combinations of CRF and AVP at concentrations relevant for intensive stress were also effective in this respect (Lim et al., 2002). The plausible in vivo applicability of these findings is also apparent. The cogent ACTH and adrenal steroid responses to certain types of physical stress such as extreme exercise (Deuster et al., 1998), haemorrhage (Thrivikraman et al., 2000) are known to be largely resistant to inhibition by dexamethasone. The common feature of these stresses is the mobilization of AVP from magnocellular hypothalamic neurons that contribute to the regulation of adenohypophyseal ACTH release (Holmes et al., 1986; Deuster et al.,
Fig. 1. Agonist-induced plasticity of cAMP signalling – multiple functional states of the adenohypophysial corticotroph cell. Abbreviations: AC, adenylyl cyclase; CaM, calmodulin; PDE, cyclic nucleotide phosphodiesterase; PKA, cAMP dependent protein kinase; PKC, protein kinase C; PPase2B, protein phosphatase 2B (alias calcineurin). Arrows indicate facilitation, T-bars indicate inhibition. The size and thickness of the lettering is proportional to the amplitude/activity of the denoted entity.
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1998; Thrivikraman et al., 2000; Wotjak et al., 2002). In another setting, sustained hypersecretion of corticosterone has been found in models of autoimmune inflammation (MacPhee et al., 1989; Sternberg et al., 1989). Significantly, extremely high levels of AVP have been demonstrated in the pituitary portal blood of rats with adjuvant-induced arthritis (Chowdrey et al., 1995). Thus vasopressinergic hypothalamic drive to the pituitary gland may cancel out the feedback inhibition by glucocorticoids and the animals survive autoimmune inflammation by way of mounting an endogenous immunosuppressant response the efficiency of which is governed by the neuroendocrine system (MacPhee et al., 1989; Mason, 1991). The final piece of information that is missing is the mechanism by which high levels of cAMP induced by AVP can reverse glucocorticoid-induced hyperpolarisation? As PKA is fully active at cAMP concentrations above 10 lM, plausible targets are CNG-channels that could counteract the K+ channel mediated hyperpolarization induced by corticosteroids (Stojilkovic et al., 2010). 4. Persistence of activated adenylyl cyclase after receptor internalization into endosomes The general schemes of signal transduction place heptahelical transmembrane receptors (7-TM – a.k.a. G protein-coupled receptors) in the plasma membrane waiting to interact with signals from the extracellular space. Upon binding the receptors activate various signalling molecules, most of which are thought to operate in physical separation of the receptor and its plasmalemmal effectors. Hence the term plasma membrane delimited function of receptors. However, work on the mitogen activated protein kinase (MAPK) cascade has shown clearly that 7-TMs may function in endosomes (see Jalink and Moolenaar, 2010, for recent review). The advent of intracellular cAMP imaging has also revealed that activated internalized receptors for thyroid stimulating hormone remain active in generating cAMP in the endosomal compartment for up to 30 min. It was also suggested that endosomal generation of cAMP is necessary for actin cytoskeleton remodelling which plays a role in the secretion of thyroid hormones (Calebiro et al., 2009). The beauty of the latter studies is that they were carried out in primary tissue obtained from mice expressing an EPAC-derived cAMP sensor. A similarly ingenious use was made of FRET technology to reveal an intracellular pathway of persistent AC activation by parathyroid hormone (PTH) (Ferrandon et al., 2009). PTH and PTH related peptide both activate the parathyroid hormone receptor but the effect of PTH is much more protracted than that of PTH related peptide. Moreover, these agents have different effects in vivo: PTH induces bone resorption whilst PTH-related peptide promotes bone formation. Using HEK293 cells as the model system it was possible to show that whilst PTH in complex with the receptor continued to stimulate cAMP biosynthesis in the endosomal compartment, the PTH related peptide ligand dissociated from the receptor and produced only short bursts of cAMP (Ferrandon et al., 2009). Whether or not these properties alone are the basis of the differential in vivo effects of these hormones, remains to be clarified. 5. Evidence for preassembled receptor-Gs–adenylyl cyclase complexes and its translational application Pre-assembled 7-TM-G protein–effector complexes have been reported previously (see Dessauer, 2009, for review). A striking example that has potential therapeutic consequences has been reported recently (Robben et al., 2009). Briefly, several missense mutations in the vasopressin 2 receptor (V2R) gene result in V2Rs that do not appear on the plasma membrane, and are
degraded in the ER. However, non-peptide vasopressin antagonists can stabilize such mutant receptors in model systems, which then appear in numbers at the plasma membrane that are sufficient to demonstrate activation by dDAVP a peptidic vasopressin analogue (see Robben et al., 2009, and references therein. Application of a recently developed non-peptide V2R agonist ligand revealed that the intracellularly retained complex of apparently immature (i.e. highmannose glycosylated) V2R respond to the cell permeant V2R agonists. The response produced cAMP levels detectable by traditional biochemical assay and was sufficient to produce functionally relevant translocation of aquaporin to the apical membranes of MDCK cells. The implication of this finding is that the receptor-Gs-tmAC complex is active in the post-Golgi vesicular compartments. Sensor based cAMP detection should clarify the issue where along the passage from ER to the plasma membrane the tmACs become responsive to stimulation by various bioactive molecules.
6. The intracellular traffic of tmAC and its unexplored consequences The studies outlined in the two previous sections conform to previous knowledge on the transduction mechanisms invoked by 7-TM receptors, but are novel with respect to cAMP signalling. However, it is worth noting that there are a number of earlier reports that collectively suggested the operation of elaborate cAMP signalling mechanism away from the plasma membrane. First, it is important to recall the structure and the functional domains of tmACs in vertebrates (Fig. 2). The protein has a quasi ABC cassette transporter design including the feature that the C1a and C2a domains come in contact to form the catalytic site. Moreover, constructs that are made from the cytosolic loops show the salient regulatory features of the holoenzyme such as bidirectional control by the alpha subunits of heterotrimeric G proteins and activation by forskolin (Tang and Gilman, 1995; Sunahara et al., 1997; Patel et al., 2001) and references therein. In the case of AC1 and AC8 the structural determinants supporting stimulation by Ca2+/calmodulin are also in the cytoplasmic loops (Ferguson and Storm, 2004; Willoughby and Cooper, 2007). Thus several facets of AC function, such as enzymatic catalysis requiring Mg2+–ATP, and its regulation by G protein alpha subunits, forskolin, Ca2+/calmodulin are all potentially operational even if the enzyme is localized to intracellular vesicles. The recent flurry of data made possible by intracellular cAMP sensors suggests that this is indeed the case: functionally relevant cAMP biosynthesis by tmACs can take place in vesicular compartments. Significantly, the laboratory of M. Rasenick reviewed in Allen et al. (2007) has been assiduously studying the redistribution of Gsa upon exposure of cells to various stimuli. These studies and those of others e.g. Schurmann et al. (1992), Muller et al. (1994) and Denker et al. (1996) have raised the possibility that upon 7-TM activation Gsa homes in on subcellular compartments to induce signalling events – are intracellularly located tmACs the target? It is important to point out here that despite 20 years of cloned ACs, a significant unresolved question is the precise subcellular localization of ACs. There is surprisingly little information on this issue, except for investigations into the preferential targeting of some isoforms into plasma membrane lipid rafts (Ostrom and Insel, 2004; Crossthwaite et al., 2005) and localization within as well as outwith synaptic densities in nerve cells (Mons et al., 1995; Sosunov et al., 2001). The limited amount of work published with immunocytochemical detection or fluorescent protein tracing indicates that in epithelial cells such as HEK293, M1CCD or MDCK plasma membrane targeting of ACs predominates (see Antoni et al., 2006, for review). In contrast, in pituitary and nerve cells no preferential localization to the plasma membrane has been found –
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CYTOPLASMIC CA2+/CAMP PROCESSOR MODULE
ER AC
Membrane bilayer
M2
M1
C1a
RyR
CICR
cAMP
Ca2+ Scaffold of: PKA PDE4 PP2B PP1 AKAP
C2b
C1b
N
CaM
Ca2+
C2a
Ca2+ stimulated AC –cAMP propagation AMPA R R AC G
Ca2+
Gs* Ca2+
cAMP
cAMP
Ca2+ AMP cAMP
Cell body: Gene regulation
Fig. 2. The molecular topography of tmAC makes it likely that it is active in intracellular vesicles – implications of a case study of Ca2+/calmodulin stimulated tmACs (AC1 and AC8) are shown. The features of the tmAC molecule in the left panel are highlighted in the text. ER vesicles containing tmAC and ryanodine activated channels: note that colocalization in the same vesicle is not a criterion of the hypothesis: AC sensing of local Ca2+ release and PKA activation by cAMP produced from the juxtaposed AC is sufficient. An increase of intracellular Ca2+ triggered, for example, by Ca2+ conducting NMDA or AMPA receptors could initiate self-propagating cAMP gradients along the dendritic tree. This could be amplified by ligands of Gs coupled 7-TM. The propagated cAMP signal eventually reaches the cell body to alter gene expression or influence the state of phosphorylation of proteins at the axon initial segment. Abbreviations: AKAP, A kinase anchoring protein; RyR, ryanodine receptor, CICR, Ca2+ induced Ca2+ release; PKA, protein kinase A; PP1, protein phosphatase 1; PPase2B, protein phosphatase 2B (alias calcineurin); PDE; cyclic nucleotide phosphodiesterase.
endogenous as well as transfected tmACs appear in intracellular punctae the nature of which has not been established with certainty (Wang et al., 2003; Chou et al., 2004; Antoni et al., 2006). The localization of tmAC in the sarcoplasmic reticulum of heart muscle cells is also reasonably well-documented (Gao et al., 1997; Zaccolo and Pozzan, 2002; Head et al., 2005). Notably, the ‘‘basal’’ – i.e. no obvious activation via cell surface receptors – level of cAMP has emerged as an important determinant of secretory vesicle dynamics at fast CNS synapses as well as in neuroendocrine cells (Sakaba and Neher, 2003; Nagy et al., 2004). Semaphorin induced turning of developing axons also is modulated by cAMP, but with no obvious activation of AC (see Pasterkamp and Verhaagen, 2006, for review). A self-propagating cAMP signal that is based on Ca2+/calmodulin stimulated tmACs and is triggered by voltage-dependent Ca2+ entry, IP3 mobilization or intracellular translocation of activated Gsa from the plasma membrane can be readily envisaged (Fig. 2). Specifically, as highlighted previously, the regulatory sites of tmACs are found in the cytoplasm. Thus, a Ca2+/calmodulin stimulated tmAC located in the vicinity of intracellular Ca2+ stores may generate cAMP sufficient to activate IP3 or ryanodine receptors by a variety of previously described cAMP-dependent mechanisms (Marx et al., 2000; Taylor and Tovey, 2010). Neuronal dendrites and cell bodies are well-endowed with both types of intracellular Ca2+ stores that support a variety of physiological functions (Berridge, 1998; Rose and Konnerth, 2001; Ludwig et al., 2002). The Ca2+ released through the activated intracellular channels can diffuse to intracellular sites to activate further populations of tmAC thus producing a self-propagating cAMP signal. The directionality of the signal could be regulated by Ca2+ as well: phosphorylation by calmodulin dependent protein kinase II and IV inhibit AC3 (Wei et al., 1998) and AC1 (Wayman et al., 1996), respectively, preventing back-propagation of the cAMP signal and enabling the restoration of the responsiveness of the putative intracellular tmAC responder system. Thus intracellularly located tmACs and their
reciprocal control by Ca2+ could underlie cAMP signals travelling relatively long distances from the dendritic tree to the cell body and the nucleus. Indeed, one of the pioneering studies of intracellular cAMP monitoring indicated that in crayfish stomatic ganglion neurons stimulus strength was proportional to the extent of the spread of an intracellular gradient of cAMP along the dendritic tree towards cell body (Hempel et al., 1996). Moreover, the rate of the intracellular diffusion of cAMP as monitored by biosensors shows marked variations between cell types (see Calebiro et al., 2010a,b, for review) suggesting intracellular interactions and signal regenerative mechanisms may be operational. In summary, we can anticipate that future work with biosensors will show that cAMP signals generated away from the plasma membrane by tmACs are the rule rather than the exception and, like other intracellularly generated messenger signals (Berridge, 1998), cAMP is elaborated at multiple sites of the internal membranes of most cell types.
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Review
New paradigms in cAMP signalling Ferenc A. Antoni ⇑ Division of Preclinical Research, EGIS PLC, Bökényföldi út 116, 1165 Budapest, Hungary
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Article history: Available online 9 November 2011 Invocation: This paper was written in honour of the 70th birthday of Prof. András Spät. Long live the spirit of collective intellectual adventure! Keywords: Adenylyl cyclase Biosensor cAMP Phosphodiesterase Plasticity Signal transduction
a b s t r a c t Signalling through adenosine 30 50 monophosphate (cAMP) is known to be important in virtually every cell. The mapping of the human genome over the past two decades has revealed an unexpected complexity of cAMP signalling, which is shared from insects to mammals. A more recent technical advance is the ability to monitor intracellular cAMP levels at subcellular spatial resolution within the time-domains of fast biochemical reactions. Thus, new light has been shed on old paradigms, some of which turn out to be multiple new ones. The novel aspects of cAMP signalling are highlighted here: (1) agonist induced plasticity – showing how the repertory of cAMP signalling genes supports homeostatic adaptation; (2) sustained cAMP signalling after endocytosis; (3) pre-assembled receptor-Gs–adenylyl cyclase complexes. Finally, a hypothetical model of propagating neuronal cAMP signals travelling form dendrites to the cell body is presented. Ó 2011 Elsevier Ireland Ltd. All rights reserved.
Contents 1.
2. 3.
4. 5. 6.
Brief synopsis of the main components of intracellular cAMP signalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Adenylyl cyclases (ACs) and cyclic nucleotide phosphodiesterases (PDEs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Downstream targets of cAMP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Methodological break-through: molecular biosensors provide direct proof of cAMP compartments and intracellular oscillations. . . . . . . . . . . . . A selection of novel paradigms in cAMP signalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Agonist induced plasticity at the level of AC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Plasticity at the level of cyclic nucleotide phosphodiesterases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Translational aspects: relevance of the plasticity of intracellular cAMP signalling at the pituitary gland for the adaptive response during stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Persistence of activated adenylyl cyclase after receptor internalization into endosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evidence for preassembled receptor-Gs–adenylyl cyclase complexes and its translational application. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The intracellular traffic of tmAC and its unexplored consequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3 3 4 4 4 4 5 5 6 6 6 7
1. Brief synopsis of the main components of intracellular cAMP signalling
1.1. Adenylyl cyclases (ACs) and cyclic nucleotide phosphodiesterases (PDEs)
The molecular toolbox of cAMP signalling has been reviewed extensively (Houslay and Milligan, 1997; Antoni, 2000; Conti and Beavo, 2007; Houslay et al., 2007), hence only a cursory treatise necessary for understanding the subsequent sections is provided.
There are 10 mammalian AC genes each of which encodes a different protein. Transmembrane domain ACs (tmACs) are differentially controlled by heterotrimeric G proteins, intracellular Ca2+ and protein phosphorylation (Taussig and Gilman, 1995; Antoni, 2000; Willoughby and Cooper, 2007). In addition, the soluble enzyme AC10 is uniquely controlled by bicarbonate ions (Kamenetsky et al., 2006). More recent results also show selective targeting of tmAC isoforms to membrane microdomains such as
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F.A. Antoni / Molecular and Cellular Endocrinology 353 (2012) 3–9
lipid rafts (Ostrom and Insel, 2004; Crossthwaite et al., 2005) and differential association with scaffolding proteins (Dodge et al., 2001), particularly A-kinase anchoring proteins (AKAPs), all contributing to the formation of intracellular cAMP compartments (Lynch et al., 2006; Dessauer, 2009). Several signalling pathways converge on ACs, which act as molecular signal integrators. The AC isoforms have distinct tissue distributions indicating nonredundant physiological roles (Sadana and Dessauer, 2009). At the single cell level, expression of multiple AC-s isotypes is the rule rather than the exception. The situation with PDEs is similar to that of ACs, an even greater genetic variability is evident for these enzymes displaying unique regulation of cAMP hydrolysis, subcellular targeting and tissue topography (Houslay and Milligan, 1997; Conti and Beavo, 2007).
1.2. Downstream targets of cAMP The classical downstream target of cAMP is the protein kinase A (PKA) family of proteins which has been extensively characterized (Taylor et al., 2004). The important advance in the field is the discovery of the multiplicity of AKAPs, that have emerged as scaffolding proteins for a bewildering variety of signallosomes (Logue and Scott, 2010). Relatively novel targets of cAMP include ion channels directly influenced by cAMP. These come in two flavours – the hyperpolarization-activated and cyclic nucleotide-modulated channels (HCN) and channels directly modulated by cyclic nucleotides (CNG channels). Both of these channel types increase the electrical activity of cells in response to cAMP. Various types of PDE are modulated by cAMP usually via the N-terminal domains (Keravis and Lugnier, 2010). Last, but not least, exchange proteins activated by cAMP (EPACs) are the most recently recognized family of proteins the functional activity of which is regulated by cAMP– EPACs modulate the activity of the small GTPase Rap1 and have important functions regulating cell shape and secretory granule dynamics (Gloerich and Bos, 2010). Importantly, the cAMP sensitivity of this panoply of targets spans a 30-fold dynamic range. The PKA isoforms are the most responsive and are saturated at 1 lM, cyclic nucleotide gated channels are close to maximally active at 10 lM whilst EPAC may require up to 40 lM cAMP to be fully active.
2. Methodological break-through: molecular biosensors provide direct proof of cAMP compartments and intracellular oscillations Arguably the most significant recent achievement in furthering our understanding of cAMP signalling has been the introduction of methodologies for monitoring intracellular cAMP levels with spatial and temporal resolutions classical biochemical approaches could not achieve. The evolution of intracellular cAMP sensors from fluorescently tagged PKA subunits delivered into cells by microinjection (Bacskai et al., 1993) to genetically engineered mice expressing EPAC-based cAMP sensor protein (Calebiro et al., 2009) has been reviewed comprehensively (Nikolaev and Lohse, 2006; Willoughby and Cooper, 2008; Calebiro et al., 2010a,b). The chief achievement of these technologies is that unique and localized patterns of cAMP concentrations have been reported that can be connected to specified elements of the cAMP signalling toolbox thus providing important clues for the biological relevance of the genetic diversity of cAMP-signalling proteins. This is not to say that the classical biochemical approaches are no longer useful, as in pharmacological screens the relatively narrow dynamic range of the biosensors restricts their applicability (Hill et al., 2010). However, where practicable, the deployment of sensor-based methods is
important for discovery research and for the full validation of the cAMP response measured. 3. A selection of novel paradigms in cAMP signalling The accounts provided below are by no means a comprehensive treatise of all the novel aspects of cAMP signalling that are beginning to emerge on the back of the biosensor revolution. Notably, cells of the immune system and the role of soluble ACs are not covered, the reader is referred to excellent recent reviews (Gloerich and Bos, 2010; Buck and Levin, 2011). There are also detailed accounts of the multiplicity of cAMP signals involved in the control of insulin release by pancreatic beta cells (Ramos et al., 2008; Leech et al., 2010). The examples highlighted here are as yet not in the mainstream of work in the field, but appear to be highly significant for physiological control. Moreover, these paradigms are amenable to further study, for example, by making use of new biosensor and RNA interference technologies. 3.1. Agonist induced plasticity at the level of AC The example cited here is the adenohypophyseal corticotrope cell. These cells produce adrenocorticotropin (ACTH) to stimulate adrenocortical steroid production and are thus the main endocrine mediators of the CNS control of the stress-response. Briefly, corticotropes are stimulated by the neuropeptides corticotropin releasing factor-41 (CRF) and arginine vasopressin (AVP) via the neurohaemal interface of the median eminence and are under feedback inhibition by adrenal corticosteroids (Antoni, 1986). With respect to the underlying signalling mechanisms, it is well established that CRF activates adenylyl cyclase (AC) and that this effect is amplified by AVP through activation of protein kinase C (PKC) (Carvallo and Aguilera, 1989; Antoni, 1993). Whilst the exact mechanism of AVP potentiation remains to be established, work over the years clearly points to the involvement of a PKC activated AC such as AC2 or AC7 (Antoni, 1993, 2000). Inhibition by adrenal corticosteroids is largely mediated by the Type 2 nuclear glucocorticoid receptor, and is characterized by distinct time domains (Dallman et al., 1992; Antoni, 1996). The steroid effect that largely determines the size of the stress response in the short and medium term (i.e. from 20 min to 4 h after the induction of stress) is called early delayed inhibition and requires the synthesis of new protein(s) (Dallman et al., 1992; Antoni, 1996). Work in the mouse pituitary cell line AtT20 showed conclusively that the effect of glucocorticoids is mediated by an alteration of the balance of kinase/phosphatase control of STREX-variant, large-conductance (BK) potassium channels (Shipston et al., 1996, 1999). These channels are inhibited by PKA activated by CRF, whilst protein phosphatase 2A recruited by glucocorticoids can override the effect of PKA and thus suppress the stimulation of ACTH release (Tian et al., 1998, 2001). Significantly, in AtT-20 cells clamping of the CRF-activation of tmAC by Ca2+ derived through voltage-operated L-channels appeared to be important for maintaining the efficacy of inhibition by glucocorticoids (Shipston et al., 1994; Antoni et al., 1995). Whilst glucocorticoid feedback inhibition of pituitary ACTH release in non-tumoral rat pituitary corticotrope cells shares the pivotal features of that observed in AtT20 cells, there was no indication for the involvement of BK channels (Lim et al., 1998). Functionally relevant BK- and SK-channels are present in rat adenohypophyseal corticotrope cells, as the ACTH release inhibiting action of atrial natriuretic peptides as well as cyclic GMP analogues is fully opposed upon blockade of these channels (Antoni and Dayanithi, 1990). However, two independent studies concur, that in rat corticotrope cells K+ channels inhibited by PKA and insensitive to
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tetraethylammonium as well as apamin, such as HERG or TREK-1 are likely to underlie glucocorticoid feedback inhibition in Lim et al. (2002) and Yamashita et al. (2009). As HERG and TREK-1 channels are thought to be largely Ca2+independent, an important question that arose from these findings was whether or not the Ca2+-clamp of cAMP synthesis is operational and functionally relevant in normal pituitary cells? Correlated molecular and pharmacological analysis showed that in rat adenohypophyseal corticotrope cells Ca2+ mobilized from ryanodine-sensitive intracellular pools suppressed cAMP synthesis induced by physiological concentrations of CRF and that the plausible target of Ca2+ is AC9 (Antoni et al., 2003). In contrast, supraphysiological concentrations of CRF (>0.3 nM) produced a dramatic escape from inhibition by intracellular free Ca2+, indicating the presence of substantial Ca2+-independent synthesizing capacity (Antoni et al., 2003). Further experiments showed that Ca2+-independent cAMP synthesis in anterior pituitary corticotropes is attributable to AC2 or AC7 and is invoked by AVP, the physiological co-agonist of CRF. Concentrations of CRF and AVP observed in the pituitary portal circulation during intensive physical stress such as endurance exercise training, haemorrhage, autoimmune inflammation (Plotsky, 1991; Antoni, 1993; Chowdrey et al., 1995) induced dramatic increases of cAMP production that were largely resistant to inhibition by Ca2+. Recent work with genetically modified mice suggests that AC7 is indeed an important molecular component of the hypothalamic–pituitary–adrenocortical system, and that overexpression of this enzyme in the CNS and the pituitary gland enhances plasma ACTH and corticosterone levels (Pronko et al., 2010). In summary, these data show that cAMP production induced by physiological concentrations of CRF is largely controlled by intracellular Ca2+ feedback impinging on AC9. Under conditions of stress that mobilize high levels of AVP, a protein kinase C activated AC, most likely AC7 becomes active, and thus the Ca2+ clamp on cAMP synthesis is removed. 3.2. Plasticity at the level of cyclic nucleotide phosphodiesterases The analysis of cAMP synthesis induced by CRF and activators of protein kinase C indicated intracellular cAMP levels far above the Km-s of PDE1C or PDE4, the high-affinity low capacity cAMP PDEs expressed in rat adenohypophyseal tissue (Carvallo and Aguilera, 1989; Ang and Antoni, 2002; Antoni et al., 2003). As upon PKC activation Ca2+ inhibition of cAMP levels was only evident in the absence of PDE blockers, the involvement of Ca2+-dependent PDEs could be inferred. Enter PDE1A, the Km of which for cAMP is around 10 lM and is prominently expressed in rat adenohypophysis (Ang and Antoni, 2002). Indeed, application of various classes of PDE inhibitor to cells exposed to combinations of CRF and AVP
5
indicated plasticity of PDE usage: at low levels of stimulation, as seen with CRF (0.3 nM<), rolipram, a selective inhibitor of PDE4 (Conti and Beavo, 2007; Houslay et al., 2007), had a prominent effect to enhance cAMP levels, whilst vinpocetine, a prototype PDE1 blocker (Conti and Beavo, 2007) had no significant impact. In contrast, vinpocetine effectively enhanced cAMP levels when the cells were exposed to CRF and AVP concentrations characteristic of intensive stress (Ang and Antoni, 2002). These data suggest than in parallel to the emergence of a PKC stimulated tmAC as the mediator of CNS stimulatory input, cAMP hydrolysis also shifts from PDE4 to PDE1A, reflecting the high levels of intracellular cAMP induced by CRF and AVP – notably, still within the concentration range that occurs during the stress response (Fig. 1). 3.3. Translational aspects: relevance of the plasticity of intracellular cAMP signalling at the pituitary gland for the adaptive response during stress The anterior pituitary corticotrope cell is an important site of integrating CNS and peripheral signals into a hormonal secretory response (Antoni, 1993, 1996). The inhibitory feedback effects of adrenal corticosteroids are clearly apparent at the pituitary level. Moreover, dexamethasone a synthetic glucocorticoid still used in clinical diagnosis, acts largely at the pituitary gland (Meijer et al., 1998). An important aspect of the dual control of corticotrope cells by CRF and AVP is the relative insensitivity of AVP-induced changes to prolonged glucocorticoid exposure (Bilezikjian et al., 1987). When the effects of glucocorticoids on ACTH release induced by CRF are examined in vitro, it transpires that high stimulus strength does not lead to proportional increases of ACTH release but instead produces resistance to early delayed inhibition by corticosterone (‘‘glucocorticoid escape’’) (Lim et al., 2002). Importantly, the amounts of CRF (3 nM or higher) required to demonstrate this exceed the highest concentrations of the neuropeptide measured in the pituitary portal circulation by 10-fold or higher (Plotsky, 1991). Augmentation of the cAMP response to physiological concentrations of CRF by rolipram also produced glucocorticoid escape of ACTH secretion and last, but not least, combinations of CRF and AVP at concentrations relevant for intensive stress were also effective in this respect (Lim et al., 2002). The plausible in vivo applicability of these findings is also apparent. The cogent ACTH and adrenal steroid responses to certain types of physical stress such as extreme exercise (Deuster et al., 1998), haemorrhage (Thrivikraman et al., 2000) are known to be largely resistant to inhibition by dexamethasone. The common feature of these stresses is the mobilization of AVP from magnocellular hypothalamic neurons that contribute to the regulation of adenohypophyseal ACTH release (Holmes et al., 1986; Deuster et al.,
Fig. 1. Agonist-induced plasticity of cAMP signalling – multiple functional states of the adenohypophysial corticotroph cell. Abbreviations: AC, adenylyl cyclase; CaM, calmodulin; PDE, cyclic nucleotide phosphodiesterase; PKA, cAMP dependent protein kinase; PKC, protein kinase C; PPase2B, protein phosphatase 2B (alias calcineurin). Arrows indicate facilitation, T-bars indicate inhibition. The size and thickness of the lettering is proportional to the amplitude/activity of the denoted entity.
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1998; Thrivikraman et al., 2000; Wotjak et al., 2002). In another setting, sustained hypersecretion of corticosterone has been found in models of autoimmune inflammation (MacPhee et al., 1989; Sternberg et al., 1989). Significantly, extremely high levels of AVP have been demonstrated in the pituitary portal blood of rats with adjuvant-induced arthritis (Chowdrey et al., 1995). Thus vasopressinergic hypothalamic drive to the pituitary gland may cancel out the feedback inhibition by glucocorticoids and the animals survive autoimmune inflammation by way of mounting an endogenous immunosuppressant response the efficiency of which is governed by the neuroendocrine system (MacPhee et al., 1989; Mason, 1991). The final piece of information that is missing is the mechanism by which high levels of cAMP induced by AVP can reverse glucocorticoid-induced hyperpolarisation? As PKA is fully active at cAMP concentrations above 10 lM, plausible targets are CNG-channels that could counteract the K+ channel mediated hyperpolarization induced by corticosteroids (Stojilkovic et al., 2010). 4. Persistence of activated adenylyl cyclase after receptor internalization into endosomes The general schemes of signal transduction place heptahelical transmembrane receptors (7-TM – a.k.a. G protein-coupled receptors) in the plasma membrane waiting to interact with signals from the extracellular space. Upon binding the receptors activate various signalling molecules, most of which are thought to operate in physical separation of the receptor and its plasmalemmal effectors. Hence the term plasma membrane delimited function of receptors. However, work on the mitogen activated protein kinase (MAPK) cascade has shown clearly that 7-TMs may function in endosomes (see Jalink and Moolenaar, 2010, for recent review). The advent of intracellular cAMP imaging has also revealed that activated internalized receptors for thyroid stimulating hormone remain active in generating cAMP in the endosomal compartment for up to 30 min. It was also suggested that endosomal generation of cAMP is necessary for actin cytoskeleton remodelling which plays a role in the secretion of thyroid hormones (Calebiro et al., 2009). The beauty of the latter studies is that they were carried out in primary tissue obtained from mice expressing an EPAC-derived cAMP sensor. A similarly ingenious use was made of FRET technology to reveal an intracellular pathway of persistent AC activation by parathyroid hormone (PTH) (Ferrandon et al., 2009). PTH and PTH related peptide both activate the parathyroid hormone receptor but the effect of PTH is much more protracted than that of PTH related peptide. Moreover, these agents have different effects in vivo: PTH induces bone resorption whilst PTH-related peptide promotes bone formation. Using HEK293 cells as the model system it was possible to show that whilst PTH in complex with the receptor continued to stimulate cAMP biosynthesis in the endosomal compartment, the PTH related peptide ligand dissociated from the receptor and produced only short bursts of cAMP (Ferrandon et al., 2009). Whether or not these properties alone are the basis of the differential in vivo effects of these hormones, remains to be clarified. 5. Evidence for preassembled receptor-Gs–adenylyl cyclase complexes and its translational application Pre-assembled 7-TM-G protein–effector complexes have been reported previously (see Dessauer, 2009, for review). A striking example that has potential therapeutic consequences has been reported recently (Robben et al., 2009). Briefly, several missense mutations in the vasopressin 2 receptor (V2R) gene result in V2Rs that do not appear on the plasma membrane, and are
degraded in the ER. However, non-peptide vasopressin antagonists can stabilize such mutant receptors in model systems, which then appear in numbers at the plasma membrane that are sufficient to demonstrate activation by dDAVP a peptidic vasopressin analogue (see Robben et al., 2009, and references therein. Application of a recently developed non-peptide V2R agonist ligand revealed that the intracellularly retained complex of apparently immature (i.e. highmannose glycosylated) V2R respond to the cell permeant V2R agonists. The response produced cAMP levels detectable by traditional biochemical assay and was sufficient to produce functionally relevant translocation of aquaporin to the apical membranes of MDCK cells. The implication of this finding is that the receptor-Gs-tmAC complex is active in the post-Golgi vesicular compartments. Sensor based cAMP detection should clarify the issue where along the passage from ER to the plasma membrane the tmACs become responsive to stimulation by various bioactive molecules.
6. The intracellular traffic of tmAC and its unexplored consequences The studies outlined in the two previous sections conform to previous knowledge on the transduction mechanisms invoked by 7-TM receptors, but are novel with respect to cAMP signalling. However, it is worth noting that there are a number of earlier reports that collectively suggested the operation of elaborate cAMP signalling mechanism away from the plasma membrane. First, it is important to recall the structure and the functional domains of tmACs in vertebrates (Fig. 2). The protein has a quasi ABC cassette transporter design including the feature that the C1a and C2a domains come in contact to form the catalytic site. Moreover, constructs that are made from the cytosolic loops show the salient regulatory features of the holoenzyme such as bidirectional control by the alpha subunits of heterotrimeric G proteins and activation by forskolin (Tang and Gilman, 1995; Sunahara et al., 1997; Patel et al., 2001) and references therein. In the case of AC1 and AC8 the structural determinants supporting stimulation by Ca2+/calmodulin are also in the cytoplasmic loops (Ferguson and Storm, 2004; Willoughby and Cooper, 2007). Thus several facets of AC function, such as enzymatic catalysis requiring Mg2+–ATP, and its regulation by G protein alpha subunits, forskolin, Ca2+/calmodulin are all potentially operational even if the enzyme is localized to intracellular vesicles. The recent flurry of data made possible by intracellular cAMP sensors suggests that this is indeed the case: functionally relevant cAMP biosynthesis by tmACs can take place in vesicular compartments. Significantly, the laboratory of M. Rasenick reviewed in Allen et al. (2007) has been assiduously studying the redistribution of Gsa upon exposure of cells to various stimuli. These studies and those of others e.g. Schurmann et al. (1992), Muller et al. (1994) and Denker et al. (1996) have raised the possibility that upon 7-TM activation Gsa homes in on subcellular compartments to induce signalling events – are intracellularly located tmACs the target? It is important to point out here that despite 20 years of cloned ACs, a significant unresolved question is the precise subcellular localization of ACs. There is surprisingly little information on this issue, except for investigations into the preferential targeting of some isoforms into plasma membrane lipid rafts (Ostrom and Insel, 2004; Crossthwaite et al., 2005) and localization within as well as outwith synaptic densities in nerve cells (Mons et al., 1995; Sosunov et al., 2001). The limited amount of work published with immunocytochemical detection or fluorescent protein tracing indicates that in epithelial cells such as HEK293, M1CCD or MDCK plasma membrane targeting of ACs predominates (see Antoni et al., 2006, for review). In contrast, in pituitary and nerve cells no preferential localization to the plasma membrane has been found –
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CYTOPLASMIC CA2+/CAMP PROCESSOR MODULE
ER AC
Membrane bilayer
M2
M1
C1a
RyR
CICR
cAMP
Ca2+ Scaffold of: PKA PDE4 PP2B PP1 AKAP
C2b
C1b
N
CaM
Ca2+
C2a
Ca2+ stimulated AC –cAMP propagation AMPA R R AC G
Ca2+
Gs* Ca2+
cAMP
cAMP
Ca2+ AMP cAMP
Cell body: Gene regulation
Fig. 2. The molecular topography of tmAC makes it likely that it is active in intracellular vesicles – implications of a case study of Ca2+/calmodulin stimulated tmACs (AC1 and AC8) are shown. The features of the tmAC molecule in the left panel are highlighted in the text. ER vesicles containing tmAC and ryanodine activated channels: note that colocalization in the same vesicle is not a criterion of the hypothesis: AC sensing of local Ca2+ release and PKA activation by cAMP produced from the juxtaposed AC is sufficient. An increase of intracellular Ca2+ triggered, for example, by Ca2+ conducting NMDA or AMPA receptors could initiate self-propagating cAMP gradients along the dendritic tree. This could be amplified by ligands of Gs coupled 7-TM. The propagated cAMP signal eventually reaches the cell body to alter gene expression or influence the state of phosphorylation of proteins at the axon initial segment. Abbreviations: AKAP, A kinase anchoring protein; RyR, ryanodine receptor, CICR, Ca2+ induced Ca2+ release; PKA, protein kinase A; PP1, protein phosphatase 1; PPase2B, protein phosphatase 2B (alias calcineurin); PDE; cyclic nucleotide phosphodiesterase.
endogenous as well as transfected tmACs appear in intracellular punctae the nature of which has not been established with certainty (Wang et al., 2003; Chou et al., 2004; Antoni et al., 2006). The localization of tmAC in the sarcoplasmic reticulum of heart muscle cells is also reasonably well-documented (Gao et al., 1997; Zaccolo and Pozzan, 2002; Head et al., 2005). Notably, the ‘‘basal’’ – i.e. no obvious activation via cell surface receptors – level of cAMP has emerged as an important determinant of secretory vesicle dynamics at fast CNS synapses as well as in neuroendocrine cells (Sakaba and Neher, 2003; Nagy et al., 2004). Semaphorin induced turning of developing axons also is modulated by cAMP, but with no obvious activation of AC (see Pasterkamp and Verhaagen, 2006, for review). A self-propagating cAMP signal that is based on Ca2+/calmodulin stimulated tmACs and is triggered by voltage-dependent Ca2+ entry, IP3 mobilization or intracellular translocation of activated Gsa from the plasma membrane can be readily envisaged (Fig. 2). Specifically, as highlighted previously, the regulatory sites of tmACs are found in the cytoplasm. Thus, a Ca2+/calmodulin stimulated tmAC located in the vicinity of intracellular Ca2+ stores may generate cAMP sufficient to activate IP3 or ryanodine receptors by a variety of previously described cAMP-dependent mechanisms (Marx et al., 2000; Taylor and Tovey, 2010). Neuronal dendrites and cell bodies are well-endowed with both types of intracellular Ca2+ stores that support a variety of physiological functions (Berridge, 1998; Rose and Konnerth, 2001; Ludwig et al., 2002). The Ca2+ released through the activated intracellular channels can diffuse to intracellular sites to activate further populations of tmAC thus producing a self-propagating cAMP signal. The directionality of the signal could be regulated by Ca2+ as well: phosphorylation by calmodulin dependent protein kinase II and IV inhibit AC3 (Wei et al., 1998) and AC1 (Wayman et al., 1996), respectively, preventing back-propagation of the cAMP signal and enabling the restoration of the responsiveness of the putative intracellular tmAC responder system. Thus intracellularly located tmACs and their
reciprocal control by Ca2+ could underlie cAMP signals travelling relatively long distances from the dendritic tree to the cell body and the nucleus. Indeed, one of the pioneering studies of intracellular cAMP monitoring indicated that in crayfish stomatic ganglion neurons stimulus strength was proportional to the extent of the spread of an intracellular gradient of cAMP along the dendritic tree towards cell body (Hempel et al., 1996). Moreover, the rate of the intracellular diffusion of cAMP as monitored by biosensors shows marked variations between cell types (see Calebiro et al., 2010a,b, for review) suggesting intracellular interactions and signal regenerative mechanisms may be operational. In summary, we can anticipate that future work with biosensors will show that cAMP signals generated away from the plasma membrane by tmACs are the rule rather than the exception and, like other intracellularly generated messenger signals (Berridge, 1998), cAMP is elaborated at multiple sites of the internal membranes of most cell types.
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