Targeting cAMP Signalling to Combat Cardiovascular Diseases
Dynamics of adenylate cyclase regulation via heterotrimeric G-proteins 1 ¨ Markus Milde*, Ruth C. Werthmann*, Kathrin von Hayn* and Moritz Bunemann*
*Department of Pharmacology and Clinical Pharmacy, University of Marburg, Karl-von-Frisch-Strasse 1, 35032 Marburg, Germany
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Abstract A wide variety of G-protein-coupled receptors either activate or inhibit ACs (adenylate cyclases), thereby regulating cellular cAMP levels and consequently inducing proper physiological responses. Stimulatory and inhibitory G-proteins interact directly with ACs, whereas Gq -coupled receptors exert their effects primarily via Ca2 + . Using the FRET-based cAMP sensor Epac1 (exchange protein directly activated by cAMP 1)–cAMPS (adenosine 3 ,5 -cyclic monophosphorothioate), we studied cAMP levels in single living VSMCs (vascular smooth muscle cells) or HUVECs (human umbilical vein endothelial cells) with subsecond temporal resolution. Stimulation of purinergic (VSMCs) or thrombin (HUVECs) receptors rapidly decreased cAMP levels in the presence of the β-adrenergic agonist isoprenaline via a rise in Ca2 + and subsequent inhibition of AC5 and AC6. Specifically in HUVECs, we observed that, in the continuous presence of thrombin, cAMP levels climbed slowly after the initial decline with a delay of a little less than 1 min. The underlying mechanism includes phospholipase A2 activity and cyclo-oxygenase-mediated synthesis of prostaglandins. We studied further the dynamics of the inhibition of ACs via Gi -proteins utilizing FRET imaging to resolve interactions between fluorescently labelled Gi -proteins and AC5. FRET between Gα i1 and AC5 developed at much lower concentration of agonist compared with the overall Gi -protein activity. We found the dissociation of Gα i1 subunits and AC5 to occur slower than the Gi -protein deactivation. This led us to the conclusion that AC5, by binding active Gα i1 , interferes with G-protein deactivation and reassembly and thereby might sensitize its own regulation.
Introduction Signalling via cAMP regulates important physiological and pharmacological responses in virtually all cells in the cardiovascular system. Both cAMP production by ACs (adenylate cyclases) and PDE (phosphodiesterase)-mediated destruction by hydrolysis contribute to the regulation of cAMP levels. The family of membrane-bound ACs is itself highly regulated. The discovery of heterotrimeric G-proteins was on the basis of the observation that they either stimulate or inhibit cAMP production [1–4]. These G-proteins were named stimulatory (Gs ) and inhibitory (Gi ), accordingly. Most isoforms of ACs can be stimulated by binding of GTP-bound Gα s subunits, whereas only AC1, AC5 and AC6 are inhibited by Gα i -GTP [5]. Even though integral membrane ACs have been very difficult to purify and characterize in cells, interaction sites of Gα s and Gα i with AC5 have been mapped to the C2 and C1 domain respectively [5]. Furthermore, Gβγ subunits can exert stimulatory or inhibitory regulation on most AC isoforms. For AC5, this interaction has been mapped to the N-terminal region upstream of the first transmembrane domain. G-proteindependent regulation of ACs in the cardiovascular system Key words: adenylate cyclase, adrenergic receptor, G-protein, prostacyclin, regulator of G-protein signalling (RGS), sensitization. Abbreviations: AC, adenylate cyclase; AR, adrenergic receptor; GIRK, G-protein-activated inwardly rectifying K + ; HUVEC, human umbilical vein endothelial cell; PKA, protein kinase A; PLC, phospholipase C; RGS, regulator of G-protein signalling; VSMC, vascular smooth muscle cell. 1
To whom correspondence should be addressed (email moritz.buenemann@staff. uni-marburg.de).
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is crucial for its functional control by the autonomous nervous system. As blood pressure and flow need to be modulated within tens of seconds, the dynamics of signalling are of great interest, but have been difficult to address in intact cardiovascular cells. The development of fluorescent biosensors that allow rapid imaging of signalling events within the cAMP-mediated signal transduction cascade has helped us to gain an insight into these processes. Specifically, the development of FRET-based sensors to measure cAMP levels [6–8], receptor activation [9], receptor–G-protein interaction [10] and G-protein activation [11], as well as, very recently, the interaction of G-proteins with ACs [12] provided interesting details about the dynamics of cAMP regulation in intact cells. G-proteins are important regulators of ACs, but there are other factors, most prominently Ca2 + , that can regulate AC activity directly in a subtype-dependent fashion. Whereas Ca2 + can bind directly to AC5 and AC6 and inhibit their activity at submicromolar levels, Ca2 + can also activate AC1 and AC8 in a calmodulin-dependent fashion. Furthermore, Ca2 + negatively regulates AC3 via activation of CaMKII (Ca2 + /calmodulin-dependent protein kinase II) [5]. Other kinases such as PKA (protein kinase A) and certain isoforms of PKC (protein kinase C) can also phosphorylate and thereby regulate the function of ACs [5]. On the basis of the expression profile of AC isoforms, Ca2 + signalling can either potentiate or antagonize cAMP production. Therefore Gq/11 -mediated Ca2 + signalling has important cAMPregulating potential via interfering with AC function. C The
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Figure 1 Regulation of ACs in HUVECs by different G-proteins Depicted is a scheme that illustrates direct and indirect regulation of endothelial ACs via different G-protein-coupled receptors. Abbreviations: AA, arachidonic acid; COX, cyclo-oxygenase; IP-R, prostacyclin receptor; Iso, isoprenaline; NE, noradrenaline (norepinephrine); PAR, protease-activated receptor (e.g. thrombin receptor); PLA2, phospholipase A2 ; Thr, thrombin.
Physiological functions of G-protein-dependent regulation of ACs On the basis of knockout and overexpression studies, some specific cardiovascular functions can be attributed to certain AC isoforms. In vascular endothelial and smooth muscle cells, as well as in cardiac myocytes, AC5 and AC6 represent functionally important isoforms [5,13,14]. Specifically, in VSMCs (vascular smooth muscle cells) and in cardiac myocytes, it is obvious that G-protein-mediated regulation of ACs is critically important for the physiological regulation controlled by the autonomous nervous system. Sympathetic control of the heart is mediated primarily via stimulatory G-proteins, which couple to ACs. At least the negative chronotropic effect induced by vagal activity involves the negative regulation of ACs by inhibitory Gproteins. This conclusion is supported by the observation that in mice which lack GIRK (G-protein-activated inwardly rectifying K + ) currents, the overall vagal control of the heart rate is intact except for the variation in beat-to-beatintervals [15]. In vascular smooth muscle, AC5 and AC6 mediate vasorelaxation in response to adrenaline [16], thereby providing further regulatory mechanisms for blood pressure.
FRET-based imaging of signal transduction in single cells reveals complex dynamics of AC activity in the cardiovascular system Most insight into the dynamics of cAMP-mediated signals was gained from studies that used FRET- or BRET (bioluminescence resonance energy transfer)-based sensors for cAMP [6–8,17]. These studies demonstrated that C The
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cAMP signals could be highly dynamic and even locally restricted depending on the receptor through which they are activated. As discovered using transgenic approaches in adult ventricular myocytes, cAMP propagation is shortranged when activated via β 2 -ARs (adrenergic receptors). In contrast, activation of β 1 -ARs results in cAMP signals reaching through the whole cell [18,19]. In both vascular endothelial cells and smooth muscle cells, Gq -mediated Ca2 + release leads to a robust negative regulation of cAMP levels if these were pre-elevated by stimulation of β 2 -AR [20,21]. As controlled by single-cell Ca2 + imaging, purinergic stimulation (VSMCs) or exposure to thrombin [HUVECs (human umbilical vein endothelial cells)] led to a rapid rise in intracellular Ca2 + that preceded the Gq -mediated decline in cAMP. On the basis of our observations that the inhibition was insensitive to pertussis toxin pre-treatment, but attenuated by pre-incubation with the Ca2 + -chelator BAPTA/AM [1,2-bis-(o-aminophenoxy)ethane-N,N,N ,N tetra-acetic acid tetrakis(acetoxymethyl ester)], we concluded that the reduction in cAMP following stimulation of Gq coupled receptors was regulated directly by Ca2 + [20,21]. Furthermore, siRNA-mediated knockdown of AC5 and AC6 attenuated the ability of UDP/ADP or thrombin to reduce cAMP levels in VSMCs and HUVECs respectively. The strength of the Ca2 + -mediated inhibition specifically in VSMCs suggests that robust Ca2 + elevation in these cells is able to put a ‘brake’ on cAMP signalling, possibly to overcome PKA-dependent relaxation of smooth muscle cells. Interestingly, in VSMCs, the attenuation of cAMP levels due to purinergic stimulation was long-lasting, whereas in HUVECs, thrombin induced a biphasic effect on cAMP levels. Initially, thrombin reduced cAMP in an
Targeting cAMP Signalling to Combat Cardiovascular Diseases
AC6-dependent manner possibly allowing a ‘breakthrough’ of the cAMP-stabilized barrier formation [22]. However, after 1 min, we observed a slow increase in cAMP levels, which reached even higher levels than those induced by the initial stimulation of β 2 -ARs [23]. Similarly to VSMCs, the Ca2 + -mediated and AC5/AC6-dependent lowering of cAMP in HUVECs may be interpreted as a counteracting mechanism of thrombin to overcome tightening of endothelial barriers induced via stimulation of Gs -coupled receptors. The pharmacological characterization of the stimulatory effect of thrombin on cAMP levels revealed that both phospholipase A2 and cyclo-oxygenases were required. Furthermore, stimulation of prostacyclin receptors with exogenous agonists easily elevated cAMP. Inhibition of these receptors with a specific antagonist (CAY10441) also attenuated the thrombin-induced elevation of cAMP. As cAMP elevation in HUVECs is considered to lead to an increase in endothelial cell barrier formation [24], the thrombin-induced slow increase in cAMP may serve as a negative-feedback mechanism to limit the well-described increase in endothelial permeability. An interesting secondary aspect of these experimental results is the fact that the thrombin-induced prostacyclin-dependent increase in cAMP occurred within single cells under continuous superfusion of these cells with buffer. This set up was well suited to wash away any mediator secreted from cells. Therefore it seems very likely that prostacyclin acts as an autocrine mediator that is produced in the identical cell in which it exerts its biological activity, as depicted in Figure 1.
Dynamics of interactions between G-proteins and ACs determine signalling sensitivity Although ACs have been known for even longer than G-proteins, it was very difficult to study the molecular mechanisms of interactions due to experimental limitations such as lack of suitable antibodies or difficulties to purify and reconstitute functional mammalian ACs. Other effectors of G-proteins in the cardiovascular system such as ion channels and PLC (phospholipase C) have been characterized successfully with respect to the dynamics of interactions with G-protein subunits. For atrial GIRK channels, many studies point towards an activity-independent complex formation of the heterotrimeric G-proteins [25]. Upon activation, this complex is thought to undergo a rearrangement, leading to a movement of GIRK channel subunits, which allow the channel to increase its open probability [26]. The dynamics of G-protein-mediated channel activation and deactivation mirror those of G-proteins themselves as measured by FRET [12,27]. Importantly, RGSs (regulators of G-protein signalling) are able to accelerate both dynamics of receptormediated GIRK currents [28] and G-protein dynamics [12]. The dynamics of the interaction of activated Gα q with PLCβ were studied in vitro. In this case, it was found that
Figure 2 Offset kinetics for Gα i –AC interaction and Gi -protein activity after agonist withdrawal determined by single-cell FRET imaging The agonist washout phase after stimulation with 10 nM noradrenaline (NE) was normalized and averaged (means ± S.E.M.). The dissociation of Gα i1 and AC5 is generally slower than the rearrangement (reflecting deactivation) of the Gi -protein subunits. RGS4 accelerates the deactivation of the Gi -protein; however, RGS4 does not significantly affect Gα i1 –AC5 dissociation kinetics. Reproduced with permission from [12]: Milde, M., Rinne, A., Wunder, F., Engelhardt, S. and Bunemann, ¨ M. (2013) Dynamics of Gα i1 interaction with type 5 adenylate cyclase reveal the molecular basis for high sensitivity of Gi -mediated inhibition of cAMP production. Biochem. J. 454, 515–523.
PLCβ influences G-protein activity by accelerating GTP hydrolysis of Gα q -GTP [29]. Later, a crystal structure of this complex [4] led to a mechanistic explanation of the observed effect [30]. G-protein interactions with ACs have been addressed recently using a FRET-based approach [12]. On the basis of previous studies from the Gilman, Dessauer and Tesmer laboratories [31–33], which all contributed to our understanding of where G-protein subunits bind on the surface of ACs, a fusion protein of YFP linked to the Nterminus of AC5 was generated. Importantly, the resulting YFP–AC5 was carefully characterized to be indistinguishable from wild-type AC5 with respect to both its ability to be activated via Gs -proteins as well as the powerful inhibition via Gi -proteins [12]. Even the kinetics of AC5-mediated alterations in cAMP were very similar for wild-type AC5 and YFP–AC5. As FRET partners for YFP–AC5, we used Gα s –CFP, Gα i1 –CFP or CFP-labelled Gβγ subunits. For AC5–G-protein FRET pairs, no specific pre-assembly before receptor activation could be detected, suggesting that assembly between G-proteins and ACs takes place only after agonist-mediated receptor activation. However, the absence of static FRET is not a final proof for the nonexistence of interaction. Agonist-mediated stimulation of both Gs -proteins via β 2 -ARs or Gi -proteins via α 2A -ARs resulted in the development of a FRET signal between AC5 and the activated G-protein subunits. Kinetics of the interaction between Gα s and AC5 were very similar to those measured between Gα s and Gβγ ([34], and C The
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Figure 3 Differential sensitivity for receptor-mediated Gi -protein activity and Gα i –AC5 interaction Representative concentration–response curves derived from single cells for noradrenaline (NE)-evoked Gα i –Gβγ or Gα i –AC5 FRET with or without co-expression of RGS4 as indicated. The lower panel shows the mean ± S.E.M. EC50 values derived from fittings of individual curves for the indicated conditions (n>11; statistics were obtained by ANOVA and Bonferroni post-hoc test; ***P < 0.001; n.s., not significant).
¨ M. Milde and M. Bunemann, unpublished work). In response to stimulation of α 2A -ARs, Gα i1 also rapidly interacted with AC5, as reflected by the developing FRET signal; however, after withdrawal of agonist, it took much longer for Gα i1 to dissociate from AC5 compared with the kinetics of Gprotein reassembly as determined by FRET between α and βγ subunits [12] (Figure 2). The most plausible explanation for this observation is an inhibition of GTP hydrolysis by AC5 or a slow dissociation of the Gα i -GDP–AC5 complex. In contrast with AC5-free Gi -proteins, RGS4 was not able to significantly accelerate dissociation of Gα i1 and AC5. To our knowledge, this is the first time that anyone has reported a negative effect of the reassembly of a heterotrimeric Gprotein due to the presence of a G-protein effector in intact cells. How relevant might this slowing of the AC– Gα i1 complex be? Any slowing of G-protein deactivation should lead to an imbalance in the G-protein cycle, resulting in a shift towards a larger portion of active G-proteins and an accordingly increased sensitivity of the signalling pathway towards agonist stimulation. Indeed, we found a remarkable sensitization of the concentration response for receptor-mediated interaction of Gα i1 with AC5 compared with the total (non-AC5-bound) Gα i1 activity (Figure 3). This 10-fold increased sensitivity was not primarily due to a massive overexpression of G-proteins over AC5 as controlled by membrane expression of the fluorescent proteins. A comparison of the agonist sensitivity of GIRK currents with the Gi -protein activity determined by FRET revealed no major difference. However, Gi/o -mediated inhibition of AC5mediated cAMP production was extremely sensitive towards C The
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receptor activation as determined in single-cell experiments using an Epac (exchange protein directly activated by cAMP)-based FRET reporter for cAMP [12]. It can therefore be concluded that, on the functional level, inhibition of AC5 by Gα i/o -proteins is more than one order of magnitude more sensitive to agonist-mediated receptor stimulation compared with activation of K + channels. It is important to note that, two decades ago, the Szabo laboratory described a similar difference in the sensitivity of two G-protein pathways in frog atrial myocytes important for chrono- and dromo-tropic effects induced via the vagal transmitter acetylcholine: AChinhibited L-type Ca2 + currents at much lower concentrations compared with its activation of inwardly rectifying K + currents [35]. At that time, the researchers already speculated that differential kinetics of the interactions of the two Gprotein effectors ACs and GIRK channels with their Gproteins may provide the basis for the different sensitivity. With our recent observations, we confirm that G-protein effectors can indeed slow G-protein kinetics. It will be interesting to learn whether other G-protein effectors also increase their sensitivity towards receptor-mediated regulation by influencing kinetics of the G-protein cycle. On the basis of the fact that prolonged interactions between Gproteins and effectors will disable these effectors from being regulated very rapidly, we like to speculate that it is more likely to observe similar effects with effectors that mediate relatively slow signals, similar to ACs.
Funding This work was funded by the Deutsche Forschungsgminschaft [grant numbers SFB487 TPA10, SFB593 TPA13 and SFB688 TBB6 (to M.B.)].
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Received 2 January 2014 doi:10.1042/BST20130280
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Dynamics of adenylate cyclase regulation via heterotrimeric G-proteins 1 ¨ Markus Milde*, Ruth C. Werthmann*, Kathrin von Hayn* and Moritz Bunemann*
*Department of Pharmacology and Clinical Pharmacy, University of Marburg, Karl-von-Frisch-Strasse 1, 35032 Marburg, Germany
Biochemical Society Transactions
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Abstract A wide variety of G-protein-coupled receptors either activate or inhibit ACs (adenylate cyclases), thereby regulating cellular cAMP levels and consequently inducing proper physiological responses. Stimulatory and inhibitory G-proteins interact directly with ACs, whereas Gq -coupled receptors exert their effects primarily via Ca2 + . Using the FRET-based cAMP sensor Epac1 (exchange protein directly activated by cAMP 1)–cAMPS (adenosine 3 ,5 -cyclic monophosphorothioate), we studied cAMP levels in single living VSMCs (vascular smooth muscle cells) or HUVECs (human umbilical vein endothelial cells) with subsecond temporal resolution. Stimulation of purinergic (VSMCs) or thrombin (HUVECs) receptors rapidly decreased cAMP levels in the presence of the β-adrenergic agonist isoprenaline via a rise in Ca2 + and subsequent inhibition of AC5 and AC6. Specifically in HUVECs, we observed that, in the continuous presence of thrombin, cAMP levels climbed slowly after the initial decline with a delay of a little less than 1 min. The underlying mechanism includes phospholipase A2 activity and cyclo-oxygenase-mediated synthesis of prostaglandins. We studied further the dynamics of the inhibition of ACs via Gi -proteins utilizing FRET imaging to resolve interactions between fluorescently labelled Gi -proteins and AC5. FRET between Gα i1 and AC5 developed at much lower concentration of agonist compared with the overall Gi -protein activity. We found the dissociation of Gα i1 subunits and AC5 to occur slower than the Gi -protein deactivation. This led us to the conclusion that AC5, by binding active Gα i1 , interferes with G-protein deactivation and reassembly and thereby might sensitize its own regulation.
Introduction Signalling via cAMP regulates important physiological and pharmacological responses in virtually all cells in the cardiovascular system. Both cAMP production by ACs (adenylate cyclases) and PDE (phosphodiesterase)-mediated destruction by hydrolysis contribute to the regulation of cAMP levels. The family of membrane-bound ACs is itself highly regulated. The discovery of heterotrimeric G-proteins was on the basis of the observation that they either stimulate or inhibit cAMP production [1–4]. These G-proteins were named stimulatory (Gs ) and inhibitory (Gi ), accordingly. Most isoforms of ACs can be stimulated by binding of GTP-bound Gα s subunits, whereas only AC1, AC5 and AC6 are inhibited by Gα i -GTP [5]. Even though integral membrane ACs have been very difficult to purify and characterize in cells, interaction sites of Gα s and Gα i with AC5 have been mapped to the C2 and C1 domain respectively [5]. Furthermore, Gβγ subunits can exert stimulatory or inhibitory regulation on most AC isoforms. For AC5, this interaction has been mapped to the N-terminal region upstream of the first transmembrane domain. G-proteindependent regulation of ACs in the cardiovascular system Key words: adenylate cyclase, adrenergic receptor, G-protein, prostacyclin, regulator of G-protein signalling (RGS), sensitization. Abbreviations: AC, adenylate cyclase; AR, adrenergic receptor; GIRK, G-protein-activated inwardly rectifying K + ; HUVEC, human umbilical vein endothelial cell; PKA, protein kinase A; PLC, phospholipase C; RGS, regulator of G-protein signalling; VSMC, vascular smooth muscle cell. 1
To whom correspondence should be addressed (email moritz.buenemann@staff. uni-marburg.de).
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is crucial for its functional control by the autonomous nervous system. As blood pressure and flow need to be modulated within tens of seconds, the dynamics of signalling are of great interest, but have been difficult to address in intact cardiovascular cells. The development of fluorescent biosensors that allow rapid imaging of signalling events within the cAMP-mediated signal transduction cascade has helped us to gain an insight into these processes. Specifically, the development of FRET-based sensors to measure cAMP levels [6–8], receptor activation [9], receptor–G-protein interaction [10] and G-protein activation [11], as well as, very recently, the interaction of G-proteins with ACs [12] provided interesting details about the dynamics of cAMP regulation in intact cells. G-proteins are important regulators of ACs, but there are other factors, most prominently Ca2 + , that can regulate AC activity directly in a subtype-dependent fashion. Whereas Ca2 + can bind directly to AC5 and AC6 and inhibit their activity at submicromolar levels, Ca2 + can also activate AC1 and AC8 in a calmodulin-dependent fashion. Furthermore, Ca2 + negatively regulates AC3 via activation of CaMKII (Ca2 + /calmodulin-dependent protein kinase II) [5]. Other kinases such as PKA (protein kinase A) and certain isoforms of PKC (protein kinase C) can also phosphorylate and thereby regulate the function of ACs [5]. On the basis of the expression profile of AC isoforms, Ca2 + signalling can either potentiate or antagonize cAMP production. Therefore Gq/11 -mediated Ca2 + signalling has important cAMPregulating potential via interfering with AC function. C The
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Figure 1 Regulation of ACs in HUVECs by different G-proteins Depicted is a scheme that illustrates direct and indirect regulation of endothelial ACs via different G-protein-coupled receptors. Abbreviations: AA, arachidonic acid; COX, cyclo-oxygenase; IP-R, prostacyclin receptor; Iso, isoprenaline; NE, noradrenaline (norepinephrine); PAR, protease-activated receptor (e.g. thrombin receptor); PLA2, phospholipase A2 ; Thr, thrombin.
Physiological functions of G-protein-dependent regulation of ACs On the basis of knockout and overexpression studies, some specific cardiovascular functions can be attributed to certain AC isoforms. In vascular endothelial and smooth muscle cells, as well as in cardiac myocytes, AC5 and AC6 represent functionally important isoforms [5,13,14]. Specifically, in VSMCs (vascular smooth muscle cells) and in cardiac myocytes, it is obvious that G-protein-mediated regulation of ACs is critically important for the physiological regulation controlled by the autonomous nervous system. Sympathetic control of the heart is mediated primarily via stimulatory G-proteins, which couple to ACs. At least the negative chronotropic effect induced by vagal activity involves the negative regulation of ACs by inhibitory Gproteins. This conclusion is supported by the observation that in mice which lack GIRK (G-protein-activated inwardly rectifying K + ) currents, the overall vagal control of the heart rate is intact except for the variation in beat-to-beatintervals [15]. In vascular smooth muscle, AC5 and AC6 mediate vasorelaxation in response to adrenaline [16], thereby providing further regulatory mechanisms for blood pressure.
FRET-based imaging of signal transduction in single cells reveals complex dynamics of AC activity in the cardiovascular system Most insight into the dynamics of cAMP-mediated signals was gained from studies that used FRET- or BRET (bioluminescence resonance energy transfer)-based sensors for cAMP [6–8,17]. These studies demonstrated that C The
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cAMP signals could be highly dynamic and even locally restricted depending on the receptor through which they are activated. As discovered using transgenic approaches in adult ventricular myocytes, cAMP propagation is shortranged when activated via β 2 -ARs (adrenergic receptors). In contrast, activation of β 1 -ARs results in cAMP signals reaching through the whole cell [18,19]. In both vascular endothelial cells and smooth muscle cells, Gq -mediated Ca2 + release leads to a robust negative regulation of cAMP levels if these were pre-elevated by stimulation of β 2 -AR [20,21]. As controlled by single-cell Ca2 + imaging, purinergic stimulation (VSMCs) or exposure to thrombin [HUVECs (human umbilical vein endothelial cells)] led to a rapid rise in intracellular Ca2 + that preceded the Gq -mediated decline in cAMP. On the basis of our observations that the inhibition was insensitive to pertussis toxin pre-treatment, but attenuated by pre-incubation with the Ca2 + -chelator BAPTA/AM [1,2-bis-(o-aminophenoxy)ethane-N,N,N ,N tetra-acetic acid tetrakis(acetoxymethyl ester)], we concluded that the reduction in cAMP following stimulation of Gq coupled receptors was regulated directly by Ca2 + [20,21]. Furthermore, siRNA-mediated knockdown of AC5 and AC6 attenuated the ability of UDP/ADP or thrombin to reduce cAMP levels in VSMCs and HUVECs respectively. The strength of the Ca2 + -mediated inhibition specifically in VSMCs suggests that robust Ca2 + elevation in these cells is able to put a ‘brake’ on cAMP signalling, possibly to overcome PKA-dependent relaxation of smooth muscle cells. Interestingly, in VSMCs, the attenuation of cAMP levels due to purinergic stimulation was long-lasting, whereas in HUVECs, thrombin induced a biphasic effect on cAMP levels. Initially, thrombin reduced cAMP in an
Targeting cAMP Signalling to Combat Cardiovascular Diseases
AC6-dependent manner possibly allowing a ‘breakthrough’ of the cAMP-stabilized barrier formation [22]. However, after 1 min, we observed a slow increase in cAMP levels, which reached even higher levels than those induced by the initial stimulation of β 2 -ARs [23]. Similarly to VSMCs, the Ca2 + -mediated and AC5/AC6-dependent lowering of cAMP in HUVECs may be interpreted as a counteracting mechanism of thrombin to overcome tightening of endothelial barriers induced via stimulation of Gs -coupled receptors. The pharmacological characterization of the stimulatory effect of thrombin on cAMP levels revealed that both phospholipase A2 and cyclo-oxygenases were required. Furthermore, stimulation of prostacyclin receptors with exogenous agonists easily elevated cAMP. Inhibition of these receptors with a specific antagonist (CAY10441) also attenuated the thrombin-induced elevation of cAMP. As cAMP elevation in HUVECs is considered to lead to an increase in endothelial cell barrier formation [24], the thrombin-induced slow increase in cAMP may serve as a negative-feedback mechanism to limit the well-described increase in endothelial permeability. An interesting secondary aspect of these experimental results is the fact that the thrombin-induced prostacyclin-dependent increase in cAMP occurred within single cells under continuous superfusion of these cells with buffer. This set up was well suited to wash away any mediator secreted from cells. Therefore it seems very likely that prostacyclin acts as an autocrine mediator that is produced in the identical cell in which it exerts its biological activity, as depicted in Figure 1.
Dynamics of interactions between G-proteins and ACs determine signalling sensitivity Although ACs have been known for even longer than G-proteins, it was very difficult to study the molecular mechanisms of interactions due to experimental limitations such as lack of suitable antibodies or difficulties to purify and reconstitute functional mammalian ACs. Other effectors of G-proteins in the cardiovascular system such as ion channels and PLC (phospholipase C) have been characterized successfully with respect to the dynamics of interactions with G-protein subunits. For atrial GIRK channels, many studies point towards an activity-independent complex formation of the heterotrimeric G-proteins [25]. Upon activation, this complex is thought to undergo a rearrangement, leading to a movement of GIRK channel subunits, which allow the channel to increase its open probability [26]. The dynamics of G-protein-mediated channel activation and deactivation mirror those of G-proteins themselves as measured by FRET [12,27]. Importantly, RGSs (regulators of G-protein signalling) are able to accelerate both dynamics of receptormediated GIRK currents [28] and G-protein dynamics [12]. The dynamics of the interaction of activated Gα q with PLCβ were studied in vitro. In this case, it was found that
Figure 2 Offset kinetics for Gα i –AC interaction and Gi -protein activity after agonist withdrawal determined by single-cell FRET imaging The agonist washout phase after stimulation with 10 nM noradrenaline (NE) was normalized and averaged (means ± S.E.M.). The dissociation of Gα i1 and AC5 is generally slower than the rearrangement (reflecting deactivation) of the Gi -protein subunits. RGS4 accelerates the deactivation of the Gi -protein; however, RGS4 does not significantly affect Gα i1 –AC5 dissociation kinetics. Reproduced with permission from [12]: Milde, M., Rinne, A., Wunder, F., Engelhardt, S. and Bunemann, ¨ M. (2013) Dynamics of Gα i1 interaction with type 5 adenylate cyclase reveal the molecular basis for high sensitivity of Gi -mediated inhibition of cAMP production. Biochem. J. 454, 515–523.
PLCβ influences G-protein activity by accelerating GTP hydrolysis of Gα q -GTP [29]. Later, a crystal structure of this complex [4] led to a mechanistic explanation of the observed effect [30]. G-protein interactions with ACs have been addressed recently using a FRET-based approach [12]. On the basis of previous studies from the Gilman, Dessauer and Tesmer laboratories [31–33], which all contributed to our understanding of where G-protein subunits bind on the surface of ACs, a fusion protein of YFP linked to the Nterminus of AC5 was generated. Importantly, the resulting YFP–AC5 was carefully characterized to be indistinguishable from wild-type AC5 with respect to both its ability to be activated via Gs -proteins as well as the powerful inhibition via Gi -proteins [12]. Even the kinetics of AC5-mediated alterations in cAMP were very similar for wild-type AC5 and YFP–AC5. As FRET partners for YFP–AC5, we used Gα s –CFP, Gα i1 –CFP or CFP-labelled Gβγ subunits. For AC5–G-protein FRET pairs, no specific pre-assembly before receptor activation could be detected, suggesting that assembly between G-proteins and ACs takes place only after agonist-mediated receptor activation. However, the absence of static FRET is not a final proof for the nonexistence of interaction. Agonist-mediated stimulation of both Gs -proteins via β 2 -ARs or Gi -proteins via α 2A -ARs resulted in the development of a FRET signal between AC5 and the activated G-protein subunits. Kinetics of the interaction between Gα s and AC5 were very similar to those measured between Gα s and Gβγ ([34], and C The
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Figure 3 Differential sensitivity for receptor-mediated Gi -protein activity and Gα i –AC5 interaction Representative concentration–response curves derived from single cells for noradrenaline (NE)-evoked Gα i –Gβγ or Gα i –AC5 FRET with or without co-expression of RGS4 as indicated. The lower panel shows the mean ± S.E.M. EC50 values derived from fittings of individual curves for the indicated conditions (n>11; statistics were obtained by ANOVA and Bonferroni post-hoc test; ***P < 0.001; n.s., not significant).
¨ M. Milde and M. Bunemann, unpublished work). In response to stimulation of α 2A -ARs, Gα i1 also rapidly interacted with AC5, as reflected by the developing FRET signal; however, after withdrawal of agonist, it took much longer for Gα i1 to dissociate from AC5 compared with the kinetics of Gprotein reassembly as determined by FRET between α and βγ subunits [12] (Figure 2). The most plausible explanation for this observation is an inhibition of GTP hydrolysis by AC5 or a slow dissociation of the Gα i -GDP–AC5 complex. In contrast with AC5-free Gi -proteins, RGS4 was not able to significantly accelerate dissociation of Gα i1 and AC5. To our knowledge, this is the first time that anyone has reported a negative effect of the reassembly of a heterotrimeric Gprotein due to the presence of a G-protein effector in intact cells. How relevant might this slowing of the AC– Gα i1 complex be? Any slowing of G-protein deactivation should lead to an imbalance in the G-protein cycle, resulting in a shift towards a larger portion of active G-proteins and an accordingly increased sensitivity of the signalling pathway towards agonist stimulation. Indeed, we found a remarkable sensitization of the concentration response for receptor-mediated interaction of Gα i1 with AC5 compared with the total (non-AC5-bound) Gα i1 activity (Figure 3). This 10-fold increased sensitivity was not primarily due to a massive overexpression of G-proteins over AC5 as controlled by membrane expression of the fluorescent proteins. A comparison of the agonist sensitivity of GIRK currents with the Gi -protein activity determined by FRET revealed no major difference. However, Gi/o -mediated inhibition of AC5mediated cAMP production was extremely sensitive towards C The
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receptor activation as determined in single-cell experiments using an Epac (exchange protein directly activated by cAMP)-based FRET reporter for cAMP [12]. It can therefore be concluded that, on the functional level, inhibition of AC5 by Gα i/o -proteins is more than one order of magnitude more sensitive to agonist-mediated receptor stimulation compared with activation of K + channels. It is important to note that, two decades ago, the Szabo laboratory described a similar difference in the sensitivity of two G-protein pathways in frog atrial myocytes important for chrono- and dromo-tropic effects induced via the vagal transmitter acetylcholine: AChinhibited L-type Ca2 + currents at much lower concentrations compared with its activation of inwardly rectifying K + currents [35]. At that time, the researchers already speculated that differential kinetics of the interactions of the two Gprotein effectors ACs and GIRK channels with their Gproteins may provide the basis for the different sensitivity. With our recent observations, we confirm that G-protein effectors can indeed slow G-protein kinetics. It will be interesting to learn whether other G-protein effectors also increase their sensitivity towards receptor-mediated regulation by influencing kinetics of the G-protein cycle. On the basis of the fact that prolonged interactions between Gproteins and effectors will disable these effectors from being regulated very rapidly, we like to speculate that it is more likely to observe similar effects with effectors that mediate relatively slow signals, similar to ACs.
Funding This work was funded by the Deutsche Forschungsgminschaft [grant numbers SFB487 TPA10, SFB593 TPA13 and SFB688 TBB6 (to M.B.)].
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Received 2 January 2014 doi:10.1042/BST20130280
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