Phosphorylation of Cav1.2 on S1928 uncouples the L‐type Ca2+ channel from the β2 adrenergic receptor
EMBO J
(2016)
35: 1330 - 1345
Abstract
Agonist‐triggered downregulation of β‐adrenergic receptors (ARs) constitutes vital negative feedback to prevent cellular overexcitation. Here, we report a novel downregulation of β2AR signaling highly specific for Cav1.2. We find that β2‐AR binding to Cav1.2 residues 1923–1942 is required for β‐adrenergic regulation of Cav1.2. Despite the prominence of PKA‐mediated phosphorylation of Cav1.2 S1928 within the newly identified β2AR binding site, its physiological function has so far escaped identification. We show that phosphorylation of S1928 displaces the β2AR from Cav1.2 upon β‐adrenergic stimulation rendering Cav1.2 refractory for several minutes from further β‐adrenergic stimulation. This effect is lost in S1928A knock‐in mice. Although AMPARs are clustered at postsynaptic sites like Cav1.2, β2AR association with and regulation of AMPARs do not show such dissociation. Accordingly, displacement of the β2AR from Cav1.2 is a uniquely specific desensitization mechanism of Cav1.2 regulation by highly localized β2AR/cAMP/PKA/S1928 signaling. The physiological implications of this mechanism are underscored by our finding that LTP induced by prolonged theta tetanus (PTT‐LTP) depends on Cav1.2 and its regulation by channel‐associated β2AR.
Synopsis
This work identifies Cav1.2 S1928 phosphorylation as negative feedback mechanism for Ca2+ channel regulation by β2AR/cAMP/PKA signaling in neurons. This desensitization is highly specific as nearby β2AR/PKA/AMPA receptor signaling is not attenuated.
•
β2AR–Cav1.2 binding at S1928 is required for channel regulation in neurons
•
Phosphorylation of S1928 specifically displaces the β2AR from Cav1.2
•
β2AR displacement prevents subsequent Cav1.2 phosphorylation and regulation
•
β2AR–Cav1.2 interaction is necessary for the induction of PTT‐LTP
Introduction
Norepinephrine in the brain is important for arousal, behavioral acuity, and learning in novel and emotionally charged situations (Cahill et al, 1994; Berman & Dudai, 2001; Hu et al, 2007; Minzenberg et al, 2008; Carter et al, 2010). It signals via β1 and β2AR–Gs–adenylyl cyclase–cAMP–PKA cascades (Sanderson & Dell'Acqua, 2011). The β2AR uniquely binds directly to the C‐terminus of α11.2, the central pore‐forming subunit of Cav1.2 (Davare et al, 2001; Balijepalli et al, 2006), and via PSD‐95 and auxiliary TARP subunits to AMPA‐type glutamate receptors (AMPARs) (Joiner et al, 2010; see also Wang et al, 2010). These complexes also contain Gs (Davare et al, 2001; Joiner et al, 2010), adenylyl cyclase (Davare et al, 2001; Efendiev et al, 2010; Joiner et al, 2010; Nichols et al, 2010), and AKAP‐anchored PKA (Davare et al, 2001; Tavalin et al, 2002; Hulme et al, 2003, 2006a; Hall et al, 2007; Oliveria et al, 2007; Joiner et al, 2010; Zhang et al, 2013; Dittmer et al, 2014). Assembly of such complexes brings all components of this cAMP cascade into close proximity with each other (Fig EV1 A and B), which results in localized cAMP signaling and regulation of β2AR‐associated Cav1.2 and AMPAR (Chen‐Izu et al, 2000; Davare et al, 2001; Hulme et al, 2003; Joiner et al, 2010). Spatial restriction of cAMP production, diffusion, and signaling is a key mechanism thought to underlie the specific cAMP effects seen for certain Gs protein‐coupled receptors (GsPCRs) (Smith et al, 2006; Leroy et al, 2008; Dai et al, 2009; Richter et al, 2013) including β2AR (Jurevicius & Fischmeister, 1996; Kuschel et al, 1999; Chen‐Izu et al, 2000; Davare et al, 2001; Balijepalli et al, 2006; Nikolaev et al, 2010). This localized signaling is in contrast to the broad non‐target selective signaling by the β1AR and other GsPCRs (Xiao et al, 1999b; Steinberg & Brunton, 2001; Balijepalli et al, 2006). Despite much effort to prove this concept, clear evidence in support of this hypothesis as provided here by the effects of acute β2AR displacement from Cav1.2 by peptide and S1928 phosphorylation (see below) has been lacking so far.
Figure EV1. Model of the β2AR–Cav1.2 and β2AR–GluA1 complexes and time course of ISO‐induced GluA1 phosphorylation and displacement of β2AR from Cav1.2
A.
The β2AR binds directly to residues 1923–1942 in the C‐terminus of α11.2 (red double‐headed arrow). S1928 phosphorylation by PKA displaces the β2AR. The second PKA site S1700 is located in a segment predicted to interact with the distal C‐terminus (Hulme et al, 2006b). The A kinase anchor protein AKAP5 interacts with the N‐terminus, loop I, and the distal C‐terminus of α11.2 (Hall et al, 2007). It links PKA and likely also AC to Cav1.2 as it does for GluA1 (Zhang et al, 2013). Peptide 2 (Pep2) mimics residues 1923–1942 and competes with the β2AR for binding to α11.2.
B.
The β2AR associates with AMPARs via binding to the third PDZ domain of PSD‐95. PSD‐95 itself binds with its first and second PDZ domain to stargazin (Stg) and its homologues (TARPs, γ2/3/4/5/7/8), which are auxiliary AMPAR subunits. AKAP5 binds to the PSD‐95 homologue SAP97 (Tavalin et al, 2002), which in turn binds to the very C‐terminus of GluA1 (Leonard et al, 1998). It recruits PKA and AC to GluA1 (Zhang et al, 2013). S845 is the PKA site on GluA1. The DSPL peptide mimics residues 894–907 at the very C‐terminus of the β2AR and competes with the β2AR for binding to the third PDZ domain of PSD‐95.
C–G.
Forebrain slices from WT mice (C–F) or S1928KI mice (G) were treated with 10 μM ISO for 0.5–10 min before solubilization, ultracentrifugation, separate IP of GluA1 (C–E) or α11.2 (F, G), and IB for pS845, followed by stripping and re‐probing for pS831 and ultimately total GluA1 (C–E) or IB for α11.2 and β2AR (F, G). Shown in (C) are IBs for pS845, pS831, and total GluA1 after GluA1 IP. For quantification of GluA1 phosphorylation, pS845 (D) and pS831 (E) signals from (C) were normalized to total GluA1 signals. Quantification of co‐IP of β2AR with α11.2 following ISO treatment is shown in (F, G). Sample IBs for WT and S1928A KI mice are depicted in bottom panels of Fig 3A and G, respectively. For quantification of coIP, β2AR signals were normalized to α11.2. Data are presented as mean ± SEM. n = 3. **P < 0.01, ***P < 0.001, one‐way ANOVA.
H.
Mouse brain was homogenized and solubilized with 1% Triton X‐100 before ultracentrifugation and IP with control IgG or β2AR antibody. IB for pS1928 was followed by stripping and reprobing for total α11.2 (top portion of blot; top two panels), for GluA1 (middle portion; third panel), or for β2AR (bottom portion; bottom panel). As illustrated here, we never observed any pS1928 signal in β2AR despite clear detection of α11.2 suggesting that S1928 phosphorylation completely prevents the β2AR–α11.2 interaction.
Cav1.2 is the most abundant L‐type Ca2+ channel in brain and heart (Hell et al, 1993a). Mutations in Cav1.2 affect many tissues indicating widespread prominent Cav1.2 functions, which include control of cardiac contractility and heart rate as well as autistic‐like behaviors (Splawski et al, 2004). Besides their prominent roles in cardiovascular function, L‐type channels are critical in the brain for long‐term potentiation (Grover & Teyler, 1990; Moosmang et al, 2005) and depression (LTD) (Bolshakov & Siegelbaum, 1994), neuronal excitability (Marrion & Tavalin, 1998; Berkefeld et al, 2006), and gene expression (Dolmetsch et al, 2001; Marshall et al, 2011; Li et al, 2012; Ma et al, 2014). Upregulation of Cav1.2 activity by β‐adrenergic signaling is a central mechanism of regulating Ca2+ influx into cardiomyocytes (Reuter, 1983; Balijepalli et al, 2006) and neurons (Gray & Johnston, 1987; Davare et al, 2001; Oliveria et al, 2007; Dittmer et al, 2014). The differential global versus local regulation of Cav1.2 by β1AR versus β2AR might be due to association of the β2AR but not β1AR with Cav1.2 (Chen‐Izu et al, 2000; Davare et al, 2001; Balijepalli et al, 2006). We now provide clear evidence for this notion by showing that acute displacement of the β2AR by a peptide and by S1928 phosphorylation prevents phosphorylation and upregulation of Cav1.2 by β2AR stimulation.
The most prominent and heavily regulated PKA phosphorylation site in Cav1.2 is S1928 in the C‐terminus of its central α11.2 subunit (Hell et al, 1993b, 1995; De Jongh et al, 1996; Davare et al, 1999, 2000; Davare & Hell, 2003; Hulme et al, 2006a; Hall et al, 2007; Dai et al, 2009). However, functional studies argue against S1928 regulating channel activity in the heart (Ganesan et al, 2006; Lemke et al, 2008). Here, we found that the β2AR binds to α11.2 residues 1923–1942 and that S1928 phosphorylation within this segment disrupts this interaction. This mechanism constitutes a particular form of downregulation of β2AR signaling upon prolonged stimulation that specifically blunts subsequent upregulation of Cav1.2 but not AMPAR phosphorylation and activity and is absent in S1928A knock‐in mice.
Results
The β2AR binds to residues 1923–1942 in the C‐terminus of Cav1.2
As the β2AR C‐terminus mediates binding to Cav1.2 (Davare et al, 2001), we utilized amylose‐immobilized maltose‐binding protein (MBP)‐tagged β2AR C‐terminus in pull‐down experiments to define its binding site in α11.2. We first tested affinity purified glutathione S‐transferase (GST) fusion proteins of the N‐terminus of α11.2, the three loops between the four homologous membrane domains of α11.2, and the three C‐terminal constructs CT1 (aa 1507–1733), CT23 (aa 1622–1905), and CT4 (aa 1909–2171), which cover the whole α11.2 C‐terminus (Fig 1A). From these constructs, only CT4 bound to the MBP‐tagged β2AR C‐terminus, indicating a highly specific interaction (Fig 1B and C). From three fragments that covered CT4 (CTC (aa 1834–1957); CTD (aa 1944–2067); CTE (aa 2054–2171)), only CTC bound to the β2AR (Fig 1D and E). These results restrict the interaction site to the overlapping region between CTC and CT4 (aa 1909–1957).
Figure 1. Identification of the β2AR binding region on Cav1.2
A.
Schematic of the α11.2 subunit of Cav1.2 (top) and the α11.2‐derived GST fusion proteins covering the C‐terminus (bottom).
B.
Pull‐down of GST‐tagged α11.2 segments (top immunoblot; IB) by immobilized MBP‐β2AR C‐terminus (residues 326–413 of human β2AR). GST fusion proteins were detected by an anti‐GST antibody and MBP fusion proteins by an anti‐MBP antibody. Middle IB shows that comparable amounts of the various GST fusion proteins had been added to the resin samples and bottom IB illustrates equal loading of all amylose resin samples with MBP‐β2AR.
C.
Quantification of (B).
D.
Pull‐down of GST‐CTC but not GST‐CTD or GST‐CTE (middle IB) by immobilized MBP‐β2AR C‐terminus (left) but not MBP alone (right), all of which were present at comparable amounts (bottom and top IBs, respectively). GST fusion proteins were detected by an anti‐GST antibody and MBP fusion proteins by an anti‐MBP antibody.
E.
Quantification of (D).
F.
IP of β2AR in the presence of 10 μM GST (Control; Ctr) or GST‐tagged C‐terminal fragments as indicated. CT4 and CTC specifically displaced α11.2 (top of IB) but not GluA1 (middle of same IB) from β2AR (bottom of same IB). Use of non‐specific IgG (left lane in right panels) indicates specificity of IP.
G, H.
For quantification of coIPs, α11.2 (G) and GluA1 (H) immunosignals from (F) were normalized to β2AR signals.
I.
Representative IB showing amounts of the GST fusion proteins that were added in (F), as detected by anti‐GST antibodies.
To test whether CT4 and CTC bind to native Cav1.2 and could be used to acutely and specifically disrupt the β2AR–Cav1.2 complex, the β2AR was immunoprecipitated in the absence and presence of CT4 and CTC and, as negative controls, CT1, CT23, and CTD. CT4 and CTC but not the other polypeptides completely displaced Cav1.2 from the β2AR (Fig 1F, G and I). To eliminate the possibility of nonspecific or secondary effects of the polypeptides on the complex, we monitored within the same samples and same immunoblot lanes co‐immunoprecipitation (co‐IP) of the AMPAR subunit GluA1, which forms a separate complex with the β2AR. In contrast to Cav1.2, this interaction is mediated by PDZ interactions with PSD‐95 (Fig EV1 A and B), and therefore, neither CT4 nor CTC affected the GluA1–β2AR co‐IP (Fig 1F and H).
To further narrow down the β2AR binding site of α11.2, synthetic peptides covering aa 1906–1925 (Pep 1), aa 1923–1942 (Pep 2), and aa 1939–1959 (Pep 3) whose N‐termini were labeled with fluorescein (FITC), were titrated with the β2AR C‐terminus, and their binding was monitored by fluorescence polarization. Pep2, but neither Pep1 nor Pep3, showed strong and saturable binding with an apparent Kd of ~1.9 μM (Fig 2A). In addition, only Pep2 displaced α11.2 (but not GluA1) from the β2AR during IP (Fig 2B–D).
Figure 2. The β2AR binds to the S1928 phosphorylation site
A.
Titration of fluorescence polarization of FITC peptides (100 nM) spanning α11.2 aa 1906–1959 with purified MBP‐β2AR C‐terminus. Kd value was obtained by fitting a nonlinear direct binding curve to Pep2.
B.
IP of β2AR in the absence (Control) or presence of 10 μM peptides, as indicated. Pep2 specifically displaced α11.2 (top of IB) but not GluA1 (middle, same IB) from β2AR (bottom, same IB). Use of non‐specific IgG (left lane) indicates specificity of (co)IPs.
C, D.
For quantification, α11.2 (C) and GluA1 (D) IB signals from (B) were normalized to β2AR signals.
E.
IP of β2AR in the absence (Control) or presence of 10 μM Pep2 or Pep2 with S1928 being phosphorylated (phPep2), which did not displace α11.2.
F.
α11.2 IB signals were normalized to β2AR signals.
Unlabeled synthetic Pep2 and synthetic Pep2 with S1928 being phosphorylated (PhPep2) were added during IP of β2AR to test whether phosphorylation of S1928 affects β2AR binding. While Pep2 removed Cav1.2 (but, once more, not GluA1), PhPep2 had no effect suggesting that S1928 phosphorylation impairs β2AR binding (Fig 2E and F).
S1928 phosphorylation displaces the β2AR from Cav1.2
To evaluate whether S1928 phosphorylation displaces the β2AR from Cav1.2, we monitored Cav1.2 phosphorylation and β2AR–Cav1.2 association in forebrain slices upon stimulation with the βAR agonist isoproterenol (ISO). S1700 has recently emerged as a PKA phosphorylation site that is important for upregulation of Cav1.2 activity in heart (Fuller et al, 2010; Hell, 2010; Fu et al, 2013). As phosphorylation of S1700 and S1928 increased (Fig 3A–C), association of Cav1.2 with the β2AR decreased (Fig 3D and E). Strikingly, no such decrease was observed in slices from S1928A KI mice, even though ISO induced S1700 phosphorylation in these mice (Fig 3G–K). Displacement of the β2AR from Cav1.2 is unique for S1928 phosphorylation, as the β2AR–GluA1 interaction was not disrupted by ISO application (Fig 3D, F, J and L), which induced phosphorylation of S845 (Fig EV1C and D), a well‐established PKA site on GluA1 (Roche et al, 1996). IP of Cav1.2 followed by IB of β2AR confirmed their dissociation upon ISO treatment in WT but not S1928A KI mice (Fig 3A, bottom; Fig EV1F and G).
Figure 3. S1928 phosphorylation displaces β2AR from Cav1.2
Forebrain slices from WT (A–F) and S1928A KI mice (G–L) were treated with vehicle (water) or 10 μM isoproterenol (ISO) for 0.5–10 min before solubilization, ultracentrifugation, IP of α11.2 (A–C, G–I) or β2AR (D–F, J–L), and sequential IB for pS1928, pS1700, and α11.2, for GluA1, or for β2AR, of corresponding regions of the blots, as indicated. All the α11.2 IPs in (A–C) and (G–I) were from the same samples (which were split in half for parallel IP) as the β2AR IPs in (D–F) and (J–L), respectively (for quantification of coIP of β2AR with α11.2 see Fig EV1F and G).
Data information: Data are presented as mean ± SEM. n = 4 (B, C, H, I, K and L) or 6 (E, F). **P < 0.01, ***P < 0.001, one‐way ANOVA.
A–F.
In WT, the time‐dependent increase in S1928 and S1700 phosphorylation (A–C) paralleled the decrease in coIP of β2AR with α11.2 (A bottom, Fig EV1F) and of α11.2 with β2AR (D–F). For quantification of α11.2 phosphorylation (B, C), pS1928 and pS1700 signals were normalized to α11.2. For quantification of coIP (E, F), α11.2 and GluA1 signals were normalized to β2AR.
G–L.
In S1928A KI mice, ISO induced S1700 phosphorylation (G, I) but did not disrupt the α11.2–β2AR interaction (G bottom, J–L, Fig EV1G). For quantification of α11.2 phosphorylation (H, I), pS1928 and pS1700 signals were normalized to α11.2. For quantification of coIP (K, L), α11.2 and GluA1 signals were normalized to β2AR.
To test whether the ISO‐induced dissociation of the β2AR from Cav1.2 results in their spatial separation, we co‐expressed α11.2 with the HA tag within an extracellular loop (α11.2‐HA) and β2AR with the FLAG tag at its extracellular N‐terminus (FLAG‐β2AR) in cultured hippocampal neurons. Line scan analysis of the fluorescence distribution of the surface labeled α11.2‐HA and FLAG‐β2AR (see Appendix Supplementary Methods) showed that the median distance between neighboring α11.2‐HA and FLAG β2AR clusters significantly increases from 0.24 μm (25–75% interquartile range: 0.15–0.44 μm) to 0.34 μm (IQR: 0.21–0.54 μm) after 5 min of ISO treatment (Fig 4). Because the distribution of distances failed two normality tests (see Appendix Supplementary Methods), we used the nonparametric Mann–Whitney Rank test for statistical analysis, which resulted in a two‐tailed P‐value of < 0.0001. The Kolmogorov–Smirnov cumulative distributions test yielded a P‐value < 0.0001. In addition, Pearson's correlation analysis yielded a coefficient 0.36 ± 0.03 (mean ± SEM) for control and 0.29 ± 0.02 for ISO treated neurons with P = 0.037. Furthermore, we calculated the fraction of overlap between CaV1.2‐HA and FLAG‐β2AR puncta. We obtained a Mander's coefficient of 0.41 ± 0.03 (mean ± SEM) for control and 0.31 ± 0.03 for ISO treated neurons with P = 0.02. These results are consistent with the idea that ISO stimulation displaces the β2AR from Cav1.2.
Figure 4. S1928 phosphorylation separates β2AR from Cav1.2
Hippocampal cultures were transfected with FLAG‐β2AR and α11.2‐HA at 6 days in vitro (DIV), treated with vehicle (water) or 1 μM isoproterenol (ISO) for 5 min at 18 DIV, fixed and surface labeled for HA and FLAG.
A, B.
Representative immunofluorescent images obtained by wide‐field microscopy at lower and higher resolutions (scale bar, 5 μm). Arrows in (A) indicate the samples enlarged in (B).
C.
Quantification of distance between centers of HA and FLAG puncta (**P < 0.001, Mann–Whitney rank sum test). The bars represent 5th (lower end) and 95th percentile (higher end).
ISO‐triggered dissociation of the β2AR from Cav1.2 prevents subsequent Cav1.2 phosphorylation
Could the displacement of the β2AR from Cav1.2 upon S1928 phosphorylation be a novel mechanism that specifically downregulates this powerful β adrenergic regulation of Ca2+ influx into neurons? To test this idea at the molecular level, forebrain slices from WT mice were treated with vehicle or ISO for 5 min, followed by washout of ISO for various time periods before re‐application of ISO. 20‐min and even 10‐min but not 3‐min washout reversed the ISO‐induced displacement of the β2AR from α11.2 (Fig 5A, lane 5 vs. 7; Fig EV2A–C). S1700 and S1928 phosphorylation returned to baseline already after 3‐min washout (Fig 5D, lanes 2–4; Fig 5E and F). As expected, phosphorylation of GluA1 on S845 behaved similarly (Fig 5D and G); phosphorylation of S831, which can be mediated by PKC and CaMKII but not PKA (Roche et al, 1996; Halt et al, 2012), served as a negative control that is inert to PKA stimulation (Fig 5D and H).
Figure 5. ISO‐induced displacement of the β2AR from Cav1.2 blunts subsequent Cav1.2 phosphorylation
Forebrain slices from WT mice were treated with vehicle (water; lanes 1, 5; numbers on bottom) or 10 μM ISO for 5 min, followed, if indicated, by 1‐min, 3‐min, or 20‐min washout of ISO (lanes 3–7) and a second application of ISO for 5 min (lane 6), before solubilization and ultracentrifugation.
Data information: Data are presented as mean ± SEM. n = 3 (B, C) or 4 (E–H). **P < 0.01, one‐way ANOVA.
A.
β2AR was IPed before IB for α11.2 (top part of IB), GluA1 (middle part of IB), and β2AR (bottom part of IB), as indicated. ISO‐induced displacement of the β2AR from α11.2 (lanes 2–6) lasted at least 3 min but not 20 min (compare lanes 6 and 7).
B, C.
For quantification, α11.2 (B) and GluA1 (C) IB signals from (A) were normalized to β2AR.
D.
α11.2 and GluA1 were concurrently IPed from same samples as in (A) by simultaneous addition of anti‐α11.2 and anti‐GluA1 antibodies before probing and stripping/re‐probing upper part of IB for pS1928, pS1700, and total α11.2 (top three panels) and middle part for pS845, pS831, and total GluA1 (bottom three panels). ISO‐induced displacement of the β2AR from α11.2 (see A) rendered α11.2 (but not GluA1) refractory to re‐phosphorylation of S1928 and S1700 upon a second ISO application of α11.2 (compare lanes 5 and 6).
E–H.
For quantification, pS1700 (E) and pS1928 (F) IB signals from (D) were normalized to total α11.2, and pS845 (G) and pS831 (H) signals from (D) to total GluA1.
Figure EV2. Time course of β2AR re‐association with α11.2 and of de‐phosphorylation of α11.2 and GluA1 after ISO treatment
Forebrain slices from WT mice were treated with vehicle or 10 μM ISO for 5 min, followed by ISO washout for 3, 10, 30, and 90 min if indicated, before solubilization and ultracentrifugation.
Data information: Data are presented as mean ± SEM. n = 4. *P < 0.05, one‐way ANOVA.
A.
IP of β2AR was followed by IB for total α11.2 (top part of blot), GluA1 (middle part), or β2AR (bottom part), as indicated. Re‐association of the β2AR with α11.2 took more than 3 min but was fully accomplished 10 min after ISO washout.
B, C.
For quantification of coIP, GluA1 (B) and α11.2 (C) signals from (A) were normalized to β2AR.
D.
α11.2 and GluA1 were simultaneously IPed before probing and re‐probing upper part of blot for pS1928, pS1700, and total α11.2 and middle part for pS845 and total GluA1.
We tested whether the acute displacement of the β2AR from α11.2 affects re‐phosphorylation and re‐stimulation of Cav1.2 after 3‐min washout. In fact, re‐application of ISO after a 3‐min washout was not able to induce a second round of phosphorylation of S1700 or S1928 (Fig 5D, lane 6). In contrast, in mock washout samples ISO was fully effective in inducing phosphorylation of these residues following initial application of vehicle instead of ISO before the 3‐min washout (Fig 5D, lane 5). The ISO‐induced displacement of the β2AR from α11.2 was specific for Cav1.2, as coIP of GluA1 with the β2AR was not affected within same samples that were analyzed for Cav1.2 coIP (Fig 5A and C). Re‐phosphorylation of S845 during the second ISO treatment was also not blunted by the first ISO application (Fig 5D, lane 6; Fig 5G). Accordingly, displacement of the β2AR from Cav1.2 selectively downregulates the signaling pathway from the β2AR to Cav1.2 without affecting another target, GluA1, which also forms a signaling complex with the β2AR. To test whether endocytosis of the β2AR is responsible for lack of re‐phosphorylation of α11.2 by the second ISO application, we blocked endocytosis with two different drugs, dynasore and pitstop. Neither affected the loss of α11.2 phosphorylation by the second ISO pulse (Fig EV3) arguing against this possibility.
Figure EV3. Blunting of ISO‐induced Cav1.2 phosphorylation by ISO pre‐treatment is not affected by endocytosis blockers
Forebrain slices from WT mice were pre‐treated for 30 min with 100 μM dynasore (lanes 4, 5; denoted at bottom of panel) or 30 μM pitstop (lanes 6, 7), treated with vehicle (water; lanes 4, 6) or 3 μM ISO for 5 min, followed by 3‐min or 20‐min washout of ISO (or of vehicle in lanes 4, 6) and a second application of ISO for 5 min (lanes 2–7), before solubilization and ultracentrifugation.
Data information: Data are presented as mean ± SEM. n = 4. ***P < 0.001, one‐way ANOVA.
A.
α11.2 and GluA1 were simultaneously IPed before probing and re‐probing upper part of IB for pS1928, pS1700, and total α11.2, and middle part for pS845 and total GluA1. Inhibiting endocytosis with either dynasore or pitstop did not affect blunting of S1700 and S1928 phosphorylation during a second ISO application (compare lanes 5 and 7 with lane 2). Note that re‐phosphorylation upon the second ISO application was blunted after 3‐min washout but restored after 20‐min washout subsequent to the first ISO treatment (compare lanes 1–3 with each other).
B–D.
For quantification, pS1700 (B) and pS1928 (C) IB signals from (A) were normalized to total α11.2, and pS845 (D) IB signals to total GluA1.
Strikingly, in S1928A KI mice, re‐phosphorylation of S1700 during the second ISO application after 3‐min washout was not decreased at all as compared to single ISO applications (Fig 6A, lane 6 vs. lanes 2 and 5). In fact, the second ISO treatment appears to have increased S1700 phosphorylation more strongly than the first treatment. These results suggest that additional, as yet to be identified, mechanisms exist that enhance phosphorylation of S1700 during repetitive activation of β2AR bound to CaV1.2. For instance, like PKA, the phosphatase PP2B/calcineurin is linked to CaV1.2 via AKAP5 to counteract CaV1.2 phosphorylation by PKA (Oliveria et al, 2007; Fuller et al, 2014; Murphy et al, 2014) but released upon elevated Ca2+ influx via CaV1.2 (Li et al, 2012; Murphy et al, 2014). Because β adrenergic stimulation will increase Ca2+ influx via CaV1.2 as occurring under basal conditions due to neuronal network activity (Hall et al, 2013), it is conceivable that PP2B is displaced from CaV1.2 for 3 min or longer, allowing for stronger phosphorylation of S1700 in S1928A KI neurons upon ISO application that is repeated within 3 min.
Figure 6. ISO pre‐treatment does not blunt Cav1.2 phosphorylation by subsequent ISO treatment in S1928A KI mice
Forebrain slices from S1928A KI mice were treated with vehicle (lanes 1, 5) or 10 μM ISO for 5 min, followed, if indicated, by 1‐min, 3‐min, or 20‐min washout of ISO (lanes 3–7) and a second application of ISO for 5 min (lane 6), before solubilization and ultracentrifugation.
Data information: Data are presented as mean ± SEM. n = 4. **P < 0.01, ***P < 0.001, one‐way ANOVA.
A.
α11.2 and GluA1 were concurrently IPed from same samples by simultaneous addition of anti‐α11.2 and anti‐GluA1 antibodies before probing and stripping/re‐probing upper part of IB for pS1928, pS1700, and total α11.2 and middle part for pS845, pS831, and total GluA1. In S1928A KI mice, S1700 re‐phosphorylation after a 3‐min washout of ISO was not blunted (lane 6) in contrast to WT mice but rather augmented (compare to lane 5).
B–E.
For quantification, pS1700 (B) and pS1928 (virtually absent) (C) IB signals from (A) were normalized to total α11.2, and pS845 (D) and pS831 (E) signals from (A) to total GluA1.
The ISO‐induced displacement of the β2AR from α11.2 is completely reversible, as 10‐min washout of ISO resulted in full coIP of α11.2 with the β2AR (Fig 5A, lane 7; Fig EV2A–C), which is preceded by dephosphorylation of S1928 and also S1700 (Fig EV2D). A 20‐min washout also restored the capability of the β2AR to induce S1700 and S1928 phosphorylation (Fig EV3A and B, compare lanes 2 and 3).
ISO‐triggered dissociation of the β2AR from Cav1.2 prevents subsequent stimulation of L‐type channel activity
To functionally test whether β2AR stimulation affects subsequent regulation of Cav1.2 by a second, closely timed pulse of β2AR stimulation, we sought to record single‐channel L‐type currents from cultured neurons in the cell‐attached patch clamp mode as in our previous work (Davare et al, 2001). Other Ca2+ channels were blocked by adding specific inhibitors (ωCTxGVIA and ωCTxMVIIC) to the patch pipette solution. We determined open probability (Po) from all channels within each patch (NPo) in recordings from neurons with either vehicle or ISO added to the patch pipette. Figure 7A shows original traces with single‐channel activity elicited by depolarizing pulses from −80 mV to several test potentials. These data were fit with a linear function that revealed a slope conductance for these channels of 27 ± 2 pS (Fig 7A), which corresponds to the expected slope conductance for an L‐type Cav1.2 channel under similar experimental conditions (Yue & Marban, 1990). As expected, ISO significantly increased NPo (Fig 7B–D) without affecting single‐channel amplitudes (see Appendix Fig S1) (Davare et al, 2001). Inclusion of the potent L‐type channel blocker nifedipine abrogated virtually all currents either with or without ISO present (Fig 7D). Hence, recordings reflect L‐type currents under either condition. The ISO effect was prevented by the highly specific PKA‐inhibitory PKI peptide, which carried 11 Arg residues to render it membrane permeant (Lu et al, 2007, 2011) (Fig 7D), thus confirming that the ISO‐induced upregulation of L‐type current is via PKA.
Figure 7. ISO‐induced displacement of the β2AR from Cav1.2 blunts subsequent stimulation of L‐type channel activity
A.
Representative single‐channel recordings from hippocampal neurons at 7–14 DIV with 500 nM BayK‐8644, 1 μM ω‐conotoxin GVIA, and 1 μM MVIIC in patch pipette upon depolarization from −80 to −30, −20, −10, and 0 mV. The right panel depicts the single‐channel current–voltage relationship. Mean amplitude of unitary currents for different membrane potential studied are −2.07 pA (−30 mV), −1.75 (−20 mV), −1.53 (−10 mV), and −1.25 (0 mV; n = 5 patches per test potential). Solid line represents best‐fit of data using a linear equation (R2 = 0.92) revealing a slope conductance for these channels of 27 ± 2 pS.
B–D.
Representative single‐channel traces and summary plot upon depolarization from −80 to 0 mV under control conditions and in the presence of ISO in the patch pipette. Cultures were pre‐incubated for 15 min with 10 μM 11R‐PKI if indicated. The patch pipette contained either vehicle for control, nifedipine (nif; 1 μM), ISO (1 μM), or ISO plus nifedipine, which blocked all currents. The ISO‐induced increase in NPo was prevented by 11R‐PKI.
E–H.
Representative single‐channel currents upon depolarization from −80 to 0 mV and summary plot after pre‐treatment of whole cultures with ISO. Cultures were pre‐incubated with vehicle (H2O, mock wash, E) or 1 μM ISO for 5 min (F, G) and washed for 3 (E, F) or 10 min (G) before forming the cell‐attached patch with ISO present in the patch pipette. The upregulation of NPo to ˜0.4 (cf. C, D) occurred only if neurons were pretreated with vehicle instead of ISO (E, H; mock wash) or if ISO washout duration was 10 min (G, H) but not if washout was only 3 min (F, H).
Most critically, when ISO was first applied to the bath for 5 min before washout and subsequent formation of a patch, the ISO included in the patch only upregulated L‐type current when the washout was at least 10 min long (Fig 7E–H). If washout was only 3 min, channel activity remained low during the cell‐attached recording with ISO in the patch pipette (Fig 7F and H). As expected, pre‐treatment with vehicle followed by a 3‐min washout (mock wash; Fig 7E and H) did not affect upregulation of channel activity by ISO in the patch pipette. Accordingly, sequential stimulation of L‐type currents by two ISO applications was only effective if the interim time period was long enough to match the time frame required for the β2AR to re‐associate with Cav1.2 (Fig 5A, lanes 6 and 7, and C; Fig EV2A and C) and re‐phosphorylate it (Fig EV3A, lane 3 vs. lane 2).
Binding of the β2AR to residues 1923–1942 is required for β adrenergic stimulation of α11.2 phosphorylation and Cav1.2 activity
To exclude the possibility that covert effects other than displacement of the β2AR from Cav1.2 might be responsible for loss of sensitivity of channel activity to a second pulse of ISO, the β2AR was acutely displaced from Cav1.2 by Myr‐Pep2, a myristoylated version of Pep2, which mimics the binding site of aa 1923–1942 on the α11.2 subunit and displaces the β2AR from Cav1.2 (Fig 2). Myristoylation renders peptides membrane permeant. We first determined at which concentration Myr‐Pep2 effectively disrupts the β2AR–Cav1.2 interaction by adding increasing amounts to brain extracts during the IP of the β2AR. 0.1–10 μM Myr‐Pep2 increasingly displaced Cav1.2 from the β2AR, with 10 μM being apparently 100% effective without affecting the β2AR–GluA1 association (Fig EV4A–C).
Figure EV4. Characterization of Myr‐Pep2 and Myr‐Pep2scr
Forebrain slices from WT mice were pre‐incubated for 30 min with vehicle (water), 0.1–10 μM Myr‐Pep2, or 10 μM Myr‐Pep2scr (i.e., scrambled Myr‐Pep2).
Data information: Data are presented as mean ± SEM. n = 3. **P < 0.01, ***P < 0.001, one‐way ANOVA.
A–C.
After incubation with Myr‐Pep2, slices were solubilized before ultracentrifugation, IP of β2AR, and IB for α11.2, GluA1, and β2AR. Increasing amounts of Myr‐Pep2 progressively displaced α11.2 but not GluA1 from the β2AR, with 10 μM resulting in near complete dissociation. For quantification, α11.2 (B) and GluA1 (C) immunosignals were normalized to β2AR signals.
D–F.
After incubation with Myr‐Pep2scr, slices were treated with ISO (10 μM, 5 min) before solubilization, ultracentrifugation, IP of β2AR, and IB for α11.2, GluA1, and β2AR. In contrast to Myr‐Pep2, Myr‐Pep2scr did not displace α11.2 (top; compare lanes 1 and 3) nor GluA1 (middle, same blot) from β2AR (bottom, same blot). ISO treatment resulted in dissociation of the α11.2–β2AR interaction, as seen before. For quantification, α11.2 (E) and GluA1 (F) immunosignals were normalized to β2AR signals.
G–J.
After incubation with Myr‐Pep2scr, slices were treated with ISO (10 μM, 5 min) before solubilization, ultracentrifugation, simultaneous IP of α11.2 and GluA1, and IB for pS1928, pS1700, and pS845. In contrast to Myr‐Pep2 (Fig 8D), Myr‐Pep2scr did not blunt ISO‐induced phosphorylation of α11.2 on S1700 nor on S1928 (top three panels reflecting sequential probings of top portion of blot) nor GluA1 phosphorylation on S845 (bottom two panels reflecting sequential probings of middle portion of same blot). For quantification, pS1928 (H) and pS1700 (I) immunosignals were normalized to α11.2, and pS845 (J) signals to GluA1 signals, respectively.
Forebrain slices were incubated for 30 min with vehicle, 10 μM Myr‐Pep2, Myr‐Pep2scr, or Myr‐DSPL. MyrPep2scr is a scrambled version of MyrPep2 and served as negative control. Myr‐DSPL consists of the 14 aa at the very C‐terminus of the β2AR, which interacts with the third PDZ domain of PSD‐95 (Joiner et al, 2010) (Fig EV1B). PSD‐95 in turn is linked to a subset of AMPARs via its binding to the auxiliary AMPAR subunits known as γ subunits or TARPs, including stargazin (Stg/γ2). Myr‐DSPL specifically disrupts the interaction of the AMPAR subunit GluA1 with the β2AR (Joiner et al, 2010) and served as a second negative control. In our experiments, Myr‐Pep2 displaced α11.2 but not GluA1 from the β2AR (Fig 8A, compare lane 5 with 3; Fig 8B and C). In contrast, Myr‐DSPL removed GluA1 but not α11.2 from the β2AR (Fig 8A, compare lane 1 with 3; Fig 8B and C). As before, ISO on its own caused a strong reduction in the coIP of α11.2, but not GluA1, with the β2AR (Fig 8A, compare lane 4 with 3; Fig 8B and C). In combination with ISO, Myr‐Pep2 (compare lane 6 with 4) but not Myr‐DSPL (compare lane 2 with 4) caused a virtually complete displacement of the β2AR from Cav1.2. As a second control, Myr‐Pep2scr had no effect on the association of the β2AR with Cav1.2 whether slices were treated with ISO or not (Fig EV4D and E).
Figure 8. β2AR binding to α11.2 is required for β‐adrenergic stimulation of α11.2 phosphorylation and L‐type currents
Forebrain slices (A–F) were pre‐incubated for 30 min with vehicle (water) or 10 μM Myr‐Pep2 or Myr‐DSPL. Like Myr‐DSPL (Joiner et al, 2010), Pep2 was myristoylated at its N‐terminus (Myr‐Pep2) to make it membrane permeant. Slices were then treated with ISO (10 μM, 5 min) or vehicle (water) before solubilization, ultracentrifugation, and IP of β2AR (A–C) or simultaneously α11.2 and GluA1 with a combination of corresponding antibodies within same samples (D–F).
Data information: Data are presented as mean ± SEM. (B, C, E, F): n = 4. **P < 0.01, ***P < 0.001, one‐way ANOVA. (H): n = 10‐12 patches; *P < 0.005, one‐way Tukey post hoc test.
A.
Myr‐Pep 2 displaced α11.2 (lane 5 vs. 3, top of blot) but not GluA1 (middle, same blot) from β2AR (bottom, same blot); the inverse was true for Myr‐DSPL (lane 1 vs. 3).
B, C.
For quantification, α11.2 (B) and GluA1 (C) immunosignals from (A) were normalized to β2AR signals.
D.
Myr‐Pep2 blunted ISO‐induced phosphorylation of α11.2 S1700 (lane 6 vs. 4, top of blot) but not GluA1 S845 (middle, same blot); the inverse was true for Myr‐DSPL (lane 2 vs. 4).
E, F.
For quantification, pS1700 (E) and pS845 (F) immunosignals from (D) were normalized to α11.2 and GluA1 signals, respectively.
G.
Representative cell‐attached recordings from hippocampal neurons as in Fig 7. In interleafed experiments, cultures were pre‐incubated for 30 min with 10 μM Myr‐Pep2 or scrambled Myr‐Pep2 (Myr‐Pep2scr). The patch pipette contained either vehicle (H2O; control) or 1 μM ISO. The ISO‐induced increase in NPo was prevented by Myr‐Pep2 but not Myr‐Pep2scr. Arrows indicate the 0‐current level (i.e., closed channel).
H.
Summary plot for (G). For statistical analysis, the NPo value was determined for each recording and pooled under each condition for comparison.
Importantly, the ISO‐induced increase in phosphorylation of α11.2 on S1700 (Fig 8D, lane 4 vs. 3, and E) was blocked by Myr‐Pep2 (lanes 6 vs. 5) but not Myr‐DSPL (lanes 1 vs. 2). The exact opposite was true for phosphorylation of GluA1 on S845 (Fig 8D and F). Accordingly, specific displacement of the β2AR from Cav1.2 affects Cav1.2 but not GluA1 phosphorylation and vice versa. Furthermore, Myr‐Pep2scr did not affect phosphorylation of either α11.2 or GluA1 (Fig EV4G–J), confirming the specific actions of Myr‐Pep2.
To define the functional consequences of disrupting the β2AR–Cav1.2 interaction, in interleaved experiments cultured neurons were pre‐incubated for 30 min with Myr‐Pep2 or Myr‐Pep2scr. Subsequent recording with ISO in the patch pipette indicated that Myr‐Pep2 but not Myr‐Pep2scr completely blocked the upregulation of channel function by ISO compared to vehicle controls (Fig 8G and H). We conclude that dissociation of the β2AR–Cav1.2 complex by Myr‐Pep2 prevents upregulation of CaV1.2 channel phosphorylation and activity.
The β2AR–Cav1.2 interaction is required for PTT‐LTP
Prolonged stimulation of the Schaffer collateral pathway at 5–10 Hz, which mimics the naturally occurring theta frequency (7 Hz), induces LTP (PTT‐LTP) if at the same time β2AR (but not β1AR) are stimulated (Thomas et al, 1996; Hu et al, 2007; Qian et al, 2012). This potentiation develops over a period of 15 min with the first 5–10 min showing an initial depression (Thomas et al, 1996; Hu et al, 2007; Qian et al, 2012). Because β2AR stimulation prominently augments Ca2+ influx through Cav1.2 at postsynaptic sites (Hoogland & Saggau, 2004), we tested whether Cav1.2 in general and specifically its upregulation by the β2AR is required for PTT‐LTP. In fact, PTT‐LTP was completely absent in conditional knockout mice in which Cav1.2 had been deleted in glutamatergic forebrain neurons when compared to WT littermate controls (Fig 9A–C). Analysis of input–output relation and paired pulse facilitation indicated that synaptic transmission is normal in both genotypes (Fig EV5). In contrast to the 5 Hz PTT‐LTP stimulus paradigm, LTP induced by a 100 Hz/1 s tetanus depends on NMDARs and not L‐type channels. In this case, potentiation is very strong immediately after the tetanus in part due to presynaptic mechanisms but typically relaxes to a significantly lower level over ~5 min. This 100 Hz LTP was normal in conditional CaV1.2 knockout mice (Fig 9D–F). Accordingly, respective NMDAR‐dependent synaptic plasticity mechanisms can be engaged in a normal manner in the CaV1.2 knockout mice when PTT‐LTP is absent. Strikingly, Myr‐Pep2 but not Myr‐Pep2scr blocked PTT‐LTP in wild‐type mice (Fig 9G–I).
Figure 9. β2AR binding to α11.2 is required for PTT‐LTP
Graphs depict fEPSP initial slopes recorded from hippocampal CA1 before and after either a 5 Hz/3 min (A–C, G–I) or 100 Hz/1 s tetanus (D–F). Arrowheads mark onset of tetani and bars perfusion with 1 μM ISO and 10 μM of Myr‐Pep2 or Myr‐Pep2scr. Inserts show sample traces immediately before (left) and ~30 min after (right) tetani.
Data information: Data are presented as mean ± SEM. **P < 0.01, one‐way ANOVA.
A–C.
Litter‐matched WT but not conditional Cav1.2 KO mice showed PTT‐LTP.
D–F.
WT as well as Cav1.2 KO mice showed NMDAR‐dependent 100 Hz LTP.
G–I.
Myr‐Pep2 but not Myr‐Pep2scr blocked PTT‐LTP.
Figure EV5. Basal synaptic transmission is normal in conditional forebrain Cav1.2 KO mice
A.
Increasing stimulus strength increased fEPSP initial slope to the same degree in Cav1.2 KO mice as in their WT littermates. Plotted are initial slopes of fEPSP against presynaptic fiber volley amplitudes. Inserts on top show sample traces for WT and KO mice for increasing stimulus strengths.
B.
Quantification of slopes of graphs in (A).
C.
Paired pulse facilitation (PPF) did not differ between WT and KO mice. Shown are summary graphs of the ratios of second to first pulse response (fEPSP initial slopes) for increasing interval lengths. The first pulse was set in each individual recording to equal 100%.
Discussion
The importance of tight control over β2AR signaling is exemplified by the existence of a complex set of distinct mechanisms for its downregulation upon prolonged activation (Shenoy & Lefkowitz, 2011), which include receptor phosphorylation by G protein‐coupled receptor kinases (GRKs) (Nobles et al, 2011) and the consequent phosphorylation‐triggered recruitment of arrestins for receptor uncoupling from Gs and endocytosis (Lohse et al, 1990; von Zastrow & Kobilka, 1992; Ferguson et al, 1996; Goodman et al, 1996; Cao et al, 1999) as well as the activity‐dependent, PKA‐mediated switching of β2AR coupling from Gs to Gi (Daaka et al, 1997; Xiao et al, 1999a). Our surprising discovery of the role of S1928 phosphorylation in displacing the β2AR unveils a novel negative feedback regulatory mechanism that targets the pervasive regulation of Cav1.2 by the β2AR to prevent excessive Ca2+ influx into neurons. This mechanism is devoted to highly specific downregulation of β2AR signaling to Cav1.2 but not AMPARs, revealing how tightly controlled the activity of Cav1.2 must be to ensure proper function.
Our first important finding is that phosphorylation of S1928 in α11.2 displaces the β2AR from the C‐terminus of Cav1.2. S1928 is the most prominent PKA phosphorylation site in Cav1.2 as determined by biochemical methods, and S1928 phosphorylation is robustly induced by β adrenergic signaling (Hell et al, 1993b, 1995; De Jongh et al, 1996; Davare et al, 1999, 2000; Davare & Hell, 2003; Hulme et al, 2006a; Hall et al, 2007; Dai et al, 2009). However, its physiological role has remained an enigma, as it does not appear to significantly augment channel function in the heart (Ganesan et al, 2006; Lemke et al, 2008), which is mediated in part by phosphorylation of S1700 (Fuller et al, 2010; Hell, 2010; Fu et al, 2013, 2014). We now identify S1928 phosphorylation as a novel negative feedback mechanism for Cav1.2 regulation by β2AR signaling. This is the first example of termination of GsPCR activity by dissociation of a receptor–substrate complex and therefore introduces a new paradigm for the regulation of cell signaling by this widely expressed class of receptors.
Our second important finding is that dissociation of the β2AR–Cav1.2 interaction by either S1928 phosphorylation during an initial ISO treatment or by Myr‐Pep2 prevents upregulation of channel phosphorylation and activity by subsequent ISO application. Could ISO‐induced displacement of the β2AR from Cav1.2 result in endocytosis of the β2AR, making it inaccessible to ISO and therefore to regulation? Evidently that is not the case, as abrogation of the re‐phosphorylation of S1700 and S1928 during a second ISO application within 3 min of the initial one was not affected by dynasore or pitstop (Fig EV3), two different endocytosis inhibitors whose efficacy is well established in our hands (Hall et al, 2013). Accordingly, preventing β2AR endocytosis, which in some cell lines is a general mechanism of downregulating signaling through the β2AR (von Zastrow & Kobilka, 1992; Cao et al, 1999; Shenoy & Lefkowitz, 2011), does not affect this Cav1.2‐specific form of downregulation. We conclude that it is the displacement of the β2AR from Cav1.2 per se that is responsible for loss of subsequent signaling and not endocytosis of this receptor. This conclusion is also in accordance with the finding that the β2AR can fully re‐associate with Cav1.2 within 10 min (Figs 5A and B, and EV2A and C). Re‐association of the β2AR with Cav1.2 was paralleled by the ability of a second ISO application to induce re‐phosphorylation of S1700 and S1928 (Fig EV3A, lane 3, and C). This finding underlines the notions that ISO‐induced displacement of the β2AR from Cav1.2 is not permanent and that the functionality of this interaction is reinstated within minutes.
Our third important finding is that displacement of the β2AR from Cav1.2 is a specific process that downregulates signaling from the β2AR to Cav1.2 without affecting β2AR‐mediated regulation of GluA1, which also forms a signaling complex with the β2AR. This mechanism is fundamentally different from the arrestin‐mediated downregulation of β2AR signaling by endocytosis and by uncoupling from Gs (Shenoy & Lefkowitz, 2011). Most strikingly, downregulation of Cav1.2 stimulation is highly specific for Cav1.2, whereas arrestin‐mediated effects are cell‐wide affecting all β2AR signaling. On a molecular level, arrestin is recruited to stimulated β2ARs upon their phosphorylation by GRKs, whereas the PKA‐mediated phosphorylation of S1928 acts to displace the β2AR from Cav1.2.
Our fourth important finding is that PTT‐LTP depends on Cav1.2 and its association of the β2AR. PTT‐LTP is induced by prolonged stimulation at 5 Hz, which approximates the naturally occurring θ rhythm in the hippocampus (Mizuseki et al, 2009). Prolonged stimulation at the naturally occurring theta tetanus induces LTP (PTT‐LTP) if at the same time β adrenergic signaling is engaged (Thomas et al, 1996; Hu et al, 2007; Qian et al, 2012). PTT‐LTP is thought to be important for contextual learning under demanding situations (Hu et al, 2007). The β2AR–Cav1.2 signaling cascade might thus be important for such learning.
The finding that the association of the β2AR with Cav1.2 is critical for β adrenergic regulation of Cav1.2 has important further functional implications. Accordingly, the β2AR must be localized in the immediate vicinity of Cav1.2 for effective signaling. This signaling is clearly mediated by the cAMP/PKA cascade as the PKA‐specific inhibitory PKI peptide prevented the ISO‐induced upregulation of L‐type currents (Fig 7D). Loss of cAMP signaling from the β2AR to Cav1.2 upon their dissociation constitutes the first clear evidence for the hypothesis that cAMP signaling by certain GsPCR, especially the paradigmatic β2AR (Kuschel et al, 1999; Chen‐Izu et al, 2000; Davare et al, 2001; Balijepalli et al, 2006; Nikolaev et al, 2010), is mediated by cAMP production that is localized within nanodomains, that is, domains smaller than 100 nm in diameter. The reasoning for this notion is that the average distance between more or less evenly distributed β2ARs on the cell surface will not allow for regions devoid of β2ARs that are larger than 100 nm; likely, such regions are much smaller. We also exclude that ISO‐triggered endocytosis is playing a role in functional uncoupling of the β2AR from regulating Cav1.2 phosphorylation (Fig EV3). The localized regulation of Cav1.2 via cAMP signaling is consistent with earlier finding that in addition to AKAP150‐anchored PKA (Hall et al, 2007; Oliveria et al, 2007; Dittmer et al, 2014), Gs and adenylyl cyclase are also associated with Cav1.2 (Davare et al, 2001; Balijepalli et al, 2006).
Downregulation of β adrenergic augmentation of Cav1.2 activity might provide a brake necessary to ensure cell integrity, which could be jeopardized by the otherwise overpowering effects of a sustained increase in Ca2+ influx. In contrast, continued upregulation of GluA1 phosphorylation by prolonged β adrenergic stimulation might not be as detrimental because these receptors primarily conduct Na+ rather than Ca2+.
In conclusion, we demonstrate that S1928 phosphorylation of Cav1.2 upon β2AR stimulation results in a temporary dissociation of the β2AR from Cav1.2 with an equally fleeting but complete loss of Cav1.2 regulation by the β2AR. This novel potent negative feedback mechanism adds to the surprisingly diverse arsenal of tools the cell developed to curb overactivation of βAR signaling and Cav1.2.
Materials and Methods
Animals
Reagents, peptides, and antibodies
Isoproterenol bitartrate salt, ICI118551, CGP20712, and microcystin LR were from Sigma. Dynasore was from Tocris and Pitstop from Abcam. Protein A‐covered beads were from Repligen and Amylose beads from New England Biolabs. Polyvinyldifluoride (PVDF) membranes were from Millipore. Horseradish peroxidase‐coupled (HRP) protein A, ECL, and ECL plus reagents were from GE Healthcare. The chemiluminescent Femto substrate, EGTA, EDTA, Tween‐20, Triton X‐100, and tris(hydroxymethyl)aminomethane (Tris) were from Fisher Thermo Scientific. Other reagents were from the typical suppliers and of the usual quality.
All synthetic peptides (Appendix Table S1) were purchased from China Peptides (Shanghai, China). Origin and other details of antibodies are given in Appendix Table S2.
Fluorescence polarization (FP)
Fluorescein (FITC)‐labeled synthetic peptides (1 μM final conc.) were added to serial dilutions (8 times twofold, i.e., each time 1:1) of recombinant MBP‐tagged β2AR C‐terminus in FP buffer (50 mM HEPES, 100 mM KCl, 1 mM MgCl2, 0.05 mM EGTA, 5 mM NTA, pH 7.4) in black 384‐well polystyrene plates (Corning). FP was measured with a Synergy 2 (BioTek) plate reader with polarization filters to determine parallel and perpendicular fluorescence intensities of exciting (485/20λ) and emitted light (528/20λ). Data were acquired with Gen5 software. FP was calculated as P = (Iv − g*Ih)/(Iv + g*Ih); Iv and Ih constitute the vertical and horizontal fluorescence intensities, respectively, and g the correction factor for fluorescein. Data were analyzed with GraphPad Prism 5 for curve fitting and Kd determination by fitting binding curves to the equation Y = B*X/(Kd + X); B is the maximal FP value that would be reached at saturation as determined by extrapolation of the fitted curve (Lim et al, 2002).
Preparation of brain slices and use for biochemical analysis
Mice (8–12 weeks) were decapitated and brains placed into ice‐cold artificial cerebrospinal fluid (ACSF; in mM: 127 NaCl, 26 NaHCO2, 1.2 KH2PO4, 1.9 KCl, 2.2 CaCl2, 1 MgSO4 and 10 D‐glucose, 290–300 mOsm/kg, saturated with 95% O2, and 5% CO2; final pH 7.3). About one‐third of the rostral and caudal ends of the brain were trimmed off. A total of 350‐μm‐thick forebrain slices containing hippocampus were prepared with a vibratome (Leica VT 1000A). Slices were equilibrated in oxygenated ACSF for 1 h at 30°C before transfer to incubation chambers, equilibration for 30 min at 32°C and treatment with vehicle (H2O), ISO (10 μM). Slices were extracted with IP buffer containing protease and phosphatase inhibitors as above before IP of β2AR, α11.2, and anti‐GluA1 and IB as above.
Methods for immunoprecipitation (IP) and immunoblotting (IB) as well as pull‐down and fluorescence microscopy are standard, and details are given in Appendix Supplementary Methods. The Cav1.2 glutathione S‐transferase (GST)‐fusion proteins of α11.2 are described in Hall et al (2006, 2007, 2013) and listed in Appendix Table S3. The maltose‐binding protein (MBP)‐tagged C‐terminus (CT) of human β2AR (residues 326–413) is as in Joiner et al (2010).
Electrophysiology
Cell‐attached patch clamp recordings were performed as previously (Davare et al, 2001) with 500 nM (S)‐(−)‐BayK‐8644, 1 μM ω‐conotoxin GVIA, and 1 μM ω‐conotoxin MCVIIC in the patch pipette. Hippocampal slice recordings were basically as described (Qian et al, 2012). Exact details are given in Appendix Supplementary Methods.
Author contributions
TP, VDB, WAC, FH, YKX, GGM, CYC, MFN, and JWH designed experiments; TP, HQ, VDB, ZAM, DC, JLP, EAH, ORB, REW, CYC, and MFN performed experiments; TP, HQ, VDB, JLP, EAH, CYC, and MFN analyzed data; TP, MFN, and JWH wrote the manuscript.
Conflict of interest
The authors declare that they have no conflict of interest.
Acknowledgements
We thank Dr. Brian K. Kobilka, Stanford University, California, USA, for the pcDNA3.1‐FLAG‐β2AR construct and Angela Steinberger for providing primary cultures of hippocampal neurons and for excellent technical assistance. This work was supported by NIH grants R01 NS078792 and R01 MH097887 (JWH), R01 HL098200 and R01 HL121059 (MFN), R01 HL085372 (WAC), and R01 HL127764 (YKX), R01 AG028488 (GGM), American Heart Association (to YKX, who is an AHA Establish Investigator), Austrian Science Fund (FWF) grant P 25085 (VDB), and grants from Deutsche Forschungsgemeinschaft (FH).
Supporting Information
Appendix (Word 2007 document, 244.18 KB)
Expanded View Figures PDF (PDF document, 1.03 MB)
References
Balijepalli RC, Foell JD, Hall DD, Hell JW, Kamp TJ (2006) From the Cover: localization of cardiac L‐type Ca2+ channels to a caveolar macromolecular signaling complex is required for beta2‐adrenergic regulation. Proc Natl Acad Sci USA 103: 7500–7505
Berkefeld H, Sailer CA, Bildl W, Rohde V, Thumfart JO, Eble S, Klugbauer N, Reisinger E, Bischofberger J, Oliver D, Knaus HG, Schulte U, Fakler B (2006) BKCa‐Cav channel complexes mediate rapid and localized Ca2+‐activated K+ signaling. Science 314: 615–620
Berman DE, Dudai Y (2001) Memory extinction, learning anew, and learning the new: dissociations in the molecular machinery of learning in cortex. Science 291: 2417–2419
Bolshakov VY, Siegelbaum SA (1994) Postsynaptic induction and presynaptic expression of hippocampal long‐term depression. Science 264: 148–152
Cahill L, Prins B, Weber M, McGaugh JL (1994) Beta‐adrenergic activation and memory for emotional events. Nature 371: 702–704
Cao TT, Deacon HW, Reczek D, Bretscher A, von Zastrow M (1999) A kinase‐regulated PDZ‐domain interaction controls endocytic sorting of the beta2‐adrenergic receptor. Nature 401: 286–290
Carter ME, Yizhar O, Chikahisa S, Nguyen H, Adamantidis A, Nishino S, Deisseroth K, de Lecea L (2010) Tuning arousal with optogenetic modulation of locus coeruleus neurons. Nature Neurosci 13: 1526–1533
Chen‐Izu Y, Xiao RP, Izu LT, Cheng H, Kuschel M, Spurgeon H, Lakatta EG (2000) G(i)‐dependent localization of beta(2)‐adrenergic receptor signaling to L‐type Ca(2+) channels. Biophys J 79: 2547–2556
Daaka Y, Luttrell LM, Lefkowitz RJ (1997) Switching of the coupling of the beta2‐adrenergic receptor to different G proteins by protein kinase A. Nature 390: 88–91
Dai S, Hall DD, Hell JW (2009) Supramolecular Assemblies and Localized Regulation of Voltage‐gated Ion Channels. Physiol Rev 89: 411–452
Davare MA, Avdonin V, Hall DD, Peden EM, Burette A, Weinberg RJ, Horne MC, Hoshi T, Hell JW (2001) A beta2 adrenergic receptor signaling complex assembled with the Ca2+ channel Cav1.2. Science 293: 98–101
Davare MA, Dong F, Rubin CS, Hell JW (1999) The A‐kinase anchor protein MAP2B and cAMP‐dependent protein kinase are associated with class C L‐type calcium channels in neurons. J Biol Chem 274: 30280–30287
Davare MA, Hell JW (2003) Increased phosphorylation of the neuronal L‐type Ca(2+) channel Ca(v)1.2 during aging. Proc Natl Acad Sci USA 100: 16018–16023
Davare MA, Horne MC, Hell JW (2000) Protein Phosphatase 2A is associated with class C L‐type calcium channels (Cav1.2) and antagonizes channel phosphorylation by cAMP‐dependent protein kinase. J Biol Chem 275: 39710–39717
De Jongh KS, Murphy BJ, Colvin AA, Hell JW, Takahashi M, Catterall WA (1996) Specific phosphorylation of a site in the full length form of the a1 subunit of the cardiac L‐type calcium channel by adenosine 3′,5′‐cyclic monophosphate‐dependent protein kinase. Biochem 35: 10392–10402
Dittmer PJ, Dell'Acqua ML, Sather WA (2014) Ca(2+)/Calcineurin‐Dependent Inactivation of Neuronal L‐Type Ca(2+) Channels Requires Priming by AKAP‐Anchored Protein Kinase A. Cell Rep 7: 1410–1416
Dolmetsch RE, Pajvani U, Fife K, Spotts JM, Greenberg ME (2001) Signaling to the nucleus by an L‐type calcium channel‐calmodulin complex through the MAP kinase pathway. Science 294: 333–339
Efendiev R, Samelson BK, Nguyen BT, Phatarpekar PV, Baameur F, Scott JD, Dessauer CW (2010) AKAP79 interacts with multiple adenylyl cyclase (AC) isoforms and scaffolds AC5 and ‐6 to alpha‐amino‐3‐hydroxyl‐5‐methyl‐4‐isoxazole‐propionate (AMPA) receptors. J Biol Chem 285: 14450–14458
Ferguson SS, Downey WE 3rd, Colapietro AM, Barak LS, Menard L, Caron MG (1996) Role of beta‐arrestin in mediating agonist‐promoted G protein‐coupled receptor internalization. Science 271: 363–366
Fu Y, Westenbroek RE, Scheuer T, Catterall WA (2013) Phosphorylation sites required for regulation of cardiac calcium channels in the fight‐or‐flight response. Proc Natl Acad Sci USA 110: 19621–19626
Fu Y, Westenbroek RE, Scheuer T, Catterall WA (2014) Basal and beta‐adrenergic regulation of the cardiac calcium channel CaV1.2 requires phosphorylation of serine 1700. Proc Natl Acad Sci USA 111: 16598–16603
Fuller MD, Emrick MA, Sadilek M, Scheuer T, Catterall WA (2010) Molecular mechanism of calcium channel regulation in the fight‐or‐flight response. Sci Signal 3: ra70
Fuller MD, Fu Y, Scheuer T, Catterall WA (2014) Differential regulation of CaV1.2 channels by cAMP‐dependent protein kinase bound to A‐kinase anchoring proteins 15 and 79/150. J Gen Physiol 143: 315–324
Ganesan AN, Maack C, Johns DC, Sidor A, O'Rourke B (2006) Beta‐adrenergic stimulation of L‐type Ca2+ channels in cardiac myocytes requires the distal carboxyl terminus of alpha1C but not serine 1928. Circ Res 98: e11–e18
Goodman OB Jr, Krupnick JG, Santini F, Gurevich VV, Penn RB, Gagnon AW, Keen JH, Benovic JL (1996) Beta‐arrestin acts as a clathrin adaptor in endocytosis of the beta2‐adrenergic receptor. Nature 383: 447–450
Gray R, Johnston D (1987) Noradrenaline and beta‐adrenoceptor agonists increase activity of voltage‐dependent calcium channels in hippocampal neurons. Nature 327: 620–622
Grover LM, Teyler TJ (1990) Two components of long‐term potentiation induced by different patterns of afferent activation. Nature 347: 477–479
Hall DD, Dai S, Tseng P‐Y, Malik ZA, Nguyen M, Matt L, Schnizler K, Shephard A, Mohapatra D, Tsuruta F, Dolmetsch RE, Christel CJ, Lee A, Burette A, Weinberg RJ, Hell JW (2013) Competition Between α‐Actinin and Ca2+‐Calmodulin Controls Surface Retention of the L‐type Ca2+ Channel CaV1.2. Neuron 78: 483–497
Hall DD, Davare MA, Shi M, Allen ML, Weisenhaus M, McKnight GS, Hell JW (2007) Critical role of cAMP‐dependent protein kinase anchoring to the L‐type calcium channel Cav1.2 via A‐kinase anchor protein 150 in neurons. Biochem 46: 1635–1646
Hall DD, Feekes JA, Arachchige Don AS, Shi M, Hamid J, Chen L, Strack S, Zamponi GW, Horne MC, Hell JW (2006) Binding of protein phosphatase 2A to the L‐type calcium channel Cav1.2 next to Ser 1928, its main PKA site, is critical for Ser1928 dephosphorylation. Biochem 45: 3448–3459
Halt AR, Dallapiazza R, Yu H, Stein IS, Qian H, Junti S, Wojcik S, Brose N, Sliva A, Hell JW (2012) CaMKII binding to GluN2B is Critical During Memory Consolidation. EMBO J 31: 1203–1216
Hell JW (2010) {beta}‐adrenergic regulation of the L‐Type Ca2+ channel CaV1.2 by PKA rekindles excitement. Sci Signal 3: pe33
Hell JW, Westenbroek RE, Warner C, Ahlijanian MK, Prystay W, Gilbert MM, Snutch TP, Catterall WA (1993a) Identification and differential subcellular localization of the neuronal class C and class D L‐type calcium channel a1 subunits. J Cell Biol 123: 949–962
Hell JW, Yokoyama CT, Breeze LJ, Chavkin C, Catterall WA (1995) Phosphorylation of presynaptic and postsynaptic calcium channels by cAMP‐dependent protein kinase in hippocampal neurons. EMBO J 14: 3036–3044
Hell JW, Yokoyama CT, Wong ST, Warner C, Snutch TP, Catterall WA (1993b) Differential phosphorylation of two size forms of the neuronal class C L‐type calcium channel a1 subunit. J Biol Chem 268: 19451–19457
Hoogland TM, Saggau P (2004) Facilitation of L‐type Ca2+ channels in dendritic spines by activation of beta2 adrenergic receptors. J Neurosci 24: 8416–8427
Hu H, Real E, Takamiya K, Kang MG, Ledoux J, Huganir RL, Malinow R (2007) Emotion Enhances Learning via Norepinephrine Regulation of AMPA‐Receptor Trafficking. Cell 131: 160–173
Hulme JT, Lin TW, Westenbroek RE, Scheuer T, Catterall WA (2003) Beta‐adrenergic regulation requires direct anchoring of PKA to cardiac CaV1.2 channels via a leucine zipper interaction with A kinase‐anchoring protein 15. Proc Natl Acad Sci USA 100: 13093–13098
Hulme JT, Westenbroek RE, Scheuer T, Catterall WA (2006a) Phosphorylation of serine 1928 in the distal C‐terminal domain of cardiac CaV1.2 channels during beta1‐adrenergic regulation. Proc Natl Acad Sci USA 103: 16574–16579
Hulme JT, Yarov‐Yarovoy V, Lin TW, Scheuer T, Catterall WA (2006b) Autoinhibitory Control of the CaV1.2 Channel by its Proteolytically Processed Distal C‐terminal Domain. J Physiol 576: 87–102
Joiner ML, Lise MF, Yuen EY, Kam AY, Zhang M, Hall DD, Malik ZA, Qian H, Chen Y, Ulrich JD, Burette AC, Weinberg RJ, Law PY, El‐Husseini A, Yan Z, Hell JW (2010) Assembly of a beta(2)‐adrenergic receptor‐GluR1 signalling complex for localized cAMP signalling. EMBO J 29: 482–495
Jurevicius J, Fischmeister R (1996) cAMP compartmentation is responsible for a local activation of cardiac Ca2+ channels by beta‐adrenergic agonists. Proc Natl Acad Sci USA 93: 295–299
Kuschel M, Zhou YY, Cheng H, Zhang SJ, Chen Y, Lakatta EG, Xiao RP (1999) G(i) protein‐mediated functional compartmentalization of cardiac beta(2)‐adrenergic signaling. J Biol Chem 274: 22048–22052
Lemke T, Welling A, Christel CJ, Blaich A, Bernhard D, Lenhardt P, Hofmann F, Moosmang S (2008) Unchanged beta‐adrenergic stimulation of cardiac L‐type calcium channels in Ca v 1.2 phosphorylation site S1928A mutant mice. J Biol Chem 283: 34738–34744
Leonard AS, Davare MA, Horne MC, Garner CC, Hell JW (1998) SAP97 is associated with the a‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid receptor GluR1 subunit. J Biol Chem 273: 19518–19524
Leroy J, Abi‐Gerges A, Nikolaev VO, Richter W, Lechene P, Mazet JL, Conti M, Fischmeister R, Vandecasteele G (2008) Spatiotemporal dynamics of beta‐adrenergic cAMP signals and L‐type Ca2+ channel regulation in adult rat ventricular myocytes: role of phosphodiesterases. Circ Res 102: 1091–1100
Li H, Pink MD, Murphy JG, Stein A, Dell'Acqua ML, Hogan PG (2012) Balanced interactions of calcineurin with AKAP79 regulate Ca2+‐calcineurin‐NFAT signaling. Nat Struct Mol Biol 19: 337–345
Lim IA, Hall DD, Hell JW (2002) Selectivity and promiscuity of the first and second PDZ domains of PSD‐ 95 and synapse‐associated protein 102. J Biol Chem 277: 21697–21711
Lohse MJ, Benovic JL, Codina J, Caron MG, Lefkowitz RJ (1990) beta‐Arrestin: a protein that regulates beta‐adrenergic receptor function. Science 248: 1547–1550
Lu Y, Allen M, Halt AR, Weisenhaus M, Dallapiazza RF, Hall DD, Usachev YM, McKnight GS, Hell JW (2007) Age‐dependent requirement of AKAP150‐anchored PKA and GluR2‐lacking AMPA receptors in LTP. EMBO J 26: 4879–4890
Lu Y, Zha XM, Kim EY, Schachtele S, Dailey ME, Hall DD, Strack S, Green SH, Hoffman DA, Hell JW (2011) A kinase anchor protein150 (AKAP150)‐associated protein kinase A limits dendritic spine density. J Biol Chem 286: 26496–26506
Ma H, Groth RD, Cohen SM, Emery JF, Li B, Hoedt E, Zhang G, Neubert TA, Tsien RW (2014) gammaCaMKII Shuttles Ca(2+)/CaM to the Nucleus to Trigger CREB Phosphorylation and Gene Expression. Cell 159: 281–294
Marrion NV, Tavalin ST (1998) Selective activation of Ca2+‐activated K+ channels by co‐localized Ca2+ channels in hippocampal neurons. Nature 395: 900–905
Marshall MR, Clark JP 3rd, Westenbroek R, Yu FH, Scheuer T, Catterall WA (2011) Functional roles of a C‐terminal signaling complex of CaV1 channels and A‐kinase anchoring protein 15 in brain neurons. J Biol Chem 286: 12627–12639
Minzenberg MJ, Watrous AJ, Yoon JH, Ursu S, Carter CS (2008) Modafinil shifts human locus coeruleus to low‐tonic, high‐phasic activity during functional MRI. Science 322: 1700–1702
Mizuseki K, Sirota A, Pastalkova E, Buzsaki G (2009) Theta oscillations provide temporal windows for local circuit computation in the entorhinal‐hippocampal loop. Neuron 64: 267–280
Moosmang S, Haider N, Klugbauer N, Adelsberger H, Langwieser N, Muller J, Stiess M, Marais E, Schulla V, Lacinova L, Goebbels S, Nave KA, Storm DR, Hofmann F, Kleppisch T (2005) Role of hippocampal Cav1.2 Ca2+ channels in NMDA receptor‐independent synaptic plasticity and spatial memory. J Neurosci 25: 9883–9892
Murphy JG, Sanderson JL, Gorski JA, Scott JD, Catterall WA, Sather WA, Dell'Acqua ML (2014). AKAP‐anchored PKA maintains neuronal L‐type calcium channel activity and NFAT transcriptional signaling. Cell Rep 7, 1577–1588
Nichols CB, Rossow CF, Navedo MF, Westenbroek RE, Catterall WA, Santana LF, McKnight GS (2010) Sympathetic stimulation of adult cardiomyocytes requires association of AKAP5 with a subpopulation of L‐type calcium channels. Circ Res 107: 747–756
Nikolaev VO, Moshkov A, Lyon AR, Miragoli M, Novak P, Paur H, Lohse MJ, Korchev YE, Harding SE, Gorelik J (2010) Beta2‐adrenergic receptor redistribution in heart failure changes cAMP compartmentation. Science 327: 1653–1657
Nobles KN, Xiao K, Ahn S, Shukla AK, Lam CM, Rajagopal S, Strachan RT, Huang TY, Bressler EA, Hara MR, Shenoy SK, Gygi SP, Lefkowitz RJ (2011) Distinct phosphorylation sites on the beta(2)‐adrenergic receptor establish a barcode that encodes differential functions of beta‐arrestin. Sci Signal 4: ra51
Oliveria SF, Dell'acqua ML, Sather WA (2007) AKAP79/150 anchoring of calcineurin controls neuronal L‐Type Ca(2+) channel activity and nuclear signaling. Neuron 55: 261–275
Qian H, Matt L, Zhang M, Nguyen M, Patriarchi T, Koval ON, Anderson ME, He K, Lee H‐K, Hell JW (2012) β2 Adrenergic Receptor Supports Prolonged Theta Tetanus ‐ induced LTP. J Neurophysiol 107: 2703–2712
Reuter H (1983) Calcium channel modulation by neurotransmitters, enzymes and drugs. Nature 301: 569–574
Richter W, Mika D, Blanchard E, Day P, Conti M (2013) beta1‐adrenergic receptor antagonists signal via PDE4 translocation. EMBO Rep 14: 276–283
Roche KW, O'Brien RJ, Mammen AL, Bernhardt J, Huganir RL (1996) Characterization of multiple phosphorylation sites on the AMPA receptor GluR1 subunit. Neuron 16: 1179–1188
Sanderson JL, Dell'Acqua ML (2011) AKAP signaling complexes in regulation of excitatory synaptic plasticity. Neuroscientist 17: 321–336
Shenoy SK, Lefkowitz RJ (2011) beta‐Arrestin‐mediated receptor trafficking and signal transduction. Trends Pharmacol Sci 32: 521–533
Smith FD, Langeberg LK, Scott JD (2006) The where's and when's of kinase anchoring. Trends Biochem Sci 31: 316–323
Splawski I, Timothy KW, Sharpe LM, Decher N, Kumar P, Bloise R, Napolitano C, Schwartz PJ, Joseph RM, Condouris K, Tager‐Flusberg H, Priori SG, Sanguinetti MC, Keating MT (2004) Ca(V)1.2 calcium channel dysfunction causes a multisystem disorder including arrhythmia and autism. Cell 119: 19–31
Steinberg SF, Brunton LL (2001) Compartmentation of G protein‐coupled signaling pathways in cardiac myocytes. Annu Rev Pharmacol Toxicol 41: 751–773
Tavalin SJ, Colledge M, Hell JW, Langeberg LK, Huganir RL, Scott JD (2002) Regulation of GluR1 by the A‐kinase anchoring protein 79 (AKAP79) signaling complex shares properties with long‐term depression. J Neurosci 22: 3044–3051
Thomas MJ, Moody TD, Makhinson M, O'Dell TJ (1996) Activity‐dependent beta‐adrenergic modulation of low frequency stimulation induced LTP in the hippocampal CA1 region. Neuron 17: 475–482
Wang D, Govindaiah G, Liu R, De Arcangelis V, Cox CL, Xiang YK (2010) Binding of amyloid beta peptide to beta2 adrenergic receptor induces PKA‐dependent AMPA receptor hyperactivity. Faseb J 24: 3511–3521
White JA, McKinney BC, John MC, Powers PA, Kamp TJ, Murphy GG (2008) Conditional forebrain deletion of the L‐type calcium channel Ca V 1.2 disrupts remote spatial memories in mice. Learn Mem 15: 1–5
Xiao RP, Avdonin P, Zhou YY, Cheng H, Akhter SA, Eschenhagen T, Lefkowitz RJ, Koch WJ, Lakatta EG (1999a) Coupling of beta2‐adrenoceptor to Gi proteins and its physiological relevance in murine cardiac myocytes. Circ Res 84: 43–52
Xiao RP, Cheng H, Zhou YY, Kuschel M, Lakatta EG (1999b) Recent advances in cardiac beta(2)‐adrenergic signal transduction. Circ Res 85: 1092–1100
Yue DT, Marban E (1990) Permeation in the dihydropyridine‐sensitive calcium channel. Multi‐ion occupancy but no anomalous mole‐fraction effect between Ba2+ and Ca2+. J Gen Physiol 95: 911–939
von Zastrow M, Kobilka BK (1992) Ligand‐regulated internalization and recycling of human beta 2‐adrenergic receptors between the plasma membrane and endosomes containing transferrin receptors. J Biol Chem 267: 3530–3538
Zhang M, Patriarchi T, Stein IS, Qian H, Matt L, Nguyen M, Xiang YK, Hell JW (2013) Adenylyl cyclase anchoring by a kinase anchor protein AKAP5 (AKAP79/150) is important for postsynaptic beta‐adrenergic signaling. J Biol Chem 288: 17918–17931
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The EMBO Journal
Volume 35,Issue 12,Jun 2016
Cover: Confocal microscopy image of a fixed NIH 3T3 cell visualizing two types of membrane‐less organelles with liquid droplet behavior, nuclear speckles (red) and GFP‐Gli31‐455/SPOP‐positive bodies (green). Depending on the conditions, SPOP can be found in a variety of nuclear bodies, but it usually does not adopt a diffuse localization. When expressed alone, SPOP co‐localizes with nuclear speckles but when co‐expressed with its substrate Gli31‐455, it co‐localizes with it. Nuclear speckles were identified using an anti‐SC‐35 antibody and GFP‐Gli31‐455 localization was identified via GFP fluorescence. DAPI was used to stain the nucleus. From Melissa R Marzahn, Suresh Marada, Stacey K Ogden, Tanja Mittag and colleagues: Higher‐order oligomerization promotes localization of SPOP to liquid nuclear speckles. For details, see the Article on p 1254. Scientific image by Suresh Marada and the Cell & Tissue Imaging Center at St. Jude Children’s Research Hospital.
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Submission history
Received: 31 October 2015
Revision received: 23 March 2016
Accepted: 24 March 2016
Published online: 21 April 2016
Published in issue: 15 June 2016
Keywords
Notes
The EMBO Journal (2016) 35: 1330–1345
Copyright
© 2016 The Authors.
Authors
Research Funding
NIH: R01 NS078792, R01 MH097887, R01 HL098200, R01 HL121059, R01 HL085372, R01 HL127764, R01 AG028488
Austrian Science Fund: P 25085
Deutsche Forschungsgemeinschaft: DFG HO 579/24‐1
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