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A β2 Adrenergic Receptor Signaling Complex Assembled with the Ca2+ Channel Cav1.2

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Science  06 Jul 2001:
Vol. 293, Issue 5527, pp. 98-101
DOI: 10.1126/science.293.5527.98

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Abstract

The existence of a large number of receptors coupled to heterotrimeric guanine nucleotide binding proteins (G proteins) raises the question of how a particular receptor selectively regulates specific targets. We provide insight into this question by identifying a prototypical macromolecular signaling complex. The β2adrenergic receptor was found to be directly associated with one of its ultimate effectors, the class C L-type calcium channel Cav1.2. This complex also contained a G protein, an adenylyl cyclase, cyclic adenosine monophosphate–dependent protein kinase, and the counterbalancing phosphatase PP2A. Our electrophysiological recordings from hippocampal neurons demonstrate highly localized signal transduction from the receptor to the channel. The assembly of this signaling complex provides a mechanism that ensures specific and rapid signaling by a G protein–coupled receptor.

Neurons contain more than a thousand G protein–coupled receptors (GPCRs). The mechanism that preserves specificity in signaling by such an abundance of GPCRs remains obscure. G proteins and their cognate receptors and immediate downstream effectors, such as adenylyl cyclases, may be colocalized in subcellular microdomains (1, 2). Spatial proximity of these components may afford specific signal transduction, but evidence for the existence of preassembled macromolecular signaling complexes that would target defined GPCRs to their ultimate effectors is lacking. We show that the β2 adrenergic receptor (β2AR) is directly linked to one of its final effectors, the class C L-type Ca2+ channel Cav1.2. This receptor-channel complex also contains a G protein, an adenylyl cyclase, cyclic adenosine monophosphate–dependent protein kinase (PKA), and a counteracting phosphatase, PP2A.

β adrenergic signaling via adenylyl cyclase and PKA acutely increases L-type channel activity in the heart and brain (3, 4). The predominant L-type channel in the heart and brain is the class C channel Cav1.2 (5, 6). It consists of the central pore-forming α1C subunit and several auxiliary subunits including α2δ and β (6). Phosphorylation of α1C on Ser1928near the COOH-terminus and β (Ser478 and Ser479 in the β2a isoform) contributes to the up-regulation of channel activity by PKA (7–10). PKA is kept in close proximity to a variety of its substrates by A kinase (PKA) anchoring proteins (AKAPs) (11). The microtubule-associated protein MAP2B is an AKAP that recruits PKA to Cav1.2 in neurons (12). We hypothesized that the channel complex may assemble signaling components upstream of PKA, including GPCRs.

Immunoprecipitation of β2ARs from Triton X-100 extracts of rat forebrain and subsequent immunoblotting for α1Crevealed that the receptor was associated with Cav1.2 (Fig. 1A). The α1C immunoreactive band was a mixture of full-length long form and COOH-terminally truncated short form; these components are not completely resolved on 10% acrylamide gels (5, 7, 13). The COOH-terminal fragment, which results from posttranslational proteolytic processing and contains the PKA phosphorylation site Ser1928 (7, 13), stays physically and functionally associated with the channel in intact cells (14). The channel was not associated with the metabotropic glutamate receptors mGluR1 and mGluR5 (two GPCRs concentrated at postsynaptic sites), nor with caveolin 1 and 3 (two caveolae markers), nor with PSD-95 and SAP102 (two postsynaptic density markers) (Fig. 1, B to D). Next, Cav1.2 was immunoprecipitated from brain extract, auxiliary subunits and other associated proteins were removed by dissociation with SDS at 60°C, and α1C was reprecipitated (7, 12). The resulting immunocomplexes were incubated with glutathione S-transferase (GST) fusion proteins of the cytosolic domains of the β2AR (15). Immunoblotting with antibodies to GST showed that the COOH terminus of the β2AR specifically bound to α1C (Fig. 1E).

Figure 1

Direct interaction of the β2AR with Cav1.2. (A toD) Crude rat forebrain membranes were extracted with Triton X-100. Immunoprecipitations with antibodies specific for the β2AR (H-20, Santa Cruz Biotechnology), α1C(12, 17), mGluR1 and mGluR5 (Upstate Biotechnology), caveolin 1 and 3 (Transduction Laboratories), and nonspecific control antibody (Control) were followed by immunoblotting for β2AR, α1C, mGluR1 and mGluR5, caveolin 1 and 3, PSD-95 (31), and SAP102 (31), as indicated (12, 17). (E) α1C was purified by double immunoprecipitation (7, 12). The resulting α1C immunocomplex was incubated with bacterial lysates containing GST or GST fusion proteins of the intracellular domains of the β2AR (i.e., loops i1, i2, and i3 and the COOH terminus) (15). Association of α1C with the receptor fragment was detected by immunoblotting with anti-GST (bottom). Upper blot was probed for α1C. (Fto H) The β2AR and Cav1.2 consisting of α1C, α2δ, and β2a were coexpressed in HEK293 cells (16). (F) Immunoprecipitations were done with anti-α1C or control antibody before immunoblotting for the receptor, which exists in differentially glycosylated isoforms in HEK293 cells. (G) Immunofluorescent confocal microscopy of α1C and β2ARs. (H) Intact HEK293 cells were surface-biotinylated 24 hours after transfection (32). Triton X-100 extract was either directly loaded (20 μl, lane 5) or used for immunoprecipitation (500 μl) with antibodies to the β2AR (lanes 1 and 3), control antibodies (lanes 2 and 4), or PP2A/C (lanes 6 and 7). Blots were probed with antibodies to α1C (lanes 1 and 2, upper part), β2AR (lanes 1 and 2, lower part), or PP2A/C (lane 7) (17), stripped with SDS, and reprobed with horseradish peroxidase–streptavidin (lanes 3 to 6).

The β2AR coprecipitated and colocalized with Cav1.2 after ectopic coexpression in human embryonic kidney (HEK) 293 cells (Fig. 1, F and G) (16). We sought to determine whether the channel interacts with the receptor in the plasma membrane. Proteins on the surface of transfected HEK293 cells were biotinylated with a membrane-impermeable reagent. Membrane proteins were then solubilized and subjected to immunoprecipitation with antibodies to the β2AR, and α1C and the β2AR were identified by immunoblotting (Fig. 1H). Stripping and reprobing the blot with streptavidin showed that not only the β2AR but also the coprecipitating α1Cwas thoroughly biotinylated (Fig. 1H). PP2A is an abundant protein present throughout the cell and also specifically interacts with α1C in brain and HEK293 cells ectopically expressing Cav1.2 (17). After immunoprecipitation of PP2A, the catalytic C subunit of PP2A showed a strong signal by immunoblotting, but biotinylation was not detectable (Fig. 1H). Accordingly, the biotinylation reagent had no access to intracellular proteins.

Assembly of a β2AR-Cav1.2 complex may locally restrict signaling from the receptor to the channel. We recorded channel activity in the cell-attached patch configuration from neurons in 2- to 3-week-old primary hippocampal cultures (18), which show largely L-type channel activity (19). When the β2 adrenergic agonist albuterol was applied outside of the recording pipette to the bath (after recording under control conditions with no agonist), no statistically significant increase in channel activity was observed (Fig. 2, A to C and G). Subsequent bath application of the general β adrenergic agonist isoproterenol raised the L-type channel open probability NPopen in the patch, presumably via β1 adrenergic receptors (β1ARs), which, as in the heart (20), may result in a more widespread activation of PKA.

Figure 2

Highly localized up-regulation of L-type channel activity by β2ARs in neurons. Cell-attached patch clamp recording of L-type channel activity from primary hippocampal cultures during 100-ms test pulses from –80 mV to 0 mV (18). (A) Sweeps show channel activity (downward deflection) before (left) and after addition of 20 μM albuterol (middle) and then 50 μM isoproterenol (right) to the bath. (D) Sweeps 1 and 12 min after seal formation with a pipette backfilled with 20 μM albuterol. For each condition, ensemble averages of 100 sweeps (B and E) and open probability NPopen versus time (C andF) are shown [center line in (C), mean NPopenaveraged over each experimental condition; center line in (F), running average calculated at each time point from 50 consecutive sweeps]. (G) Current sizes from an average of 100 sweeps after bath (left, n = 9) or pipette (right, n = 14) application of albuterol normalized to those before the application. Mean values are significantly different (p < 0.05, t test). (H) Sweeps 1 min (black) and 12 min (red) after seal formation with a pipette backfilled with albuterol in the presence of 100 nM ωCTx GVIA, 100 nM ωCTx MVIIC, and 1 μM BayK8644. Amplitude histograms from 50 sweeps at the beginning (black) and end (red) of the experiment are shown at bottom. (I) Ensemble averages of 50 consecutive sweeps over the time course of the experiment shown in (H). (J) Current amplitudes measured from ensemble averages with albuterol either applied to the bath (n = 10; open circles) or by pipette backfilling (n = 10; closed circles) versus time with 100 nM ωCTx GVIA, 100 nM ωCTx MVIIC, and 1 μM BayK8644 present. Current amplitudes were normalized to the first value (1 at t = 0) obtained after bath application of albuterol or after seal formation in backfilling experiments (bars, SEM). Mean values after 700 s were significantly different (p < 0.05,t test).

To evaluate whether localized stimulation of the β2AR within the patch would increase channel activity, we partially filled the tip of the recording pipette without agonist and then backfilled it with agonist. During 10 of 14 recordings, channel activity significantly increased within the first few minutes of seal formation as the drug diffused to the pipette tip (Fig. 2, D to G). If no agonist was present, channel activity was either stable or declined slightly (Fig. 2C, control condition) (21). Openings of the Ca2+ channels observed in our experiments were prolonged by the L-type channel agonist BayK8644 in the presence of non–L-type channel blockers ωCTx GVIA and ωCTx MVIIC (Fig. 2H), confirming the identity of these Ca2+ channels as L-type. The channel activity observed with BayK8644 was significantly up-regulated over time by albuterol when applied inside the recording pipette, but bath application of albuterol had no effect (Fig. 2, H to J). Single-channel amplitudes were not affected by albuterol (Fig. 2H, bottom). These results show that β2 adrenergic stimulation can strongly increase L-type channel activity only if it occurs in the vicinity of the channel.

We next sought to determine where β2ARs and Cav1.2 are colocalized in vivo. Rat hippocampal sections were double-labeled for immunofluorescence microscopy (22); in the CA1 region, most cells and dendrites in which Cav1.2 was detectable also had β2ARs (Fig. 3A). In dendrites stained with the membrane tracer DiA (blue) in addition to the antibody labeling, β2AR (red) and α1C (green) colocalized at tips of dendritic spines (postsynaptic sites of excitatory synapses; Fig. 3B). Extensive colocalization was also observed on the surface of the soma (Fig. 3C). Earlier immunoelectron microscopy had suggested a postsynaptic localization for both proteins (13,23). Triple labeling with antibodies to the presynaptic marker synaptophysin confirmed that the observed colocalization of the receptor and Cav1.2 occurs at axodendritic and axosomatic synapses (Fig. 3D) (21); in dendrites, 80 ± 3% of synaptic β2 AR clusters contained Cav1.2 and 77 ± 3% of synaptic Cav1.2 clusters had β2ARs (22). This observation suggests that Cav1.2 may often, but not always, be associated with β2ARs.

Figure 3

Synaptic colocalization of the β2AR with Cav1.2. Rat hippocampal sections were stained with antibodies to the β2AR (red) and α1C (green) (22). (A) Colocalization of the β2AR and Cav1.2 in pyramidal cell bodies and apical dendrites in the CA1 region, as indicated by the yellow color from overlay of the red and green signal. (B to D) Apical dendrite (B) and cell body [(C) and (D); asterisk marks cell interior] in CA1 stained for the receptor and α1C and with the membrane tracer DiA to outline the dendritic shaft studded with spines [blue in (B)] or for synaptophysin [blue in (D)] to identify synapses. Arrowheads indicate spines (B) or axosomatic synapses [(D); arrow on right indicates an adjacent axodendritic synapse] on which both β2AR and α1C are detectable. Arrowheads in (C) point to examples of puncta surrounding a cell body in CA1 that are immunoreactive for both β2AR and α1C. Scale bars, 50 μm (A), 2 μm (B), 5 μm (C), 1 μm (D).

Like Cav1.2 and β2ARs (13, 23), adenylyl cyclase is concentrated at postsynaptic sites in the hippocampus (24). We tested whether adenylyl cyclase and G proteins are also components of the β2AR-Cav1.2 complex. Immunoprecipitation of α1C and adenylyl cyclase was followed by immunoblotting with an antibody to adenylyl cyclase (25). The antibody recognized several isoforms indicated by multiple bands around the 200-kD region of the blot, including a doublet at the top of the blot (Fig. 4). This doublet was also present in the channel complex. We have not identified the adenylyl cyclase isoforms in the channel complex.

Figure 4

Association of G proteins and adenylyl cyclase with the β2AR-Cav1.2 complex. Triton X-100 extracts of rat forebrain membranes were used for immunoprecipitations (12, 17) with anti-α1C, nonspecific control IgG from rabbit, and an antibody that recognizes multiple adenylyl cyclases (25) as indicated, followed by immunoblotting (12, 17) with the adenylyl cyclase antibody (top) or antibodies (28) to Gαs (middle) and Gβ (bottom).

Earlier findings indicated that Gαs and Gβ copurify with adenylyl cyclase (26) and that the Gβγ dimer can directly bind to various GPCRs (27, 28). Beyond short catalytic interactions of G proteins with GPCRs and adenylyl cyclases, G proteins may be tied up in stable interactions that keep them in the immediate vicinity of signaling complexes for rapid and specific signal transduction (1, 2). Indeed, Gαs and Gβ specifically coprecipitated not only with the adenylyl cyclase but also with Cav1.2 (Fig. 4). In addition, PKA and the phosphatase PP2A are associated with Cav1.2 and phosphorylate and dephosphorylate α1C, respectively (7, 8, 12, 17). Accordingly, Cav1.2 assembles a signaling complex that consists of the β2AR, trimeric G proteins, adenylyl cyclase, PKA, and the counterbalancing PP2A. A similar complex might exist in the heart, where activation of β1ARs results in PKA-mediated phosphorylation of substrates distributed throughout the cell, whereas stimulation of β2ARs acts more selectively on Cav1.2 (29). Our electrophysiological studies indicate that signaling from the β2AR to the channel is spatially highly restricted in neurons. Colocalization of GPCRs with their ultimate targets in macromolecular complexes could be a general mechanism to ensure that signaling is both specific and fast.

  • * Present address: Vollum Institute, Oregon Health Sciences University, Portland, OR 97201, USA.

  • Present address: Department of Pharmacology, University of Iowa, Iowa, City, IA 52242, USA.

  • To whom correspondence should be addressed. E-mail: johannes-hell{at}uiowa.edu

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