The PDZ-binding motif of the β₂-adrenoceptor is essential for physiologic signaling and trafficking in cardiac myocytes

Abstract

β₁- and β₂-adrenergic receptors (AR) regulate cardiac myocyte function through distinct signaling pathways. In addition to regulating cardiac rate and contractility, β₁AR and β₂AR may play different roles in the pathogenesis of heart failure. Studies on neonatal cardiac myocytes from β₁AR and β₂AR knockout mice suggest that subtype-specific signaling is determined by subtype-specific membrane targeting and trafficking. Stimulation of β₂ARs has a biphasic effect on contraction rate, with an initial increase followed by a sustained G_i-dependent decrease. Recent studies show that a PDZ domain-binding motif at the carboxyl terminus of human β₂AR interacts with ezrin-binding protein 50/sodium–hydrogen exchanger regulatory factor, a PDZ-domain-containing protein. The human β₂AR carboxyl terminus also binds to N-ethylmaleimide-sensitive factor, which does not contain a PDZ domain. We found that mutation of the three carboxyl-terminal amino acids in the mouse β₂AR (β₂AR-AAA) disrupts recycling of the receptor after agonist-induced internalization in cardiac myocytes. Nevertheless, stimulation of the β₂AR-AAA produced a greater contraction rate increase than that of the wild-type β₂AR. This enhanced stimulation of contraction rate can be attributed in part to the failure of the β₂AR-AAA to couple to G_i. We also observed that coupling of endogenous, wild-type β₂AR to G_i in β₁AR knockout myocytes is inhibited by treatment with a membrane-permeable peptide representing the β₂AR carboxyl terminus. These studies demonstrate that association of the carboxyl terminus of the β₂AR with ezrin-binding protein 50/sodium–hydrogen exchanger regulatory factor, N-ethylmaleimide-sensitive factor, or some related proteins dictates physiologic signaling specificity and trafficking in cardiac myocytes.

The β-adrenergic receptors (ARs) are essential for the physiologic regulation of cardiac function by the sympathetic nervous system (1), and may play important roles in the pathogenesis of heart failure (2). The three known subtypes of βARs (β₁AR, β₂AR, and β₃AR) belong to the large family of G protein-coupled receptors. Although all three subtypes have been detected in mammalian hearts (3), most of the functional responses to β-agonists can be attributed to β₁AR and β₂AR (3, 4). Although β₁AR and β₂AR respond to the same physiologic ligands (the hormone epinephrine and the neurotransmitter norepinephrine), they have distinct functional properties in vivo (5, 6). Specifically, β₁AR knockout mice lack the normal inotropic and chronotropic response to the adrenergic agonist isoproterenol (6), whereas these responses are preserved in β₂AR knockout mice (5).

When expressed in undifferentiated fibroblast cells, β₁AR and β₂AR exhibit similar signaling properties (7). However, recent studies suggest that these subtypes activate different signaling pathways in differentiated cells in vivo (8, 9). We have recently reported that β₁AR and β₂AR regulate the intrinsic contraction rate in mouse neonatal myocytes through different signal-transduction pathways (4). Activation of β₁ARs leads to a PKA-dependent increase in contraction rate. In contrast, activated β₂ARs undergo sequential coupling to G_s and G_i, which has a biphasic effect on contraction rate, with an initial PKA-independent increase followed by a sustained decrease that can be blocked by pertussis toxin (PTX) (4). The functional differences between β₁AR and β₂AR in cardiac myocytes are mediated in part by subtype-specific targeting of the receptors on the myocyte plasma membrane (10, 11). Immunofluorescence and membrane fractionation studies show that the β₂ARs are concentrated in caveolar structures, whereas the β₁ARs are mainly distributed in noncaveolar membrane in cardiac myocytes (11, 12). Disruption of caveolar structures in cardiac myocytes selectively enhances and prolongs the increase in myocyte contraction rate mediated by β₂AR activation, but has no effect on signaling by the β₁AR (12). Moreover, studies show that β₂AR signaling can modulate L-type Ca^2– channel activity in distinct subcellular microdomains in hippocampal neurons and cardiac myocytes (13–15). These observations suggest that subtype-specific signaling complexes conduct β₁AR and β₂AR signaling in cardiac myocytes.

A growing body of evidence from in vitro studies supports the notion that G protein-coupled receptors can form complexes with downstream effectors to facilitate signaling. Activated β₂AR recruits β-arrestin that scaffolds many trafficking molecules such as clathrin, AP-2, and ARF, and signaling molecules including the tyrosine kinase Src and mitogen-activated kinases c-Jun amino-terminal kinase and extracellular signal-regulate kinase 1/2 (16). β₂ARs can also associate with AKAP79 and Gravin, which are scaffolding proteins that connect β₂AR to PKA, PKC, PP2A, and L-type Ca^– channels (17–19). Moreover, interactions mediated by carboxyl-terminal PDZ-domain-binding motifs serve as another general mechanism to recruit G protein-coupled receptors into signaling complexes. Studies show that the β₁AR PDZ motif can interact with postsynaptic scaffolding proteins PSD-95 (synaptic associated protein 90) and membrane-associated guanylate-inverted 2 in HEK293 cells (20, 21). In cardiac myocytes, the interaction between β₁AR PDZ motif with PSD-95 or related proteins dictates signaling specificity by retaining the receptor at the cell surface and preventing interaction with G_i proteins (22).

It has been shown that the β₂AR carboxyl-terminal PDZ motif interacts with the Na^–/H^– exchanger regulatory factor [NHERF, also known as ezrin-binding protein 50 (EBP50) (23, 24)], which affects Na^–/H^– exchange in HEK293 cells (23). The β₂AR carboxyl terminus also binds to N-ethylmaleimide-sensitive factor (NSF) (25), which does not contain a PDZ domain. This interaction is critical for receptor recycling to the plasma membrane after agonist-induced internalization. However, the functional role of an interaction between the β₂AR carboxyl terminus and its associated protein(s) in cardiac myocytes has not been reported. To examine the role of the β₂AR carboxyl terminus on receptor trafficking and signaling in neonatal cardiac myocytes, we generated recombinant adenovirus expressing a mutant β₂AR in which the three carboxyl-terminal amino acids are mutated to alanine (β₂AR-AAA). This mutation is predicted to disrupt interactions with NSF- and PDZ-domain-containing proteins. We compared the functional properties of wild-type β₂AR and β₂AR-AAA in neonatal myocytes from β₁/β₂AR knockout (KO) mice. Our results demonstrate that the carboxyl terminus of the β₂AR is essential for physiologic signaling and trafficking in cardiac myocytes.

Materials and Methods

Culture and Adenovirus Infection of Neonatal Mouse Ventricular Myocytes. Spontaneously beating neonatal cardiac myocytes were prepared from hearts of 1-day-old mouse pups (from wild-type, β₁AR-KO, β₂AR-KO, and β₁/β₂AR-KO mice) as described (4). The myocyte-enriched cells remaining in suspension after preplating were plated in 35-mm dishes for contraction-rate studies, in 12-well plates for ELISA, in 12-well plates with coverslips for immunocytochemistry, or in 10-cm dishes for Western blot and ligand-binding assays as described (22).

Recombinant adenovirus encoding amino-terminal Flag-tagged mouse β₂AR-AAA (the β₂AR carboxyl terminus DSPL was mutated into DAAA) was generated with the pAdEasy system (Q.biogen, Carlsbad, CA); adenoviruses encoding HA-β₁AR-PDZmut and Flag-β₂AR were described (12, 22). Neonatal myocytes were infected with viruses at a multiplicity of infection of 100 after being cultured for 24 h as described (12).

Measurement of Myocyte Contraction Rate and cAMP Accumulation. Measurement of spontaneous contraction rate was carried out as described (4). In the time-course experiments, statistical significance between groups was analyzed with two-way analysis of variance with prism software (GraphPad, San Diego). Myocyte cAMP accumulation was determined by using a RIA as described (12).

Tat peptide, Tat-β₂-DSPL consisting of Tat linked to GRQGFSSDSPL of β₂AR, Tat-β₂-DAAA consisting of Tat linked to GRQGFSSDAAA, Tat-β₂-DSAL consisting of Tat linked to GRQGFSSDSAL, and Tat-β₂-ASPL consisting of Tat linked to GRQGFSSASPL through a cysteine bridge were synthesized in the Stanford Core facility. Neonatal myocytes were preincubated at 37°C with 1 μM peptide (Tat, Tat-β₂-DSPL, Tat-β₂-DAAA, Tat-β₂-DSAL, or Tat-β₂-ASPL) for 25 min, or filipin (2 μg/ml; Sigma) for 30 min before isoproterenol (10 μM; Sigma) exposure. In some assays, PTX (0.75 μg/ml; Sigma) or PKI (20 μM; Calbiochem) was used as described (12) before isoproterenol stimulation.

Immunofluoresence Microscopy and ELISA. Myocytes cultured on coverslips were infected with the Flag-β₂AR, Flag-β₂AR-AAA, or HA-β₁AR-PDZmut adenoviruses as described above. Myocytes were pulsed with 10 μM isoproterenol for 10 min. The cells were either fixed with 1× PBS containing 5% paraformaldhyde or washed three times before being chased for 15, 30, or 60 min with medium containing 1 μM aprenelol. The fixed cells were permeablized with 1× PBS containing 1% Nonidet P-40, and stained with anti-Flag M1 antibody (mouse monoclonal IgG_2b, 1:600 dilution; Sigma) or anti-HA 16B12 antibody (mouse monoclonal IgG₁, 1:600 dilution; Covance, Berkeley, CA). The primary antibodies were detected with FITC-conjugated goat anti-mouse IgG_2b (1:200) and Texas red-conjugated goat anti-mouse IgG₁ (1:400; Fisher). The images were acquired with a Zeiss Axioplan 2 microscope. Quantitative analysis of myocyte cell-surface receptors was carried out with ELISA as described (22).

Ligand Binding and Western Blot Analysis. Membrane proteins were prepared from adenovirus-infected β₁/β₂AR-KO myocytes as described (22). Saturation binding was carried out with 20 nM nonselective βAR antagonist [³H]dihydroalprenolol (NEN). Alprenolol (1 μM) was used to define nonspecific binding. In competition binding experiments, assay tubes contained 5 μg of membrane protein, 2 nM [³H]dihydroalprenolol, and different concentrations of the β₂AR antagonist ICI118551 or the βAR agonist isoproterenol. Membrane proteins were subjected to Western blot analysis with anti-Flag M1 antibody as described (22).

Results

Mutation of the Carboxyl-Terminal Three Amino Acids of the β₂AR Inhibits Receptor Coupling to G_i. To study the role of the β₂AR PDZ motif in receptor function in neonatal cardiac myocytes, we generated a recombinant adenovirus expressing a mutant β₂AR that lacks a functional PDZ motif (Flag-β₂AR-AAA). This mutation would also be predicted to disrupt interactions between the β₂AR and NSF (25). Flag-β₂AR and Flag-β₂AR-AAA were expressed in β₁/β₂AR-KO neonatal myocytes by recombinant adenovirus infection at a multiplicity of infection of 100. Both receptors could be efficiently expressed in neonatal myocytes, and the levels of the functional receptor expression were similar (Fig. 1A). Moreover, β₂AR-AAA and the wild-type β₂AR displayed similar binding affinities to the agonist isoproterenol and to the β₂AR-selective antagonist ICI118551 (Fig. 1B).

Fig. 1.

Functional expression of Flag-β₂AR or Flag-β₂AR-AAA in β₁/β₂AR-KO myocytes. (A) The expression of Flag-β₂AR and Flag-β₂AR-AAA protein in the β₁β₂AR-KO myocytes was examined by Western blot analysis (Left) and saturation binding (Right) to determine B_max.(B) The expression of Flag-β₂AR and Flag-β₂AR-AAA was characterized by competition binding assays with β₂AR agonist isoproterenol (IC₅₀ = 284 and 294 nM, respectively) and the selective antagonist ICI118551 (IC₅₀ = 3 and 3.5 nM, respectively). The data represent the mean ± SE of five experiments (triplicates) from different myocyte preparations. [³H]DHA, [³H]dihydroalprenolol; Iso, isoproterenol; ICI, ICI118551.

Fig. 2 shows the effect of isoproterenol on the contraction rate of β₁AR-KO myocytes or β₁/β₂AR-KO myocytes expressing either Flag-β₂AR or Flag-β₂AR-AAA. The activated Flag-β₂ARs induce a contraction rate response in the β₁/β₂AR-KO myocytes similar to that induced by the activated endogenous β₂AR in the β₁AR-KO myocytes. The response displayed an initial increase followed by a decrease to below the basal level (Fig. 2 A) (4). In comparison with the wild-type β₂AR, the activated β₂AR-AAA induced a much more robust contraction-rate increase, and the contraction rate did not drop below the basal level (Fig. 2B). We have previously shown that β₂ARs are enriched in the caveolar structures on myocyte plasma membrane. Filipin, a reagent that disrupts caveolar structure by binding cholesterol, selectively enhances and prolongs the contraction-rate increase mediated by β₂ARs, but not by β₁ARs (4). Disruption of caveolar structures with filipin also enhanced the contraction rate increase induced by the activated β₂AR-AAA (Fig. 2C). We also examined cAMP accumulation in the cultures of β₁/β₂AR-KO myocytes expressing either the Flag-β₂AR or the Flag-β₂AR-AAA. In contrast to the different contraction-rate responses, the cAMP accumulations by the activated receptors were not significantly different in the myocytes expressing either β₂ARs (basal, 10.6 ± 2.9 pmol per dish; isoproterenol, 35.6 ± 5.5 pmol per dish) or β₂AR-AAA (basal, 10 ± 3.8 pmol per dish; isoproterenol, 37.7 ± 5.8 pmol per dish).

Fig. 2.

The β₂AR PDZ mutation alters receptor regulation of contraction rate in cardiac myocytes. (A) The effect of 10 μM isoproterenol on the contraction rate of the β₁/β₂AR-KO myocytes expressing Flag-β₂AR or of the β₁AR-KO myocytes with endogenous β₂AR. (B) The effect of 10 μM isoproterenol on the contraction rate of β₁/β₂AR-KO myocytes expressing Flag-β₂AR-AAA or of the β₁AR-KO myocytes with endogenous β₂AR. (C) Disruption of caveolae with filipin enhances the increase in contraction rate after β₂AR-AAA stimulation. The data represent the mean ± SE of N experiments from at least three different myocyte preparations. *, P < 0.05; time-course curves were found to be significantly different by two-way analysis of variance.

The biphasic contraction-rate response mediated by the activated β₂AR is due to sequential coupling of the receptor to G_s and G_i (4). PTX, a G_i protein inhibitor, efficiently prevents the secondary decrease in contraction rate induced by activation of the β₂AR (Fig. 3A). The larger, monophasic contraction-rate response to stimulation of the β₂AR-AAA suggests a limited role of G_i signaling. Furthermore, pretreatment of myocytes with PTX had no significant effect on the contraction rate induced by the β₂AR-AAA (Fig. 3B). Thus, the β₂AR-AAA appears to couple only to G_s in β₁/β₂AR-KO neonatal myocytes. We also examined the role of PKA in the contraction-rate response induced by the wild-type β₂AR and the β₂AR-AAA. We have previously shown that PKI, a selective PKA inhibitor, has no significant effect on the β₂AR-mediated contraction-rate increase at concentrations that markedly inhibit β₁AR signaling (4). Thus, the β₂AR normally stimulates contraction rate through a PKA-insensitive mechanism, possibly through activation of the cAMP-gated, nonselective cation channel. PKI partially inhibits the maximum contraction-rate increase after β₂AR-AAA stimulation (Fig. 3C).

Fig. 3.

Disruption of the β₂AR PDZ motif inhibits receptor coupling to G_i. PTX (0.75 μg/ml) treatment selectively affected the contraction rate of the β₁/β₂AR-KO myocytes expressing Flag-β₂AR (A) but not Flag-β₂AR-AAA (B). (C) PKI partially inhibits the myocyte contraction-rate increase mediated by Flag-β₂AR-AAA, but not by Flag-β₂AR. The data represent the mean ± SE of four experiments from at least three different myocyte preparations. *, P < 0.05; time-course curves were found to be significantly different by two-way analysis of variance.

Mutation of the Carboxyl-Terminal Three Amino Acids of the β₂AR Disrupts Receptor Recycling After Endocytosis in Neonatal Myocytes. Immunostaining shows that both Flag-β₂AR and Flag-β₂AR-AAA are localized predominantly on the cell surface of neonatal myocytes at steady state (Fig. 4A). On isoproterenol stimulation, both receptors undergo significant internalization, as indicated by punctate intracellular staining (Fig. 4A). Quantitative ELISAs confirm that isoproterenol stimulation causes an equivalent (≈21–24%) decrease in cell-surface receptor density in the myocytes expressing either the Flag-β₂AR or the Flag-β₂AR-AAA (Fig. 4C). However, whereas the wild-type β₂AR efficiently recycles back to the cell surface, the mutant Flag-β₂AR-AAA fails to do so (Fig. 4 B and C). These results are consistent with previously published studies (24) and suggest that the β₂AR PDZ motif is required for the receptor recycling to the cell surface and for the receptor coupling to G_i in the myocytes. In contrast, mutation of the PDZ-binding motif in the β₁AR has very different consequences. Disruption of the PDZ motif in the HA-β₁AR (HA-β₁AR-PDZmut) dramatically enhances agonist-induced internalization and promotes coupling to G_i in the neonatal myocytes. In contrast to the β₂AR-AAA, the β₁AR-PDZmut efficiently recycles back to the cell surface after isoproterenol-induced internalization in myocytes (Fig. 4 B and C).

Fig. 4.

Agonist-induced internalization of Flag-β₂AR, Flag-β₂AR-AAA, and HA-β₁AR-PDZmut in neonatal cardiac myocytes. (A) Flag-β₂AR and Flag-β₂AR-AAA are localized on the cell surface in neonatal myocytes at steady state. Punctate intracellular staining is observed after agonist stimulation of both receptors. (B) Flag-β₂AR and HA-β₁AR-PDZmut efficiently recycle back to the myocyte cell surface after removal of isoproterenol, while the Flag-β₂AR-AAA remains inside the cell. (C) The cell-surface receptor level was measured by ELISAs after agonist-induced internalization and recycling. Cell-surface HA-β₁AR-PDZmut (11%), Flag-β₂AR (22%), and Flag-β₂AR-AAA (23%) decreased significantly after isoproterenol stimulation. Although the surface density of HA-β₁AR-PDZmut and Flag-β₂AR was restored by 60 min after removal of isoproterenol, the surface density of Flag-β₂AR-AAA remained low. Iso, isoproterenol; Alp, alprenolol.

A Membrane-Permeable Peptide Containing the β₂AR Carboxyl Terminus Alters β₂AR Signaling in Cardiac Myocytes. The experiments using adenovirus to express β₂AR-AAA in β₁/β₂AR-KO myocytes provide strong evidence for a functionally important interaction between the β₂AR and a PDZ-domain-containing protein, such as EBP50/NHERF, or another protein that binds to the β₂AR carboxyl terminus, such as NSF. To confirm these observations with endogenously expressed β₂AR, we used a membrane-permeable peptide representing the wild-type β₂AR carboxyl terminus (Tat-β₂-DSPL) to interfere with the interaction between the endogenous β₂ARs and their binding partners in β₁AR-KO myocytes. In comparison with the control cells, the Tat-β₂-DSPL-treated myocytes showed a greater maximum contraction-rate increase, and the contraction rate did not decrease below the basal level (Fig. 5A). In contrast, the control peptide, Tat-Flag-β₂-DAAA, had no significant affect on the contraction rate (Fig. 5B). Previous studies have shown that disruption of the carboxyl-terminal PDZ motif of the human β₂AR could alter binding to NHERF/EBP50, a PDZ-domain-containing protein, and NSF, a protein that lacks a PDZ domain (25). Interactions between these proteins could be distinguished by more selective mutations of the carboxyl terminus (25). We therefore examined the effect of two additional Tat-peptides, Tat-β₂-ASPL and Tat-β₂-DSAL. Based on the D410A mutation in the human β₂AR (25), Tat-β₂-ASPL would not be expected to bind NHERF/EBP50. We found that, like Tat-β₂-DAAA, Tat-β₂-ASPL had no effect on myocyte contraction rates (Fig. 5C). Based on the L412A mutation in the human β₂AR (25), Tat-β₂-DSAL would be expected to bind NHERF/EBP50, but not NSF. The carboxyl terminus of the mouse β₂AR (-DSPL) differs from the human (-DSLL), and it is not known whether the wild-type mouse β₂AR binds to NSF. Myocytes treated with Tat-β₂-DSAL exhibited the same behavior as myocytes treated with Tat-β₂-DSPL, showing a greater agonist-stimulated contraction rate (Fig. 5D). Thus, our results suggest that peptides that can bind NHERF/EBP50 can selectively modulate β₂AR-mediated signaling in cardiac myocytes by competing with the endogenous β₂ARs for binding to one or more PDZ-domain-containing proteins.

Fig. 5.

Cell-permeable β₂AR carboxyl-terminal peptides affect the contraction rate in β₁AR-KO myocytes. β₁AR-KO neonatal myocytes were cultured and treated with peptide (1 μM) for 25 min before contraction-rate experiments. The basal contraction rate of myocytes was not altered significantly by peptide treatment. Pretreatment with Tat-β₂-DSPL (A) and Tat-β₂-DSAL (D), but not Tat-β₂-DAAA (B) or Tat-β₂-ASPL (C), significantly changed the isoproterenol (Iso)-stimulated contraction-rate increase in β₁AR-KO myocytes. *, P < 0.05; time-course curves were found to be significantly different by two-way analysis of variance.

Discussion

β-Adrenergic receptors play critical roles in mediating physiologic responses to the hormone epinephrine and the neurotransmitter norepinephrine in animal hearts. Both β₁AR and β₂AR are expressed in cardiac myocytes and respond to the same stimuli, but they possess distinct functions in vivo. We have previously shown that β₁AR and β₂AR display different trafficking and signaling properties in neonatal myocytes. Activated β₁ARs remain on the cell surface and couple to G_s (22). In contrast, activated β₂ARs undergo robust endocytosis, and the receptors couple sequentially to G_s and G_i in cardiac myocytes (4). A mutation disrupting the interaction between the β₁AR PDZ motif and its binding partners enables agonist-induced receptor internalization (22). The same mutation also enables the receptor to couple sequentially to G_s and G_i after activation (22).

In this study, we examined the effect of mutating the carboxyl-terminal amino acids of the mouse β₂AR. Based on previous studies (25), β₂AR-AAA should not bind to NSF or PDZ domain proteins such as NHERF/EBP50. Stimulation of wild-type β₂AR induces a biphasic contraction-rate response in neonatal myocytes, with an initial G_s-dependent increase in contraction rate and a secondary G_i-dependent decrease in contraction rate below baseline. In contrast, stimulation of β₂AR-AAA leads to a larger, more sustained increase in contraction rate that does not decrease below baseline and is not altered by pretreatment of cells with PTX. These results are most consistent with the failure of β₂AR-AAA to couple to G_i. One must also consider the possibility that G_i coupling is masked by much more efficient coupling of β₂AR-AAA to G_s, thereby exceeding the cAMP levels needed for maximum contractionrate response. However, we found that it is possible to further stimulate contraction rate with forskolin after isoproterenol stimulation of β₂AR-AAA (data not shown). Thus, we feel our results are most consistent with a failure of β₂AR-AAA to couple to G_i. Moreover, the results of the Tat-peptide studies (Fig. 5) suggest that the failure of β₂AR-AAA to couple to G_i is caused by disruption of an interaction with a PDZ-domain-containing protein, such as NHERF/EBP50, and not by disruption of an interaction with NSF.

It has recently been reported that activation of G_s by βARs is involved in inducing myocyte apoptosis, whereas activation of G_i confers a protective effect against myocyte apoptosis (26, 27). The dual signaling mediated by β₂ARs in cardiac myocytes may represent a sophisticated mechanism enabling enhanced cardiac function in response to acute increases in sympathetic tone, but protecting myocytes from the detrimental effects of chronic catecholamine stimulation. Indeed, moderate overexpression of β₂AR in the heart of transgenic mice leads to an enhanced cardiac contractility without developing cardiomyopathy (28). In contrast, overexpression of the β₁AR leads to heart failure (29). Because the coupling of β₂ARs to G_i is inhibited by mutation of the PDZ binding motif, we might expect that a similar mutant expressed in vivo would have a significant and possibly detrimental effect in animal hearts under chronic stress.

Filipin is a reagent that disrupts caveolar structures in cardiac myocytes. Filipin does not affect the basal contraction rate and has no effect on β₁AR stimulation of contraction rate; however, it selectively enhances the contraction-rate increase after stimulation of β₂AR (12). In this study, the β₂AR-AAA-induced contraction-rate increase under isoproterenol stimulation is further enhanced by filipin. Thus, the PDZ motif may not be required for the targeting of the β₂AR to caveolae in cardiac myocytes. In contrast to the wild-type β₂AR, the contractionrate increase induced by β₂AR-AAA is partially inhibited by the PKA inhibitor PKI (Fig. 3C). This inhibition suggests that the enhanced coupling of the β₂AR-AAA to an increase in contraction rate is due in part to activation of a PKA-dependent pathway that is not activated by the wild-type β₂AR. This altered signaling may be explained by the inability of the β₂AR-AAA to associate with regulatory components of a signaling complex. β₂ARs associate with different scaffold proteins, including arrestin, A-kinase associate protein (AKAP), NSF, and the PDZ-domain-containing protein NHERF/EBP50 (16). EBP50 associates with an A-kinase-anchoring protein (AKAP) ezrin (23). A muscle-specific mAKAP can scaffold both PKA and a phospho-diesterase PDE4D in cardiac myocytes (30). Therefore, the signaling complexes containing the β₂ARs can orchestrate tight regulation of the second messenger cAMP within a plasma membrane microdomain (31). For example, disruption of the PDZ-mediated interaction between β₂ARs and PDE4 may allow cAMP to diffuse over a larger subcellular volume, thereby activating signaling pathways not normally activated by the wild-type β₂AR. We did not observe detectable differences in the whole-cell cAMP accumulation stimulated by the wild-type β₂AR and β₂AR-AAA. However, the cAMP studies were performed in myocytes that are preincubated with a phospho-diesterase inhibitor (3-isobutyl-1-methylxanthine).

In contrast to the inhibitory effect of mutating the β₂AR PDZ-binding motif on receptor coupling to G_i, mutation of the PDZ-binding motif in the β₁AR promotes coupling to G_i in the neonatal myocytes (12). The ability of these receptors to couple to G_i correlates with their propensity to undergo agonist-induced internalization and recycle back to the plasma membrane. The wild-type β₁AR does not undergo agonist-induced internalization, and the β₂AR-AAA cannot recycle back to the cell surface after internalization. In contrast, both the wild-type β₂AR and β₁AR-PDZmut undergo agonist-induced internalization and efficient recycling to the plasma membrane (Fig. 4C). The role of the PDZ-binding motif in subtype-specific trafficking has previously been demonstrated in HEK293 cells. The β₁AR PDZ motif has been shown to interact with two postsynaptic scaffold proteins, PSD-95 (synaptic associated protein 90) (20) and membrane-associated guanylate-inverted 2 (21), in HEK293 cells. In contrast, the β₂AR PDZ motif selectively associates with EBP50/NHERF (23, 24) and NSF (25). Although overexpression of PSD-95 in HEK293 cells inhibits agonist-induced internalization of the β₁AR (20), mutations disrupting of the interaction between the human β₂AR and NSF inhibit receptor recycling (25). However, it is noteworthy that a key residue (leucine 410) in human β₂AR necessary for NSF binding is not conserved in the mouse β₂AR used in this study. Therefore, we cannot be certain that disruption of interactions between NSF and the mouse β₂AR account for the failure of β₂AR-AAA to efficiently recycle after agonist-induced internalization (Fig. 4).

In conclusion, we present evidence that the β₂AR PDZ motif is essential for the physiologic signaling of this receptor subtype in neonatal mouse cardiac myocytes. Disruption of the β₂AR PDZ motif inhibited receptor recycling after isoproterenol-induced internalization and inhibited the receptor coupling to G_i. These results, together with our previous studies on the β₁AR, demonstrate that interactions between β-adrenergic receptors and specific PDZ-domain-containing proteins play an essential role in subtype-specific signaling of β-adrenergic receptors in cardiac myocytes. These interactions may serve to organize receptors, G proteins, effectors, and regulatory proteins into discrete signaling complexes in the plasma membrane (13). The formation of signaling complexes facilitates local, rapid, and highly specific cellular responses in vivo.