Targeting of Diacylglycerol Degradation to M1 Muscarinic Receptors by ß-Arrestins

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Science  02 Feb 2007:
Vol. 315, Issue 5812, pp. 663-666
DOI: 10.1126/science.1134562


Seven-transmembrane receptor (7TMR) signaling is transduced by second messengers such as diacylglycerol (DAG) generated in response to the heterotrimeric guanine nucleotide–binding protein Gq and is terminated by receptor desensitization and degradation of the second messengers. We show that β-arrestins coordinate both processes for the Gq-coupled M1 muscarinic receptor. β-Arrestins physically interact with diacylglycerol kinases (DGKs), enzymes that degrade DAG. Moreover, β-arrestins are essential for conversion of DAG to phosphatidic acid after agonist stimulation, and this activity requires recruitment of the β-arrestin–DGK complex to activated 7TMRs. The dual function of β-arrestins, limiting production of diacylglycerol (by receptor desensitization) while enhancing its rate of degradation, is analogous to their ability to recruit adenosine 3′,5′-monophosphate phosphodiesterases to Gs-coupled β2-adrenergic receptors. Thus, β-arrestins can serve similar regulatory functions for disparate classes of 7TMRs through structurally dissimilar enzymes that degrade chemically distinct second messengers.

Stimulation of 7TMRs activates heterotrimeric guanine nucleotide–binding proteins (G proteins), initiating the production of second messenger molecules. For 7TMRs coupled to the G protein family member Gs, activation of adenylyl cyclase increases intracellular adenosine 3′,5′-monophosphate (cAMP) concentrations; 7TMRs that couple to Gq/11 stimulate phospholipase C and consequent hydrolysis of phosphatidylinositol 4,5-bisphosphate to produce inositol 1,4,5-trisphosphate and diacylglycerol (DAG) (1). Proper regulation of signal transduction requires G protein inactivation, degradation of second messengers, and silencing of activated receptors (desensitization) to return the cell to a basal state. Deactivation of G proteins is achieved through the intrinsic guanosine triphosphatase (GTPase) activity of the α subunit with subsequent reassembly of the inactive heterotrimeric complex. However, unlike the autocatalytic G proteins, most second messenger molecules require specific enzymes for metabolism to an inactive form. For DAG, regulation is particularly crucial because dysregulation leading to prolonged DAG signaling is tumorigenic (2, 3).

The main pathway of DAG metabolism is phosphorylation by members of the family of diacylglycerol kinases (DGKs) (4). DGKs are predominantly cytoplasmic and translocate to the plasma membrane upon stimulation of many receptors, including 7TMRs. These enzymes catalyze the adenosine triphosphate (ATP)–dependent creation of phosphatidic acid (PA) through phosphorylation of the sn-3 position of DAG, thus negatively regulating DAG-dependent proteins such as protein kinase C (PKC). However, PA itself is a signaling molecule that influences vesicle trafficking (5), promotes translocation of the protein kinase Raf to the plasma membrane (6), and affects the activity of multiple enzymes, including type I phosphatidylinositol 5-kinases (7, 8), PKCζ (9), and small GTPase proteins (10).

Homologous desensitization of 7TMRs occurs through a highly conserved mechanism. The phosphorylation of cytoplasmic 7TMR serine and threonine residues by G protein–coupled receptor kinases promotes the translocation and binding of β-arrestin proteins, sterically hindering receptor–G protein coupling and diminishing G protein signaling (11). β-Arrestins interacting with AP-2 (12) and clathrin (13) then internalize activated receptors from the cell surface via clathrin-coated pits. β-Arrestins also recruit PDE4 phosphodiesterases to stimulated β2-adrenergic receptors, thereby accelerating the rate of cAMP degradation in a concerted mechanism of receptor desensitization and second messenger inactivation (14). Here, we investigated whether β-arrestins might function more generally to coordinate second messenger inactivation and assist DGKs to quench DAG signaling.

To test whether β-arrestins interact with DGKs, we transiently overexpressed hemagglutinin (HA) epitope–tagged DGK α, β, γ, δ, ϵ, ζ, or ι in COS7 cells along with FLAG epitope–tagged β-arrestin 1, β-arrestin 2, or pcDNA3 vector (Fig. 1A). Overnight incubation of cell lysates with resin coated with antibody to the FLAG epitope revealed coimmunoprecipitation of all seven HA-tagged DGKs with FLAG-tagged β-arrestin 1 and β-arrestin 2, but not with vector controls (Fig. 1A). Coimmunoprecipitation of endogenous β-arrestin 1 and 2 with HA-DGKζ verified this interaction (Fig. 1B). Stimulation of endogenous M1 receptors (M1R) with carbachol (50 μM) or direct stimulation of PKC with 100 nM phorbol ester had no effect on the amount of β-arrestin immunoprecipitated, which indicates that β-arrestins and DGKs can exist in a constitutively formed protein complex.

Fig. 1.

Coimmunoprecipitation of DGK isoforms and DAG kinase activity with β-arrestins. (A) Top and middle: Western blots of FLAG immunoprecipitates from COS7 cells overexpressing HA-tagged DGK isoforms cotransfected with FLAG β-arrestin 1 (βarr1), FLAG β-arrestin 2 (βarr2), or pcDNA3 vector. Bottom: Cell lysate immunoblots normalized for total protein, confirming construct expression. Images are representative of five independent experiments. (B) Western blots of immunoprecipitated HA-DGKζ (top) and coimmunoprecipitated endogenous β-arrestins (bottom) after stimulation with 50 μM carbachol or 1 μM phorbol ester, as indicated. Data are representative of three independent experiments (ev, empty vector; PMA, phorbol 12-myristate 13-acetate; Lys, HEK293 cell lysate). (C) In vitro DAG kinase assays were performed on FLAG–β-arrestin immunocomplexes as described (15). Data were normalized to empty vector transfections and represent the mean ± SE of four independent experiments. (D) Endogenous β-arrestins were immunoprecipitated with rabbit polyclonal antibodies A1CT or A2CT and analyzed as above. Data were normalized versus preimmune rabbit antiserum and represent the mean ± SE of five independent experiments. Statistical significance was determined by repeated-measures analysis of varriance (ANOVA) with a Bonferroni post hoc test to correct for multiple comparisons (*P < 0.05, **P < 0.01).

To examine the activity of β-arrestin–bound DGKs, we transiently transfected FLAG–β-arrestins and HA-DGKζ into human embryonic kidney HEK293 cells and tested FLAG immunoprecipitates for DAG kinase activity in vitro (15). The two β-arrestin isoforms were associated with similar amounts of DGK enzymatic activity (Fig. 1C). Endogenous β-arrestins immunoprecipitated with rabbit polyclonal antibodies to β-arrestin 1 and 2 [A1CT (16) and A2CT (16)] also showed significantly more associated endogenous DGK activity than did controls (Fig. 1D).

To map the site of β-arrestin binding to DGKs, we used a panel of DGKζ deletion mutants (17) to immunoprecipitate endogenous β-arrestins from HEK293 cells. Preliminary mapping implicated the N terminus in β-arrestin binding (fig. S1), and finer mapping of this region showed a requirement for the cysteinerich domains (CRDs) in this interaction (Fig. 2, A to C). This agrees with our data showing that β-arrestin interacts with multiple DGKs, as the CRDs are one of two elements conserved across the entire DGK family (the catalytic domain being the other). The CRDs are necessary for the translocation of DGKζ (18), and although DAG does not induce DGK translocation, receptor stimulation induces robust membrane recruitment (19); these observations are consistent with β-arrestin–mediated DGK trafficking.

Fig. 2.

Interaction of β-arrestins with the cysteinerich domains of DGKζ. (A) Diagram showing truncation sites of the DGKζ deletion mutants. CRD, cysteine-rich domain; MARCKS, myristoylated alaninerich C-kinase substrate domain. (B) A1CT blot of endogenous β-arrestins immunoprecipitated with DGKζ mutants. (C) The same immunoblot reprobed with antibody to DGKζ (15). (D) Western blot of β-arrestin 2 truncation mutants coimmunoprecipitated with HA-DGKζ. (E) Anti-HA probe of HA-DGKζ samples confirming comparable protein immunoprecipitation. (F) Expression levels of FLAG-tagged β-arrestin 2 mutants in HEK293 cell lysates. All panels are representative of at least four independent experiments.

The critical regions of β-arrestin required for DGKζ binding were determined using a similar approach with multiple β-arrestin 2 truncation mutants (Fig. 2, D to F). Deletion of either the N or C terminus of β-arrestin 2 was of little consequence. However, the mutant composed of amino acids 1 to 176 was not observed in HA-DGKζ immunoprecipitates. Therefore, the critical elements of DGKζ binding within β-arrestin 2 appear to be between residues 177 and 253, corresponding to an outer loop in the C-terminal half of β-arrestin 1 and 2.

To investigate the consequences of β-arrestin–DGK complexation in cells, we quantified carbachol-induced production of phosphatidic acid by whole-cell 32P labeling. Labeled lipid species were extracted from HEK293 cells transfected with pcDNA3 vector, FLAG–β-arrestin 1 or FLAG–β-arrestin 2 (Fig. 3A), then separated by thin-layer chromatography (16) (Fig. 3B). Quantification of 32P incorporation (Fig. 3C) revealed that, relative to nonstimulated cells, vector-transfected cells produced 4.8 ± 0.4 times as much radiolabeled PA with 5 min of endogenous M1R stimulation. Overexpression of either β-arrestin 1 or β-arrestin 2 increased agonist-induced PA levels to 7.2 ± 0.6 and 8.5 ± 0.9 times the basal state, respectively.

Fig. 3.

Positive influence of β-arrestin expression on carbachol-stimulated phosphatidic acid production. (A) Immunoblot for β-arrestins from HEK293 cells transfected with pcDNA3, FLAG-βarr1, or FLAG-β arr2. (B) Phosphorimager screen image of radiolabeled lipids extracted and separated by thin-layer chromatography from β-arrestin– and control-transfected cells with and without 50 μM carbachol stimulation of endogenous M1Rs for 5 min (CBC, carbachol; PI, phosphatidylinositol; PA, phosphatidic acid). (C) Quantification of carbachol-stimulated [32P]PA normalized to nonstimulated controls. Values shown represent the mean ± SE from eight independent experiments. Statistical significance was determined by one-way ANOVA with a Bonferroni post hoc test to correct for multiple comparisons (**P < 0.01 versus stimulated pcDNA3; ***P < 0.001 vs. stimulated pcDNA3). (D) Western blot of β-arrestins from HEK293 cells treated with β-arrestin–specific siRNA oligonucleotides. (E) Summary data of carbachol-stimulated [32P]PA production across β-arrestin siRNA treatments. Values shown represent the mean ± SE of five independent experiments. Statistical significance was determined by one-way ANOVA with a Bonferroni post hoc test to correct for multiple comparisons (*P < 0.05 versus stimulated control cells; ***P < 0.001 versus stimulated control cells; †P < 0.01 versus nonstimulated control cells). (F) Anti-GFP immunoblot showing expression of GFP-tagged rat β-arrestin 1 in HEK293 cells treated with siRNA specific for human β-arrestin 1. (G) Summary data of rescued [32P]PA production normalized to nonstimulated control siRNA transfections. Values shown represent the mean ± SE of three independent experiments. Statistical significance was determined by one-way ANOVA with a Bonferroni post hoc test to correct for multiple comparisons (*P < 0.05 versus nonstimulated cells; **P < 0.01 versus nonstimulated cells).

To further analyze the role of β-arrestins in M1R stimulation of DGK activity, we used small interfering RNA (siRNA) to deplete endogenous β-arrestins in HEK293 cells (16). Specific siRNAs reduced expression of β-arrestin 1 by ∼80% and β-arrestin 2 by ∼90% (Fig. 3D) and significantly affected the [32P]PA generated in response to carbachol stimulation (Fig. 3E). Relative to untreated, nonstimulated cells, cells transfected with nonsilencing control siRNA showed a factor of 2.8 ± 0.4 increase in the amount of PA produced after agonist treatment, whereas β-arrestin 2 siRNA-treated cells produced only 1.7 ± 0.2 times as much radioactive PA. The effects of depleting β-arrestin 1 were even greater, reducing basal PA concentrations to less than half (0.43 ± 0.08) of control and limiting the carbachol-stimulated response to 0.8 ± 0.1 of the control basal state.

To validate the RNA interference results, we investigated the effect of replenishing cells with exogenously expressed β-arrestin 1 after siRNA treatment. Rat β-arrestin 1 fused to green fluorescent protein (GFP) was expressed in cells transfected with siRNA targeting the human β-arrestin 1 sequence. Although β-arrestin 1 proteins from these species are 90% identical, siRNA directed against human β-arrestin 1 has no effect on expression of rat β-arrestin 1 because of six nucleotide mismatches within the 21-base sequence to which the siRNA hybridizes. With an increasing titration of rat GFP–β-arrestin 1 plasmid, there was a proportional rise in fusion protein expression (Fig. 3F) (to about 5 times the amount of endogenous β-arrestin 1) and a concomitant augmentation in the incorporation of 32P into PA (Fig. 3G and fig. S2B). GFP–β-arrestin 1 rescued 71% of the carbachol-stimulated PA (100 ± 0.5% increase over basal control cells) (Fig. 3G).

Next, we sought to dissociate the roles of β-arrestins in receptor desensitization from their DGK scaffolding function. We screened the β-arrestin 2 deletion mutants used previously for use as a dominant negative protein to inhibit DGK activity. The β-arrestin 2 163–410 mutant, henceforth referred to as βarr0N (β-arrestin 2, no N terminus), was robustly overexpressed, bound all isoforms of DGK tested in coimmunoprecipitation experiments (fig. S3A), and also competed with endogenous β-arrestins for binding to DGKs in a dose-dependent manner (fig. S3B). However, this mutant lacks N-terminal residues that are critical for receptor binding (20) and thus, as expected, failed to interact with agonist-stimulated 7TMRs (fig. S3C). Thus, βarr0N appeared to act as a dominant negative β-arrestin, retaining DGKs in the cytoplasm and prohibiting translocation of the β-arrestin–DGK complex to activated 7TMRs. Immunoblotting muscarinic receptor immunoprecipitates for cross-linked DGKζ revealed a carbachol-dependent association that was eliminated by βarr0N overexpression (Fig. 4A and fig. S3D). We compared HEK293 cells transfected with βarr0N to cells transfected with an equal amount of pcDNA3 vector. Extracts from control cells showed a factor of 4.0 ± 0.4 increase in DAG phosphorylation after agonist stimulation, whereas the βarr0N-transfected cells generated only a factor of 1.9 ± 0.3 increase (Fig. 4B and fig. S2C).

Fig. 4.

Dominant negative effect of βarr0N on DGK translocation to the M1 muscarinic receptor. (A) Western blots of dithiobis-maleimidoethane–cross-linked HA-M1R immunoprecipitates from HEK293 cells transfected with FLAG-DGKζ and βarr0N as described, with and without 50 μM carbachol stimulation for 5 min. (B) Summary data of three experiments showing the effect of βarr0N overexpression on agonist-induced [32P]PA production. Statistical significance was determined by a paired t test (*P < 0.05 versus stimulated pcDNA3).

Our results demonstrate the ability of β-arrestins to recruit DGKs to a ligand-activated Gq-coupled 7TMR. This finding is analogous to their previously discovered function in recruiting cAMP phosphodiesterases to the Gs-coupled β2-adrenergic receptor. However, the second messenger degrading enzymes involved in the Gs and Gq pathways are structurally and functionally unrelated. Thus, the data suggest a very general role for β-arrestins in coordinating second messenger degradation as well as receptor desensitization. Unlike adenosine 5′-monophosphate (the degradative product of phosphodiesterase action on cAMP), which is biologically inactive, PA resulting from DAG phosphorylation by DGKs has been implicated as an effector in many signaling pathways. Consequently, β-arrestin–mediated targeting of DGKs to 7TMRs may also serve as a critical molecular switch, turning off DAG-dependent signaling (such as PKC) while activating PA-sensitive pathways.

Supporting Online Material

Materials and Methods

Figs. S1 to S3

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