The α2 adrenergic receptor (AR) subtypes are important for blood pressure control. When activated, the α2A subtype elicits a hypotensive response whereas the α2B subtype mediates a hypertensive effect that counteracts the hypotensive response by the α2A subtype. We have previously shown that spinophilin attenuates the α2AAR-dependent hypotensive response; in spinophilin null mice, this response is highly potentiated. In this study, we demonstrate that spinophilin impedes arrestin-dependent phosphorylation and desensitization of the α2BAR subtype by competing against arrestin binding to this receptor subtype. The Del301-303 α2BAR, a human variation that shows impaired phosphorylation and desensitization and is linked to hypertension in certain populations, exhibits preferential interaction with spinophilin over arrestin. Furthermore, Del301-303 α2BAR-induced ERK signaling is quickly desensitized in cells without spinophilin expression, showing a profile similar to that induced by the wild type receptor in these cells. Together, these data suggest a critical role of spinophilin in sustaining α2BAR signaling. Consistent with this notion, our in vivo study reveals that the α2BAR-elicited hypertensive response is diminished in spinophilin deficient mice. In arrestin 3 deficient mice, where the receptor has a stronger binding to spinophilin, the same hypertensive response is enhanced. These data suggest that interaction with spinophilin is indispensable for the α2BAR to elicit the hypertensive response. This is opposite of the negative role of spinophilin in regulating α2AAR-mediated hypotensive response, suggesting that spinophilin regulation of these closely related receptor subtypes can result in distinct functional outcomes in vivo. Thus, spinophilin may represent a useful therapeutic target for treatment of hypertension.
Citation: Che P, Chen Y, Lu R, Peng N, Gannon M, Wyss JM, et al. (2015) Spinophilin Is Indispensable for the α2B Adrenergic Receptor-Elicited Hypertensive Response. PLoS ONE 10(8): e0135030. doi:10.1371/journal.pone.0135030
Editor: Adriano Marchese, Loyola University Chicago, Stritch School of Medicine, UNITED STATES
Received: April 16, 2015; Accepted: July 16, 2015; Published: August 5, 2015
Copyright: © 2015 Che et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Data Availability: All relevant data are within the paper.
Funding: This research is supported by National Institute of Mental Health grant MH081917 to QW. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The discovery of a plethora of G protein-coupled receptor (GPCR) interactions with non-G protein partners has led to the view that GPCRs do not function in isolation, but in complex protein networks which impact receptor trafficking, signaling and pharmacology. Among GPCR interacting proteins, G protein-coupled receptor kinases (GRKs) and arrestins are considered “universal regulators” and are the most extensively studied (e.g. recently reviewed in [1–6]). GRK-catalyzed phosphorylation, which leads to subsequent arrestin binding, represents a major mechanism for homologous desensitization of GPCRs. In addition to terminating G protein coupling, β-arrestins (arrestin 2 and 3) mediate receptor trafficking and scaffold cellular signaling cascades. Our previous studies identified spinophilin as an endogenous antagonist of arrestin, impeding multiple arrestin-mediated regulations of the receptor in cultured cells and in vivo . Spinophilin is a ubiquitously expressed protein, and competes against arrestin for binding to the α2A adrenergic receptor (AR) third intracellular (3i) loop . Interestingly, multiple α2AAR-elicited central responses are dampened in arrestin 3 deficient mice, but these responses are potentiated in spinophilin deficient mice where arrestin functions are unimpeded [7, 9, 10]. This suggests that arrestin 3 promotes, whereas spinophilin attenuates, α2AAR-dependent processes in vivo.
Although closely related to the α2AAR, the α2BAR subtype exhibits distinct trafficking profiles [11–14] and induces separate physiological responses [15–17] from the α2AAR. In response to α2 adrenergic ligands, the α2BAR mediates the hypertensive effect , which counteracts the hypotensive response elicited by the α2A subtype . The α2BAR is also required for development of salt-induced hypertension [20–22]. In humans, a common polymorphism of the α2BAR gene, Del301-303, has been linked to early onset hypertension in a Swedish population [23, 24]. Unlike the α2A subtype, the α2BAR 3i loop contains a highly acidic stretch of amino acids (aa294-309 ), which promotes GRK phosphorylation [25, 26]. Consistently, the Del301-303 α2BAR exhibits reduced phosphorylation and desensitization profiles . The α2BAR interacts with both β-arrestins [13, 28] and spinophilin . How spinophilin and β-arrestins regulate the α2BAR-mediated in vivo responses and how Del301-303 may affect the receptor’s interaction with these proteins remain to be investigated.
In the present study, we demonstrated that spinophilin impeded β arrestin-dependent α2BAR phosphorylation and desensitization by competing against arrestin binding to the receptor. Compared to the wild type (WT) α2BAR, the Del301-303 α2BAR exhibited diminished binding affinity to arrestin 3 but enhanced interaction with spinophilin. Moreover, ERK signaling induced by this polymorphic variant was prolonged. Intriguingly, Del301-303 α2BAR-induced ERK signaling was quickly desensitized in cells without spinophilin expression, showing a profile similar to that induced by the WT receptor in these cells. Together, these data suggest a critical role of spinophilin in sustaining α2BAR signaling. Furthermore, the α2BAR-elicited hypertensive response is diminished in spinophilin deficient mice, but the same response is enhanced in arrestin 3 deficient mice where the α2BAR has a stronger binding to spinophilin. These data strongly suggest that the interaction with spinophilin is indispensable for α2BAR to elicit the hypertensive response.
Methods and Materials
Reagents and drugs
Rat anti-HA rat monoclonal antibody (Roche); mouse HA.11 monoclonal antibody (Covance); rabbit anti-spinophilin antibody (Upstate); phospho- and total-p42/44 antibodies (Cell Signaling Technology); rabbit anti-GFP monoclonal antibody (Santa Cruz Biotechnology); mouse anti-myc antibody (Clontech); rabbit anti-GRK2 polyclonal antibody (Santa Cruz Biotechnology); anti-mouse IRDye 800CW and anti-rabbit IRDye 680RD (LI-COR) immobilized protein G-agarose (Pierce); arrestin 3 polyclonal antibodies (generously provided by Dr. Benovic, Thomas Jefferson University). All other chemicals were reagent-grade, and were purchased from Sigma-Aldrich or Fisher Chemicals.
Spinophilin deficient (Sp-/-), arrestin 3 deficient (Arr3-/-) and their respective corresponding wild type (WT) mice in the same genetic background were obtained and maintained as described previously . Mice were housed in the Association for Assessment and Accreditation of Laboratory Animal Care-accredited Animal Resources Program at the University of Alabama at Birmingham. Experimental procedures are in accordance with Animal Welfare Act and the 1989 amendments to this Act. All protocols were approved by University of Alabama Institutional Animal Care and Use Committee.
HEK293 and CosM6 cells were originally from American Type Culture Collection (ATCC). Immortalized arrestin 2 and 3 double knockout (Arr2,3-/-) and the corresponding WT (Arr2,3+/+) mouse embryonic fibroblasts (MEFs) were generated  and generously provided by Dr. Lefkowitz’s laboratory. Sp-/- and the corresponding Sp+/+ MEFs were generated previously as described in . Cells were cultured in 5% CO2 at 37°C in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 100 units/ml of penicillin and 10 μg/ml of streptomycin (Invitrogen). For mouse embryonic fibroblasts (MEFs), 2mM glutamine was added to the culture medium. Parental cell lines used in this study have no detectable endogenous expression of α2AR subtypes.
Plasmid and primers
Constructions of plasmids pGFP-Arr3 , pCMV4-Myc-Sp [29, 32, 33], and pcDNA3-GRK2  were described previously. The pGFP-Arr3-R170E construct expressing arrestin 3 with R to E mutation at R170 was generated by quickchange PCR mutagenesis using a primer pair, 5’-CTCAGGAGCAAACTGTACCTTCTCGATGATAAGCCGCACGGAGTT and 5’- AACTCCGTGCGGCTTATCATCGAGAAGGTACAGTTTGCTCCTGAG. pcDNA3.1- HA-α2B expressing wild type human α2BAR with N-terminal 3xHA tag was purchased from UMR cDNA Resource Center (clone ID: AR0A2BTN00). pcDNA3.1-HA-Del301-303 expressing N-terminal tagged human α2BAR with deletion of amino acid 301–303 was generated by overlap PCR mutagenesis using two primer pairs, the 5’-CGGGGTACCACCATGTACCCATACGATGTT and 5’-ACACTCTTCCTCCTCCTCCTCCTCCTCTTCAGCTTCATCCT pair, and the 5’-GAGGATGAAGCTGAAGAGGAGGAGGAGGAGGAGGAAGAGTG and 5’-ATACCGCTCGAGTCACCAGGCCGTCTGGGTCC pair. All constructs were confirmed by sequencing.
Cells were transfected with indicated plasmid using Lipofectamine 2000 (invitrogen) according to manufacturer’s instruction. MEFs were transduced with a retroviral construct encoding HA-α2BAR as described previously [31, 34].
Intact cell receptor phosphorylation
Intact cell phosphorylation was described previously . Briefly, cells were incubated for 1 hr with [32p] orthophosphate (0.1mCi/ml) in phosphate-free, serum-free DMEM at 37°C. Following agonist treatment, cells were harvested in lysis buffer (1% Triton x-100, 0.05% SDS, 1mM EDTA, 1mM EGTA, 10mM NaF, 10mM sodium pyrophosphoate, and protease inhibitors). Detergent-soluble extracts were then subjected to immunoprecipitation assay with a rat anti-HA antibody.
Co-immunoprecipitation was performed as described [8, 35]. In brief, cells were harvested in ice-cold lysis buffer (10 mM Tris HCl, pH 8.0, 0.3% Nonidet P-40, 10% glycerol, 5 mM EDTA, 5 mM EGTA supplemented with protease inhibitors). Soluble extracts were then subjected to immunoprecipitation assays using a rat anti HA antibody.
Kinase activation for ERK1/2 was determined by measuring the level of phosphorylated kinase and normalizing the value to the total protein level of ERK1/2. Phospho- and total-ERK1/2 were detected and analyzed by LI-COR odyssey Fc dual-mode western blot system. Mouse anti-phospho-p44/42 MAPK (T202/Y204) (Cell signaling) and donkey anti-mouse IRDye 800CW (LI-COR) were used to detect activation of ERK1/2 by green fluorescence channel. Rabbit anti-p44/42 MAPK (Cell signaling) and goat anti-rabbit IRDye 680RD (LI-COR) were used to detect total ERK1/2 by red fluorescence channel.
Measurement of cardiovascular responses
Measurement of cardiovascular responses was performed as described previously . Mice were anesthetized with 100mg/kg ketamine and 10 mg/kg xylazine. Catheterized left femoral artery was used to measure arterial pressure while the right jugular vein was used for anesthetic administration. Arterial blood pressure was recorded with a pressure transducer (BIOPAC’s AcqKnowledge 3.8.2, BioPac, Goleta, CA) continually in conscious and free moving mice 24 hr after the surgery. Twenty minutes after the baseline measurement, 0.1mg/kg UK14,304 was administered through bolus injection into the right jugular vein.
Interactions of the α2BAR with spinophilin and β-arrestins are mutually exclusive
We first confirmed that β-arrestins and spinophilin compete for interaction with the α2BAR in cells. MEFs express endogenous arrestins and spinophilin, and we readily detected interactions of the α2BAR with endogenous arrestin 3 (Fig 1A, left) and spinophilin (Fig 1C, left), which were enhanced by epinephrine stimulation. In spinophilin deficient (Sp-/-) MEFs, the epinephrine-promoted interaction between the α2BAR and arrestin 3 was markedly increased, as compared to that in the corresponding Sp+/+ MEFs prepared from WT mice with the same genetic background (Fig 1A, right, and Fig 1B). Similarly, the association of the α2BAR with spinophilin in response to epinephrine treatment was significantly enhanced in MEFs with no β-arrestin expression (Arr2,3-/-), as compared to that in the corresponding Arr2,3+/+ MEFs (Fig 1C, right, and Fig 1D). We obtained similar results with other α2 agonists, including clonidine and UK14,304 (data not shown). Additionally, in the absence of arrestin, the basal interaction between α2BAR and spinophilin was also dramatically enhanced (Fig 1C and 1D). Together, these data demonstrate that interactions of the α2BAR with spinophilin and β-arrestins are mutually exclusive.
(A) Interaction between α2BAR and the endogenous arrestin 3 was enhanced in Sp-/- MEFs. Sp-/- and corresponding WT (Sp+/+) MEFs expressing HA-α2BAR were stimulated with 100μM epinephrine (plus 1 μM propranolol to block βARs) for indicated time points. Cell lysates were subjected to IP assays using an HA antibody. (B) Quantitation of the fold change of arrestin 3 in the IP complex isolated from cells with or without stimulation. Data were mean ± SEM. n = 4 for each condition. *, p<0.05 by unpaired Student’s t test, Sp-/- vs. Sp+/+. (C) Interaction between α2BAR and the endogenous spinophilin was enhanced in Arr2,3-/- MEFs. Arr2,3-/- and corresponding WT (Arr2,3+/+) MEFs expressing HA-α2BAR were stimulated with 100μM epinephrine (plus 1μM propranolol to block βARs). Lane C (control) refers to MEFs (Arr2,3+/+ or Arr2,3-/-) without HA-α2BAR overexpression. (D) Quantitation of the fold change of spinophilin in the IP complex isolated from cells with or without stimulation. Data were mean ± SEM. n = 4 for each condition. **, p<0.01, Arr2,3-/- vs. Arr2,3+/+.
Spinophilin attenuates α2BAR phosphorylation through competition against arrestin in cells
We previously demonstrated that stable phosphorylation of the α2BAR requires β-arrestins . Consistently, when arrestin 3 was overexpressed in CosM6 cells, which have a relatively low level of endogenous arrestins compared to other cell lines such as HEK cells and MEFs [7, 36, 37], the levels of α2BAR phosphorylation following epinephrine stimulation were dramatically enhanced compared to the control (Fig 2A and 2B). On the other hand, when spinophilin was overexpressed in HEK293 cells, epinephrine-induced α2BAR phosphorylation was markedly reduced compared to the control (Fig 2C and 2D). This result is consistent with the notion that spinophilin competes against β-arrestins for binding to the α2BAR and impedes the arrestin effect in promoting receptor phosphorylation.
(A) CosM6 cells co-expressing HA-α2BAR together with GFP-tagged arrestin 3 (GFP-Arr3) or GFP alone (vector) were stimulated with 100μM epinephrine (plus 1μM propranolol to block βARs) for indicated time points. Overexpression of GFP-Arr3 increased the phosphorylation level of α2BAR following epinephrine stimulation. (B) Quantitation of agonist-induced fold change in α2BAR phosphorylation. Data were mean ± SEM. n = 5 for each condition. ***, p<0.001 by unpaired Student’s t test, GFP-Arr3 vs. vector control. (C) HEK293 cells co-expressing HA-α2BAR with or without Myc-spinophilin were stimulated. Overexpression of Myc-spinophilin (Myc-Sp) reduced the phosphorylation level of α2BAR following epinephrine stimulation. (D) Quantitation of agonist-induced fold change in α2BAR phosphorylation. Data were mean ± SEM. n = 3 for each condition. **, p<0.01, Myc-Sp vs. vector control.
However, spinophilin may regulate α2BAR phosphorylation through mechanisms other than competing against arrestin, given that it contains multiple functional domains in addition to the receptor binding region [38, 39]. To address this, we examined whether the receptor binding region of spinophilin (Sp156-444) alone can sufficiently regulate α2BAR phosphorylation. In HEK293 cells overexpressing Myc-Sp156-444, the levels of α2BAR phosphorylation in response to agonist stimulation were significantly reduced compared to those in control cells expressing the empty vector (Fig 3A and 3B). This effect on α2BAR phosphorylation caused by the receptor binding region of spinophilin is comparable to that caused by the full length spinophilin (comparing Fig 2D and Fig 3B). Furthermore, in CosM6 cells (which express a low level of endogenous β-arrestins), overexpression of Myc-Sp156-444 failed to alter agonist-induced α2BAR phosphorylation (Fig 4C). This suggests that the inhibitory effect of the receptor binding domain of spinophilin on α2BAR phosphorylation requires a relatively high level of arrestin expression to be detected. Taken together, these data strongly support that spinophilin attenuates α2BAR phosphorylation through competition against β-arrestins in cells.
(A) HEK293 cells co-expressing HA-α2BAR t.ogether with or without Myc-Sp151-444 were stimulated with 100μM epinephrine (plus 1μM propranolol to block βARs) for indicated time points. (B) Quantitation of agonist-induced fold change in α2BAR phosphorylation. Data were mean ± SEM. n = 4 for each condition. **, p<0.01; ***, p<0.001 by unpaired Student t test, Myc-Sp151-444 vs. vector control. (C) Overexpression of Sp151-444 showed no effect on α2BAR phosphorylation in CosM6 cells, which have a low level of endogenous arrestin expression. CosM6 cells co-expressing HA-α2BAR together with or without Sp151-444 were stimulated. Representative blots from multiple independent experiments are shown.
(A) Arr2,3-/- and corresponding WT (Arr2,3+/+) MEFs expressing HA-α2BAR were stimulated with 1μM clonidine for indicated time points. Phospho- and total-ERK1/2 were detected by Western blots. Representative blots from multiple independent experiments are shown. (B) Quantitation of ERK1/2 activation in Arr2,3+/+ or Arr2,3-/- MEFs at indicated time points. The relative ERK1/2 activation at each time point was expressed as a ratio to the peak level of ERK1/2 activation in the same experiment, which was arbitrarily defined as 1.0. Data were mean ± SEM. n = 4 for each condition. *, p<0.05, Arr2,3-/- vs. Arr2,3+/+. (C) Sp-/- and corresponding WT (Sp+/+) MEFs expressing HA-α2BAR were stimulated. Representative blots for phospho- and total ERK1/2 from multiple independent experiments are shown. (D) Quantitation of ERK1/2 activation in Sp+/+ or Sp-/- MEFs at indicated time points. Data were mean ± SEM. n = 7 for data collected in Sp+/+ cells and n = 4 for Sp-/- cells. *, p<0.05; ***, p<0.001, Sp-/- vs. Sp+/+.
Spinophilin counteracts arrestin-dependent desensitization of α2BAR-induced ERK1/2 activation
We next examined the effect of spinophilin and β-arrestins on α2BAR signaling. In MEFs expressing both spinophilin and arrestins, activation of the α2BAR by clonidine induced transient ERK1/2 activation, which was desensitized after 20 min of stimulation (Fig 4). In MEFs without β-arrestin expression (Arr2,3-/-), ERK1/2 signaling was prolonged after a 30-min treatment (Fig 4A and 4B) as compared to that in the corresponding Arr2,3+/+ MEFs, suggesting that β-arrestins are required for terminating α2BAR signaling. On the other hand, in cells without spinophilin expression (Sp-/-), α2BAR-induced ERK1/2 activation was quickly desensitized at the 10 min time point (Fig 4C and 4D). This desensitization of ERK1/2 signaling is much faster than that in the corresponding Sp+/+ MEFs, suggesting an opposing effect of spinophilin on arrestin-dependent desensitization. We obtained similar results with other α2 agonists, including epinephrine and UK14,304 (data not shown). The activation rate of α2BAR-induced ERK1/2 signaling seemed not altered in Arr2,3-/- or Sp-/- cells. This is different from what we have previously observed for the α2AAR-induced ERK1/2 activation, which was accelerated in Sp-/- cells but slowed in Arr2,3-/- cells . Taken together, our data suggest that reciprocal regulation by spinophilin and β-arrestins can have differential impacts on signaling evoked by the closely related α2AR subtypes.
The Del301-303 α2BAR exhibits preferential interaction with spinophilin over arrestin
The human variation Del301-303 α2BAR displays reduced phosphorylation and desensitization following agonist treatment . Given the importance of β-arrestins for α2BAR phosphorylation and desensitization demonstrated above, we predicted that this receptor would show impaired interaction with β-arrestins. Indeed, while epinephrine markedly enhanced the amount of arrestin 3 associated with the WT α2BAR, such treatment failed to increase the association of arrestin 3 with the Del301-303 α2BAR (Fig 5A and 5B). Impaired arrestin binding to the Del301-303 α2BAR may be a consequence of the decreased phosphorylation level of this receptor. Alternatively, Del301-303 may cause conformational changes that reduce its binding to arrestins independent of receptor phosphorylation. To address this possibility, we examined the ability of Del301-303 α2BAR to interact with the phosphorylation-insensitive arrestin 3 (Arr3R170E). Replacement of Arg170 with a Glu results in constitutive binding of arrestin 3 to agonist-activated GPCRs even in the absence of receptor phosphorylation [40, 41]. Epinephrine treatment significantly enhanced interaction of Arr3R170E with the WT α2BAR (Fig 5C and 5D). However, such treatment had no effect on Arr3R170E interaction with the Del301-303 α2BAR (Fig 5C and 5D). These data suggest that Del301-303 changes the conformation of the receptor leading to a diminished affinity for β-arrestin binding.
(A) Agonist treatment failed to promote interaction of the Del301-303 α2BAR with arrestin 3. Cells co-expressing GFP-tagged arrestin3 (GFP-Arr3) with HA-tagged WT α2BAR or Del301-303 α2BAR were stimulated with 100μM epinephrine (plus 1μM propranolol to block βARs), and the interaction between arrestin and either WT or Del301-303 α2BAR was examined by co-IP assays. (B) Quantitation of the agonist-induced fold change of GFP-Arr3 in the IP complex with the WT or Del301-303 α2BAR. n = 3–4 for each condition. **, p<0.01, WT vs. Del301-303. (C) Del 301–303 α2BAR was unable to interact with constitutively active mutant arrestin3 R170E following agonist stimulation. Cells co-expressing GFP-Arr3R170E together with WT or Del301-303 α2BAR were stimulated with 100μM epinephrine (plus 1μM propranolol). (D) Quantitation of the agonist-induced fold change of GFP-Arr3R170E in the IP complex with the WT or Del301-303 α2BAR. n = 3–4 for each condition. *, p<0.05, WT vs. Del301-303.
Based on the reciprocal effect of spinophilin and arrestin on receptor interaction, we predicted that the Del301-303 α2BAR would have a higher affinity for spinophilin binding. As expected, interaction of spinophilin with the Del301-303 α2BAR was significantly increased when compared to that with the WT α2BAR (Fig 6). Taken together, our data suggest biased interaction of the Del301-303 α2BAR with spinophilin.
(A) Cells co-expressing Myc-spinophilin together with HA-tagged WT or Del301-303 α2BAR were stimulated with 100μM epinephrine (plus 1μM propranolol to block βARs). (B) Quantitation of the agonist-induced fold change of Myc-spinophilin in the IP complex with the WT α2B or Del301-303 α2BAR. n = 3–5 for each condition. **, p<0.01, WT vs. Del301-303 α2B.
Spinophilin is essential for sustaining the prolonged ERK1/2 signaling elicited by the Del301-303 α2BAR
The above data suggest that the diminished affinity of the Del301-303 α2BAR for β-arrestin binding likely underlies impaired desensitization of signaling elicited by this receptor variant. Since the Del301-303 α2BAR showed increased interaction with spinophilin, we further sought to address whether spinophilin binding to this receptor plays a role in sustaining its prolonged signaling. We therefore examined the kinetics of ERK1/2 signaling elicited by the WT or Del301-303 α2BAR in Sp-/- and the corresponding Sp+/+ MEFs. Consistent with the reduced desensitization of the Del301-303 α2BAR signaling reported previously , ERK1/2 activation elicited by the Del301-303 α2BAR was prolonged when compared to that elicited by the WT α2BAR in Sp+/+ MEFs (Fig 7A and 7B). Strikingly, in Sp-/- MEF, we failed to observe any difference in ERK1/2 activation kinetics induced by the Del301-303 versus the WT α2BAR (Fig 7C and 7D). In both cases, ERK1/2 signaling was quickly desensitized (Fig 7C and 7D). These data suggest that spinophilin is required for sustaining the prolonged ERK1/2 signaling elicited by the Del301-303 α2BAR.
(A) Sp+/+ MEFs expressing the WT or Del301-303 α2BAR were stimulated with 1μM clonidine for indicated time points. Representative blots show phospho- and total-ERK1/2. (B) Quantitation of ERK1/2 activation in Sp+/+ MEFs. The relative ERK1/2 activation at each time point was expressed as a ratio to the peak level of ERK1/2 activation in the same experiment, which was arbitrarily defined as 1.0. n = 7 for the WT α2B group and n = 6 for the Del301-303 α2B group. *, p<0.05; ***, p<0.001, WT α2B vs. Del301-303 α2B. (C) Sp-/- MEFs expressing the WT or Del301-303 α2BAR were stimulated with 1μM clonidine for indicated time points. Representative blots show phospho- and total-ERK1/2. (D) Quantitation of ERK1/2 activation in Sp-/- MEFs. n = 4 for the WT α2B group and n = 3 for the Del301-303 α2B group.
The α2BAR-elicited hypertensive response is enhanced in arrestin 3 null mice but diminished in spinophilin null mice
Given the reciprocal effects of spinophilin and β-arrestins in regulating α2BAR phosphorylation and desensitization, we sought to further determine the role of these proteins in regulating the α2BAR-elicted hypertensive response. We first compared the level of increase in blood pressure (a response known to be elicited by the α2BAR ) following administration of an α2 agonist, UK14,304, in Arr3-/- and the corresponding WT (Arr3+/+) mice in the same genetic background. In Arr3-/- mice, UK14,304 induced a significantly higher increase in the mean arterial pressure (MAP) compared to that in Arr3+/+ mice (Fig 7A and 7B). Furthermore, this α2BAR-elicited hypertensive response appeared to last for a longer time in Arr3-/- mice compared to WT mice (Fig 7A), and the peak area under the hypertensive curve (AUC) was more than twice of that in WT mice (Fig 7C). These data suggest that the α2BAR-elicited hypertensive response is enhanced and prolonged in the absence of arrestin 3, supporting an in vivo role of arrestin 3 in desensitizing this effect.
We then compared the level of increase in blood pressure induced by UK14,304 in Sp-/- and the corresponding WT (Sp+/+) mice in the same genetic background. UK14,304-induced change in MAP in Sp+/+ mice were somewhat higher than that in Arr3+/+ mice (7.4 ± 2.5 vs 5.4 ± 2.9, p = 0.28), likely due to the difference in genetic background between these lines. In Sp-/- mice, the α2BAR-elicited hypertensive response was greatly diminished (Fig 8A and 8B) and the AUC in these mice was dramatically reduced as compared to those in Sp+/+ mice (Fig 8C). These data strongly suggest that the in vivo α2BAR responsiveness requires the presence of spinophilin; in mice without spinophilin expression, the α2BAR cannot elicit an effective hypertensive response to α2 ligands.
(A) Mean arterial pressure (MAP) measured in Arr3-/-and corresponding Arr3+/+ mice in the same genetic background after injection of UK14,304 (0.1mg/kg i.v.). (B) Quantitation of agonist-induced changes in MAP(ΔMAP) over the basal level. (C) Quantitation of area under curve of the hypertensive response curve. n = 5 for each group. *, p <0.05, Arr3+/+ vs. Arr3-/-.
Using the α2AAR subtype as a model, we previously identified competition between spinophilin and β-arrestins for interaction with the 3i loop of the receptor . In this study, we confirmed the mutually exclusive binding of these proteins to the 3i loop of the α2BAR subtype (Fig 1), and validated that β-arrestins promoted, whereas spinophilin attenuated, agonist-induced α2BAR phosphorylation (Fig 2). In addition, we demonstrated that the receptor-binding domain of spinophilin sufficiently attenuated α2BAR phosphorylation (Fig 3), which further supports the notion that spinophilin regulates receptor phosphorylation through competition against arrestin binding to the receptor.
Consistent with a role of β-arrestins in desensitizing receptor signaling, we found that α2BAR-mediated ERK1/2 activation was prolonged in cells without β-arrestins expression (Fig 4A and 4B). In cells without spinophilin expression, this signaling process was quickly desensitized (Fig 4C and 4D), presumably due to enhanced arrestin binding to the receptor in these cells. In our previous studies, activation of ERK1/2 signaling by the α2A subtype was accelerated in spinophilin deficient cells but slowed in arrestin deficient cells . However, we did not observe these changes for α2BAR-induced ERK1/2 activation, suggesting that the signaling profiles induced by closely related α2AR subtypes are differentially regulated by spinophilin and β-arrestins.
The α2BAR mediates the hypertensive response to α2 ligands . The human Del301-303 α2BAR variant, which exhibits reduced phosphorylation and desensitization profiles in vitro  (also Fig 7A and 7B) and in vivo , has been associated with early onset hypertension in a Swedish population [23, 24]. We found that this variant showed impaired interaction with arrestin 3 (Fig 5A). In particular, the Del301-303 α2BAR failed to interact with the constitutive form of arrestin 3 that is insensitive to receptor phosphorylation (Fig 5B), suggesting that the conformational change caused by Del301-303 alters the intrinsic affinity of the receptor to arrestins. By contrast, the Del301-303 α2BAR showed a much stronger interaction with spinophilin (Fig 6). Strikingly, in cells without spinophilin expression, ERK1/2 activation induced by the Del301-303 α2BAR was quickly desensitized with a profile similar to that induced by the WT receptor (Fig 7C and 7D). These data suggest that spinophilin interaction is essential for maintaining the prolonged signaling profile induced by this receptor variant, and further indicate that spinophilin may represent an attractive target in manipulating functions of this polymorphic variant.
We previously found that multiple in vivo responses elicited by the α2AAR subtype are potentiated in spinophilin deficient mice, but dampened in arrestin 3 deficient mice where spinophilin binding to the receptor is enhanced [7, 9, 10]. Particularly, we have found that spinophilin attenuates the α2AAR-dependent hypotensive response; in spinophilin null mice, this response is highly potentiated . This is the opposite of what we have observed for responses elicited by the α2BAR subtype in the current study. In spinophilin null mice, the α2BAR-elicited hypertensive response was nearly abolished (Fig 9), whereas in arrestin 3 deficient mice, this response was enhanced and prolonged (Fig 8). Our current data suggest that interaction with spinophilin is indispensable for α2BAR to elicit the hypertensive response. Collectively, our previous and current studies suggest that spinophilin regulation of the closely related α2AR subtypes can result in distinct functional outcomes in vivo. Diminished regulation by spinophilin enhances the hypotensive effect elicited by the α2A subtype  while reducing the counteracting hypertensive effect by the α2B subtype (Fig 9). Hence, reducing spinophilin binding to the α2AR subtypes may represent a useful therapeutic strategy for treatment of hypertension. This strategy may be particularly beneficial to the hypertensive population with the spinophilin-biased variation of α2BAR, Del301-303, given the essential role of spinophilin in sustaining signaling by this receptor variant (Fig 7C and 7D).
(A) Mean arterial pressure (MAP) measured in Sp+/+ and Sp-/- mice in the same genetic background after UK14,304 injection (0.1mg/kg i.v.). (B) Quantitation of agonist-induced changes in MAP(ΔMAP) over the basal level. (C) Quantitation of area under curve of the hypertensive response curve. n = 5 for each group. *, p <0.05, Sp+/+ vs. Sp-/-.
We tested multiple α2 ligands in this study. Although all these ligands promoted binding of both arrestin and spinophilin and gave similar results in our experimental readouts, it should be noted that these ligands likely exhibit different biases for the arrestin pathway, as described for the α2CAR subtype previously . Further investigation is needed to quantitatively compare the functional selectivity of these ligands to the α2BAR, including arrestin and spinophilin recruitment. Our studies suggest that ligands that lead to stronger biased interaction of the α2AR subtypes with β-arrestins over spinophilin would be more beneficial for hypertension treatment than traditional ligands that can enhance binding of both proteins to the receptor. Identifying such ligands may represent a new direction of therapeutic development for treatment of hypertension.
We thank Dr. Paul Greengard for generously providing the Sp-/- mouse line, thank Dr. Robert Lefkowitz for generously providing the Arr3-/- mouse line and Arr2,3-/- MEFs, and thank Dr. Jeffrey Benovic for generously providing the arrestin 3 antibody. This study has been supported by the National Institute of Mental Health (MH081917 to QW).
Conceived and designed the experiments: JMW KJ QW. Performed the experiments: PC YC RL NP MG. Analyzed the data: PC JMW KJ QW. Wrote the paper: PC MG KJ QW.
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