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Spinophilin Is Indispensable for the α2B Adrenergic Receptor-Elicited Hypertensive Response.

Che P, Chen Y, Lu R, Peng N, Gannon M, Wyss JM, Jiao K, Wang Q - PLoS ONE (2015)

Bottom Line: 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.Thus, spinophilin may represent a useful therapeutic target for treatment of hypertension.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, 1918 University Boulevard, Birmingham, AL 35294, United States of America.

ABSTRACT
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 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.

No MeSH data available.


Related in: MedlinePlus

Spinophilin and arrestin reciprocally regulate α2BAR-induced ERK1/2 activation kinetics in MEFs.(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+/+.
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pone.0135030.g004: Spinophilin and arrestin reciprocally regulate α2BAR-induced ERK1/2 activation kinetics in MEFs.(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+/+.

Mentions: 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.


Spinophilin Is Indispensable for the α2B Adrenergic Receptor-Elicited Hypertensive Response.

Che P, Chen Y, Lu R, Peng N, Gannon M, Wyss JM, Jiao K, Wang Q - PLoS ONE (2015)

Spinophilin and arrestin reciprocally regulate α2BAR-induced ERK1/2 activation kinetics in MEFs.(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+/+.
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pone.0135030.g004: Spinophilin and arrestin reciprocally regulate α2BAR-induced ERK1/2 activation kinetics in MEFs.(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+/+.
Mentions: 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.

Bottom Line: 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.Thus, spinophilin may represent a useful therapeutic target for treatment of hypertension.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, 1918 University Boulevard, Birmingham, AL 35294, United States of America.

ABSTRACT
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 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.

No MeSH data available.


Related in: MedlinePlus