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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

The endogenous arrestin and spinophilin competes for interaction with the α2BAR.(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+/+.
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pone.0135030.g001: The endogenous arrestin and spinophilin competes for interaction with the α2BAR.(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+/+.

Mentions: 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.


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)

The endogenous arrestin and spinophilin competes for interaction with the α2BAR.(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+/+.
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pone.0135030.g001: The endogenous arrestin and spinophilin competes for interaction with the α2BAR.(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+/+.
Mentions: 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.

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