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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

Arrestin 3 and spinophilin reciprocally regulate agonist-induced α2BAR 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.
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pone.0135030.g002: Arrestin 3 and spinophilin reciprocally regulate agonist-induced α2BAR 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.

Mentions: We previously demonstrated that stable phosphorylation of the α2BAR requires β-arrestins [7]. 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.


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)

Arrestin 3 and spinophilin reciprocally regulate agonist-induced α2BAR 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.
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Related In: Results  -  Collection

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pone.0135030.g002: Arrestin 3 and spinophilin reciprocally regulate agonist-induced α2BAR 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.
Mentions: We previously demonstrated that stable phosphorylation of the α2BAR requires β-arrestins [7]. 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.

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