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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 Del301-303 α2BAR shows impaired interaction with arrestin 3.(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.
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pone.0135030.g005: The Del301-303 α2BAR shows impaired interaction with arrestin 3.(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.

Mentions: The human variation Del301-303 α2BAR displays reduced phosphorylation and desensitization following agonist treatment [27]. 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.


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 Del301-303 α2BAR shows impaired interaction with arrestin 3.(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.
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pone.0135030.g005: The Del301-303 α2BAR shows impaired interaction with arrestin 3.(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.
Mentions: The human variation Del301-303 α2BAR displays reduced phosphorylation and desensitization following agonist treatment [27]. 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.

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