8 research outputs found
The α<sub>2B</sub>AR dependent hypertensive response is diminished in spinophilin deficient mice.
<p>(A) Mean arterial pressure (MAP) measured in Sp<sup>+/+</sup> and Sp<sup>-/-</sup> 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. *, <i>p</i> <0.05, Sp<sup>+/+</sup><i>vs</i>. Sp<sup>-/-</sup>.</p
Del301-303 α<sub>2B</sub>AR has a stronger interaction with spinophilin than WT α<sub>2B</sub>AR.
<p>(A) Cells co-expressing Myc-spinophilin together with HA-tagged WT or Del301-303 α<sub>2B</sub>AR 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 α<sub>2B</sub> or Del301-303 α<sub>2B</sub>AR. n = 3–5 for each condition. **, <i>p<</i>0.01, WT <i>vs</i>. Del301-303 α<sub>2B</sub>.</p
Arrestin 3 and spinophilin reciprocally regulate agonist-induced α<sub>2B</sub>AR phosphorylation.
<p>(A) CosM6 cells co-expressing HA-α<sub>2B</sub>AR 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 α<sub>2B</sub>AR following epinephrine stimulation. (B) Quantitation of agonist-induced fold change in α<sub>2B</sub>AR phosphorylation. Data were mean ± SEM. n = 5 for each condition. ***, <i>p</i><0.001 by unpaired Student’s <i>t</i> test, GFP-Arr3 <i>vs</i>. vector control. (C) HEK293 cells co-expressing HA-α<sub>2B</sub>AR with or without Myc-spinophilin were stimulated. Overexpression of Myc-spinophilin (Myc-Sp) reduced the phosphorylation level of α<sub>2B</sub>AR following epinephrine stimulation. (D) Quantitation of agonist-induced fold change in α<sub>2B</sub>AR phosphorylation. Data were mean ± SEM. n = 3 for each condition. **, <i>p</i><0.01, Myc-Sp <i>vs</i>. vector control.</p
The α<sub>2B</sub>AR dependent hypertensive response is enhanced in arrestin 3 deficient mice.
<p>(A) Mean arterial pressure (MAP) measured in Arr3<sup>-/-</sup>and corresponding Arr3<sup>+/+</sup> 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. *, <i>p</i> <0.05, Arr3<sup>+/+</sup><i>vs</i>. Arr3<sup>-/-</sup>.</p
Spinophilin is required for maintaining the sustained ERK1/2 activation induced by the Del301-303 α<sub>2B</sub>AR in MEFs.
<p>(A) Sp<sup>+/+</sup> MEFs expressing the WT or Del301-303 α<sub>2B</sub>AR 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<sup>+/+</sup> 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 α<sub>2B</sub> group and n = 6 for the Del301-303 α<sub>2B</sub> group. *, <i>p</i><0.05; ***, <i>p</i><0.001, WT α<sub>2B</sub><i>vs</i>. Del301-303 α<sub>2B</sub>. (C) Sp<sup>-/-</sup> MEFs expressing the WT or Del301-303 α<sub>2B</sub>AR 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<sup>-/-</sup> MEFs. n = 4 for the WT α<sub>2B</sub> group and n = 3 for the Del301-303 α<sub>2B</sub> group.</p
Spinophilin and arrestin reciprocally regulate α<sub>2B</sub>AR-induced ERK1/2 activation kinetics in MEFs.
<p>(A) Arr2,3<sup>-/-</sup> and corresponding WT (Arr2,3<sup>+/+</sup>) MEFs expressing HA-α<sub>2B</sub>AR 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<sup>+/+</sup> or Arr2,3<sup>-/-</sup> 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. *, <i>p</i><0.05, Arr2,3<sup>-/-</sup><i>vs</i>. Arr2,3<sup>+/+</sup>. (C) Sp<sup>-/-</sup> and corresponding WT (Sp<sup>+/+</sup>) MEFs expressing HA-α<sub>2B</sub>AR were stimulated. Representative blots for phospho- and total ERK1/2 from multiple independent experiments are shown. (D) Quantitation of ERK1/2 activation in Sp<sup>+/+</sup> or Sp<sup>-/-</sup> MEFs at indicated time points. Data were mean ± SEM. n = 7 for data collected in Sp<sup>+/+</sup> cells and n = 4 for Sp<sup>-/-</sup> cells. *, <i>p</i><0.05; ***, <i>p</i><0.001, Sp<sup>-/-</sup><i>vs</i>. Sp<sup>+/+</sup>.</p
Overexpression of spinophilin aa151-444 sufficiently attenuates α<sub>2B</sub>AR phosphorylation.
<p>(A) HEK293 cells co-expressing HA-α<sub>2B</sub>AR 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 α<sub>2B</sub>AR phosphorylation. Data were mean ± SEM. n = 4 for each condition. **, <i>p</i><0.01; ***, <i>p</i><0.001 by unpaired Student <i>t</i> test, Myc-Sp151-444 <i>vs</i>. vector control. (C) Overexpression of Sp151-444 showed no effect on α<sub>2B</sub>AR phosphorylation in CosM6 cells, which have a low level of endogenous arrestin expression. CosM6 cells co-expressing HA-α<sub>2B</sub>AR together with or without Sp151-444 were stimulated. Representative blots from multiple independent experiments are shown.</p
The Del301-303 α<sub>2B</sub>AR shows impaired interaction with arrestin 3.
<p>(A) Agonist treatment failed to promote interaction of the Del301-303 α<sub>2B</sub>AR with arrestin 3. Cells co-expressing GFP-tagged arrestin3 (GFP-Arr3) with HA-tagged WT α<sub>2B</sub>AR or Del301-303 α<sub>2B</sub>AR were stimulated with 100μM epinephrine (plus 1μM propranolol to block βARs), and the interaction between arrestin and either WT or Del301-303 α<sub>2B</sub>AR 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 α<sub>2B</sub>AR. n = 3–4 for each condition. **, <i>p<</i>0.01, WT <i>vs</i>. Del301-303. (C) Del 301–303 α<sub>2B</sub>AR was unable to interact with constitutively active mutant arrestin3 R170E following agonist stimulation. Cells co-expressing GFP-Arr3R170E together with WT or Del301-303 α<sub>2B</sub>AR 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 α<sub>2B</sub>AR. n = 3–4 for each condition. *, <i>p<</i>0.05, WT <i>vs</i>. Del301-303.</p