Dephosphorylation of juxtamembrane serines and threonines of the NPR2 guanylyl cyclase is required for rapid resumption of oocyte meiosis in response to luteinizing hormone

AbstractThe meiotic cell cycle of mammalian oocytes starts during embryogenesis and then pauses until luteinizing hormone (LH) acts on the granulosa cells of the follicle surrounding the oocyte to restart the cell cycle. An essential event in this process is a decrease in cyclic GMP in the granulosa cells, and part of the cGMP decrease results from dephosphorylation and inactivation of the natriuretic peptide receptor 2 (NPR2) guanylyl cyclase, also known as guanylyl cyclase B. However, it is unknown whether NPR2 dephosphorylation is essential for LH-induced meiotic resumption. Here, we prevented NPR2 dephosphorylation by generating a mouse line in which the seven regulatory serines and threonines of NPR2 were changed to the phosphomimetic amino acid glutamate (Npr2–7E). Npr2–7E/7E follicles failed to show a decrease in enzyme activity in response to LH, and the cGMP decrease was attenuated; correspondingly, LH-induced meiotic resumption was delayed. Meiotic resumption in response to EGF receptor activation was likewise delayed, indicating that NPR2 dephosphorylation is a component of the pathway by which EGF receptor activation mediates LH signaling. We also found that most of the NPR2 protein in the follicle was present in the mural granulosa cells. These findings indicate that NPR2 dephosphorylation in the mural granulosa cells is essential for the normal progression of meiosis in response to LH and EGF receptor activation. In addition, these studies provide the first demonstration that a change in phosphorylation of a transmembrane guanylyl cyclase regulates a physiological process, a mechanism that may also control other developmental events

Regulation of Constitutive GPR3 Signaling and Surface Localization by GRK2 and β-arrestin-2 Overexpression in HEK293 Cells.

G protein-coupled receptor 3 (GPR3) is a constitutively active receptor that maintains high 3'-5'-cyclic adenosine monophosphate (cAMP) levels required for meiotic arrest in oocytes and CNS function. Ligand-activated G protein-coupled receptors (GPCRs) signal at the cell surface and are silenced by phosphorylation and β-arrestin recruitment upon endocytosis. Some GPCRs can also signal from endosomes following internalization. Little is known about the localization, signaling, and regulation of constitutively active GPCRs. We demonstrate herein that exogenously-expressed GPR3 localizes to the cell membrane and undergoes internalization in HEK293 cells. Inhibition of endocytosis increased cell surface-localized GPR3 and cAMP levels while overexpression of GPCR-Kinase 2 (GRK2) and β-arrestin-2 decreased cell surface-localized GPR3 and cAMP levels. GRK2 by itself is sufficient to decrease cAMP production but both GRK2 and β-arrestin-2 are required to decrease cell surface GPR3. GRK2 regulates GPR3 independently of its kinase activity since a kinase inactive GRK2-K220R mutant significantly decreased cAMP levels. However, GRK2-K220R and β-arrestin-2 do not diminish cell surface GPR3, suggesting that phosphorylation is required to induce GPR3 internalization. To understand which residues are targeted for desensitization, we mutated potential phosphorylation sites in the third intracellular loop and C-terminus and examined the effect on cAMP and receptor surface localization. Mutation of residues in the third intracellular loop dramatically increased cAMP levels whereas mutation of residues in the C-terminus produced cAMP levels comparable to GPR3 wild type. Interestingly, both mutations significantly reduced cell surface expression of GPR3. These results demonstrate that GPR3 signals at the plasma membrane and can be silenced by GRK2/β-arrestin overexpression. These results also strongly implicate the serine and/or threonine residues in the third intracellular loop in the regulation of GPR3 activity

Primer sets used for GPR3-HA mutagenesis.

<p>Primer sets used for GPR3-HA mutagenesis.</p

Inhibition of endocytosis increases intracellular cAMP.

<p><b>A)</b> HEK293 cells were co-transfected with GPR3-HA and pcDNA or with Dyn WT or Dyn K44A. Twenty-four hr after transfection, cells were harvested for cAMP EIA. Bars with different letters are significantly different. Significance was determined by One-way ANOVA followed by Newman-Keuls multiple comparison test (<i>p</i><0.01).Results are presented as mean ± S.E.M from 4 separate experiments. <b>B–C</b>) HEK293 cells were co-transfected with GPR3-RFP and CFP-Epac1(δDEP-CD)-YFP and pcDNA, or Dyn WT or Dyn K44A. The YFP/CFP ratios were measured before and after isoproterenol and IBMX treatment. <b>B)</b> Percent change in the YFP/CFP ratio. <b>C</b>) FRET measurements were converted to cAMP levels (µM) as described in the experimental protocol. Bars with different letters are significantly different. Significance was determined by One-way ANOVA followed by Newman-Keuls Multiple Comparison Test (<i>p</i><0.01). Results were obtained from 15 different GPR3-RFP expressing cells per group.</p

PKC inhibition increases intracellular cAMP and PKC activation decreases intracellular cAMP levels.

<p><b>A-B).</b> HEK293 cells were transfected with GPR3-HA WT and 4 hr later treated with 5 µM Bis I (<b>A</b>) or 10 nM PMA (<b>B</b>). Following 18 to 24 hr treatment, cells were harvested for cAMP EIA. <b>C–D</b>) HEK293 cells were transfected with GPR3-HA ST/A and 4 hr later treated with 5 µM Bis I (<b>C</b>) or 10 nM PMA (<b>D</b>). Following 18 to 24 hr treatment, cells were harvested for cAMP EIA. Results are presented as mean ± S.E.M from 3–4 separate experiments. (*) indicates a significant difference in cAMP levels from “-” DMSO treated cells as determined by two-tailed paired Student’s <i>t</i> test (<i>p</i><0.05). ∼170,000 cells lysed in 0.1 M HCl were used in the EIA assay, except for the GPR3-HA ST/A treated with Bis I and DMSO where ∼90,000 cells were used in order to keep the levels of cAMP on the standard curve.</p

Overexpression of GRK2 and β-arrestin-2 decreases cell surface GPR3 expression.

<p><b>A</b>) HEK293 cells were co-transfected with GPR3-HA and pcDNA (<b>−</b>) or GPR3-HA and GRK2 and β-arrestin-2 (<b>+</b>). Twenty-four hr after transfection, cell surface proteins were biotinylated, precipitated, and cell surface expression of GPR3-HA was detected by Western blotting. 0.5 µg of total lysate was used to detect total GPR3 and GAPDH expression. Blot is representative of 3 separate experiments. <b>B–C</b>) Bands corresponding to surface GPR3 and total GPR3 expression were analyzed using densitometry. Densitometric values for total GPR3 expression were normalized to GAPDH. Densitometric values for surface GPR3 was divided by densitometric values for total GPR3 and normalized to GAPDH to compare the amount of GPR3 at the surface vs. total GPR3 expression. (*) indicates a significant decrease in surface/total GPR3 expression as a result of GRK2 and β-arrestin-2 overexpression. Significance was determined by Student’s <i>t</i> test (<i>p</i><0.05). Results are presented as mean ± S.E.M. from 3 separate experiments.</p

Mutation of S and T residues in the third intracellular loop increases intracellular cAMP.

<p><b>A</b>) Schematic identifying potential serine and threonine residues in the third intracellular loop and C-terminus that could be targeted for regulation by phosphorylation. These residues were mutated to alanine to create 3 mutants: ST/A, S1-6A, and ST/A+S1-6A. <b>B</b>) GPR3-HA WT and mutants were transfected into HEK293 cells and harvested 24 hr later for cAMP EIA. (*) indicates a significant increase in cAMP level compared to “WT”. Significance was determined by Repeated Measures ANOVA followed by Dunnett’s Multiple Comparison Test (<i>p</i><0.01). Results are presented as mean ± S.E.M. from 6 separate experiments. <b>C)</b> 24 hr after transfection, cell surface proteins were biotinylated, precipitated, and surface expression of GPR3-HA was detected by Western blotting. 0.5 µg of total lysate was used to detect total GPR3 and β-actin expression. Blot is representative of 3 separate experiments: WT, lane 1; ST/A, lane 2; S1-6A, lane 3; ST/A+S1-6A, lane 4. <b>D–E</b>) Bands corresponding to surface GPR3 and total GPR3 expression were analyzed using densitometry. Densitometric values for total expression of GPR3 WT and mutants were normalized to β-actin (<b>D</b>). The densitometric value for surface GPR3 was divided by the densitometric value for total GPR3 expression and normalized to β-actin to compare the amount of GPR3 at the surface vs. total GPR3 (<b>E</b>). (*) indicates a significant decrease in surface expression of GPR3 mutants compared to “WT”. Significance was determined by One-way ANOVA followed by Newman-Keuls Multiple Comparison Test (<i>p</i><0.05). Results are presented as mean ± S.E.M from 3 separate experiments.</p

Inhibition of endocytosis increases cell surface GPR3.

<p>(<b>A and B</b>) HEK293 cells were transfected with Dyn WT (<b>A</b>) or Dyn K44A (<b>B</b>) and 24 hr later, the cells were incubated with Alexa Fluor 488-labelled transferrin and imaged with a confocal microscope. Images are representative of 3 separate experiments. (<b>C–E</b>) Cells were co-transfected with GPR3-RFP and pcDNA (<b>C</b>), or Dyn WT (<b>D</b>) or Dyn K44A (<b>E</b>) and imaged using a confocal microscope 24 hr later. Bar = 10 µm. (<b>F</b>) One µg of HEK293 lysate was used to detect Dyn WT and Dyn K44A overexpression by Western blot. (<b>G</b>) HEK293 cells were co-transfected with GPR3-HA and Dyn WT or Dyn K44A and 24 hr after transfection cell surface proteins were biotinylated, precipitated, and surface expression of GPR3-HA was detected by Western blot analysis. 0.5 µg of total lysate was used to detect total GPR3 and GAPDH expression. Blot is representative of 3 separate experiments. (<b>H–I</b>) Bands corresponding to surface GPR3 and total GPR3 expression were analyzed using densitometry. <b>H</b>) Densitometric values for total GPR3 expression in cell lysates were normalized to GAPDH. <b>I</b>) The densitometric value for surface GPR3 was divided by the densitometric value for total GPR3 expression and normalized to GAPDH to compare the amount of GPR3 at the surface vs. total GPR3 expression. (*) indicates a significant increase in cell surface GPR3 compared to “GPR3+Dyn WT”. Significance was determined by Student’s <i>t</i> test (<i>p</i><0.05). Results are presented as mean ± S.E.M from 3 separate experiments.</p