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

Abstract

The 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.

Natriuretic peptide type C induces sperm attraction for fertilization in mouse

“…2c). Two immunoreactive bands (~130 kDa and ~117 kDa) correspond to NPR2 with different amounts of glycosylation as observed in previous studies of mouse and rat granulosa cells353637.…”

Dephosphorylation is the mechanism of fibroblast growth factor inhibition of guanylyl cyclase-B

“…Because inhibition of GC-B by multiple hormones and signaling factors is correlated with receptor dephosphorylation [23, 24, 27, 35, 39, 40], we investigated whether dephosphorylation contributes to the FGF2-dependent inhibition of GC-B in RCS cells. To determine GC-B phosphate levels, we employed two different phosphate detection methods, Phos-tag and ProQ Diamond.…”

“…Unlike the wild type enzyme, GC-B-7E cannot be inactivated by dephosphorylation of the mutated phosphorylation sites [19, 24]. Therefore, we hypothesized that if FGF2 inhibits GC-B through dephosphorylation, then FGF2 should not inhibit GC-B-7E.…”

“…Recently, luteinizing hormone (LH) was shown to stimulate GC-B dephosphorylation and inactivation in ovarian follicles by a process that requires a PPP family serine and threonine protein phosphatase [23]. Importantly, a knock-in mouse (GC-B 7E/7E ) expressing GC-B-7E at the normal GC-B genetic locus was immune to LH-dependent GC-B inactivation and displayed a 5-h delay in the resumption of meiosis in the oocyte [24]. These findings indicate that hormones that activate G protein coupled receptors inactivate GC-B by dephosphorylation.…”

Forkhead box O member FOXO1 regulates the majority of follicle-stimulating hormone responsive genes in ovarian granulosa cells

“…Nppc encodes natriuretic peptide precursor C that, upon proteolytic cleavage, generates C-type natriuretic peptide that functions as the agonist of the guanylyl cyclase membrane receptor NPR2. In ovarian follicles, mural GCs (that line the basement membrane) produce C-type natriuretic peptide that binds NPR2 on the surface of mural GCs and of nearby cumulus GCs that surround the oocyte to trigger production of cGMP (Shuhaibar et al, 2016). cGMP then transits into the oocyte and maintains meiotic arrest of fully grown oocytes until the ovulatory surge of LH that reduces oocyte levels of cGMP to trigger the resumption of meiosis (Lee et al, 2013; Norris et al, 2009; Vaccari et al, 2009; Zhang et al, 2010).…”