Using knock-in mice expressing the cGMP FRET sensor cGi500 , we determined that the cGMP decrease begins in the outer layers of granulosa cells where the G-protein-coupled receptors for LH are located . In these cells, the concentration of cGMP decreases to a plateau level with a half time of ~2 min. Because the cells of the follicle are connected by gap junctions , cGMP also decreases in the inner layers of the follicle, and finally in the oocyte, where the [cGMP] decreases with a half time of ~10 minutes. This rapid cGMP decrease throughout the follicle correlates with our previous finding that the guanylyl cyclase activity of NPR2 has decreased by ~50% at 20 minutes after applying LH .
Because NPR2 activity depends on phosphorylation of several juxtamembrane serines and threonines , we investigated whether the LH-induced decrease in NPR2 activity might be due to dephosphorylation. The decrease in guanylyl cyclase activity measured at 20 minutes after LH application was inhibited by preincubating the follicles with the phosphatase inhibitor okadaic acid, suggesting that LH signaling decreases NPR2 activity by dephosphorylating the protein. To determine if dephosphorylation occurred, we immunoprecipitated NPR2 from rat follicle membranes, and separated phosphorylated forms of NPR2 using 6% polyacrylamide gels containing 25 µM Mn2+-Phos-tag-acrylamide, which retards the migration of phosphorylated proteins . Immunoblotting of these gels showed that a 20 minute treatment of the follicles with LH reduced the amount of the more slowly migrating species of NPR2, which is consistent with dephosphorylation.