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Dev Biol
2007 Jul 15;3072:195-201. doi: 10.1016/j.ydbio.2007.04.021.
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Guanylate cyclase and cyclic GMP-dependent protein kinase regulate agrin signaling at the developing neuromuscular junction.
Godfrey EW
,
Longacher M
,
Neiswender H
,
Schwarte RC
,
Browning DD
.
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During formation of the neuromuscular junction (NMJ), agrin secreted by motor axons signals the embryonic muscle cells to organize a postsynaptic apparatus including a dense aggregate of acetylcholine receptors (AChRs). Agrin signaling at the embryonic NMJ requires the activity of nitric oxide synthase (NOS). Common downstream effectors of NOS are guanylate cyclase (GC), which synthesizes cyclic GMP, and cyclic GMP-dependent protein kinase (PKG). Here we show that GC and PKG are important for agrin signaling at the embryonic NMJ of the frog, Xenopus laevis. Inhibitors of both GC and PKG reduced endogenous AChR aggregation in embryonic muscles by 50-85%, and blocked agrin-induced AChR aggregation in cultured embryonic muscle cells. A cyclic GMP analog, 8-bromo-cyclic GMP, increased endogenous AChR aggregation in embryonic muscles to 3- to 4-fold control levels. Overexpression of either GC or PKG in embryos increased AChR aggregate area by 60-170%, whereas expression of a dominant negative form of GC inhibited endogenous aggregation by 50%. These results indicate that agrin signaling in embryonic muscle cells requires the activity of GC and PKG as well as NOS.
Fig. 1. Inhibitors of GC and PKG reduce endogenous AChR aggregation at the embryonic neuromuscular junction, and a cyclic GMP analog greatly increases AChR aggregate area compared to untreated control embryos (A). Embryos were exposed to the GC inhibitor ODQ (50 μM, B), the PKG inhibitor Rp-8-pCPT-cGMPS (0.5 mM, C), or the cGMP analog 8-Br-cGMP (100 μM, D) during the period of neuromuscular junction formation (stages 24–31). In the experiments shown here, skin was removed from myotomes of embryos treated with Rp-8-pCPT-cGMPS and 8-Br-cGMP and their respective controls, but not from embryos treated with ODQ or corresponding controls (A). AChR aggregates were labeled and imaged by confocal microscopy, and area of aggregates was quantified (Table 1) with Metamorph image analysis software as described in Materials and methods. Images shown are maximum projections of stacks of 4 images taken at 1 μm intervals. In the image stacks projected in this figure, AChR aggregate area was inhibited 86% by ODQ (B) and 65% by Rp-8-pCPT-cGMPS (C), and increased 460% by 8-Br-cGMP (D), compared to the respective untreated controls (e.g., A).
Fig. 2. Inhibitors of GC and PKG block agrin-induced AChR aggregation on cultured Xenopus embryo myotomal muscle cells. Cells were pretreated with inhibitors 2.5 h before 17 h incubation with 7 ng/ml agrin (half-maximal dose, 4.4 ng/ml) with or without inhibitors. AChR aggregates (white) were then labeled with fluorescent α-bungarotoxin, cells were fixed, mounted, and aggregates counted as described in Materials and methods. In the experiment from which these images were taken, the average inhibition of agrin-induced AChR aggregation was 99% with ODQ (2 μM), 75% with KT5823 (1 μM; data not shown), and 75% with Rp-8-pCPT-cGMPS (0.5 μM). The number of aggregates per cell in the images shown was 1.8 in untreated control cells (A), 3.4 after agrin treatment (B), 2.5 after agrin plus ODQ (66% inhibition, C), and 0.2 after agrin plus Rp-8-pCPT-cGMPS (88% inhibition, D).
Fig. 3. Overexpression of guanylate cyclase (GC) increases AChR aggregation in embryonic muscles, but a dominant negative GC inhibits endogenous aggregation at NMJs. Embryos were injected with RNA encoding GFP (A), GC α and β subunits (B), GC α only (C), or a dominant negative GC-α construct (D) encoding an inactive point mutant. GC α + β increased AChR aggregation 2–3-fold, GC α alone did not affect aggregation, but the dominant negative GC α reduced aggregate area by about 50%. Quantitative data from the experiments shown here are found in Table 3 (Experiment 1 for panels A–C; Experiment 2 for panel D). Dashed lines in panel C indicate boundaries of muscle fibers. Scale bar, 20 μm.
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