Strittmatter S M, Igarashi M, Fishman M C
Developmental Biology Laboratory, Massachusetts General Hospital, Charlestown 02129.
J Neurosci. 1994 Sep;14(9):5503-13. doi: 10.1523/JNEUROSCI.14-09-05503.1994.
The neuronal growth-associated protein GAP-43 is expressed maximally during development and regeneration, and is enriched at the cytosolic surface of the growth cone membrane. GAP-43 can activate the GTP-binding protein G(o) which is also a major component of the growth cone membrane. These findings have led to the hypothesis that GAP-43 might modulate neurite outgrowth by altering G-protein activity. Here we define the sequence requirements for GAP-43 amino terminal peptide stimulation of G(o), and test these peptides as potential modulators of neurite outgrowth. The first 10 amino acids of GAP-43, Met-Leu-Cys-Cys-Met-Arg-Arg-Thr-Lys-Gln, stimulate G(o). Substitutions at particular residues reveal that cys3, cys4, arg6, and lys9 are critical, but arg7 is not. Both the GAP-43(1-10) peptide and the G-protein-activating peptide mastoparan induce growth cone collapse and inhibit neurite extension from embryonic chick dorsal root ganglion and retinal neurons. This is likely to be mediated by G-proteins: pertussis toxin blocks the inhibition, and mutant peptides that do not activate G(o) do not alter outgrowth. In contrast to the case with embryonic chick dorsal root ganglion cells, neurite outgrowth from N1E-115 neuroblastoma cells is stimulated by GAP-43(1-10). This is probably also a G-protein-mediated event because it is blocked by pertussis toxin, because the sequence requirements match those for G(o) stimulation, and because mastoparan stimulates outgrowth from these cells. The longer GAP-43(1-25) peptide does not alter neurite outgrowth unless the cells are permeabilized, suggesting an intracellular site of action. These data identify a novel set of compounds that modulate neurite outgrowth, and also support the notion that GAP-43 can alter neurite extension by modulating pertussis toxin-sensitive G-protein activity in the growth cone.
神经元生长相关蛋白GAP - 43在发育和再生过程中表达量最高，且在生长锥膜的胞质表面富集。GAP - 43可激活GTP结合蛋白G(o)，而G(o)也是生长锥膜的主要成分。这些发现引发了一个假说，即GAP - 43可能通过改变G蛋白活性来调节神经突生长。在此，我们确定了GAP - 43氨基末端肽刺激G(o)的序列要求，并将这些肽作为神经突生长的潜在调节剂进行测试。GAP - 43的前10个氨基酸，即甲硫氨酸 - 亮氨酸 - 半胱氨酸 - 半胱氨酸 - 甲硫氨酸 - 精氨酸 - 精氨酸 - 苏氨酸 - 赖氨酸 - 谷氨酰胺，可刺激G(o)。特定残基的替换表明，半胱氨酸3、半胱氨酸4、精氨酸6和赖氨酸9至关重要，但精氨酸7并非如此。GAP - 43(1 - 10)肽和G蛋白激活肽蜂毒素均能诱导生长锥塌陷，并抑制胚胎鸡背根神经节和视网膜神经元的神经突延伸。这可能是由G蛋白介导的：百日咳毒素可阻断这种抑制作用，而不能激活G(o)的突变肽不会改变神经突生长。与胚胎鸡背根神经节细胞的情况不同，GAP - 43(1 - 10)可刺激N1E - 115神经母细胞瘤细胞的神经突生长。这可能也是一个由G蛋白介导的事件，因为它被百日咳毒素阻断，因为序列要求与刺激G(o)的要求相符，并且因为蜂毒素可刺激这些细胞的神经突生长。更长的GAP - 43(1 - 25)肽不会改变神经突生长，除非细胞被通透化，这表明其作用位点在细胞内。这些数据确定了一组新型的可调节神经突生长的化合物，也支持了GAP - 43可通过调节生长锥中对百日咳毒素敏感的G蛋白活性来改变神经突延伸的观点。
