Uchida H, Banba S, Wada M, Matsumoto K, Ikeda M, Naito N, Tanaka E, Honjo M
Life Sciences Laboratory, Mitsui Chemicals, Inc., 1144 Togo, Mobara, Chiba 297-0017, Japan.
J Mol Endocrinol. 1999 Dec;23(3):347-53. doi: 10.1677/jme.0.0230347.
It has recently been shown that 20 kDa human growth hormone (hGH) forms the 1:2 hGH:hGH receptor (hGHR) complex and expresses full agonistic activity, although it hardly forms the 1:1 GH:GHR complex as compared with 22 kDa hGH. To clarify this mechanism, we analyzed the mode of receptor dimerization of 20 kDa hGH using the intact form and mutants. Complex formation analysis between hGHR extracellular domain (hGHBP) and either site1 mutant (K157A) or site2 mutant (G105R) by gel-filtration showed that the site1 mutant apparently formed no 1:1 complex and that the site2 mutant formed only the 1:1 complex. Cell proliferation analysis revealed that the activity curve (vs ligand concentration) of 20 kDa hGH showed a bell-shaped pattern. This indicates that the receptor dimerization of 20 kDa hGH proceeds in a sequential manner. Based on this sequential binding we have produced a mathematical model for receptor dimerization as a function of [hGH], [hGHBP], K(d) values for the first hGHBP binding (K(d1)) and the second hGHBP binding (K(d2)). The result of 20 kDa hGH binding to (S201C) hGHBP immobilized on biosensor tip showed that the K(d1) value was 1. 6x10(-8) M. Adopting this value as a constant in the function described above, we have obtained calculative hGHR dimerization curves vs hGH concentration. Since the K(d2) value could not be experimentally determined, the curves were simulatively obtained with varied K(d2) values. The simulated curve pattern coincided with the experimental result of the cell proliferation in Ba/F3-hGHR when the value 2.5x10(-10) M was adopted as K(d2). In conclusion, although the affinity of 20 kDa hGH for the first hGHR binding is reduced to one-tenth, that for the second binding is increased ten-fold in comparison with those of 22 kDa hGH, indicating that 20 kDa hGH can be an effective hGH isoform in the presence of hGHBP.
最近研究表明，20 kDa的人生长激素（hGH）可形成1:2的hGH:hGH受体（hGHR）复合物并表现出完全激动活性，尽管与22 kDa的hGH相比，它几乎不形成1:1的GH:GHR复合物。为阐明这一机制，我们使用完整形式和突变体分析了20 kDa hGH的受体二聚化模式。通过凝胶过滤分析hGHR细胞外结构域（hGHBP）与位点1突变体（K157A）或位点2突变体（G105R）之间的复合物形成情况，结果显示位点1突变体明显不形成1:1复合物，而位点2突变体仅形成1:1复合物。细胞增殖分析表明，20 kDa hGH的活性曲线（相对于配体浓度）呈钟形。这表明20 kDa hGH的受体二聚化以顺序方式进行。基于这种顺序结合，我们构建了一个受体二聚化的数学模型，该模型是[hGH]、[hGHBP]、第一个hGHBP结合的K(d)值（K(d1)）和第二个hGHBP结合的K(d)值（K(d2)）的函数。20 kDa hGH与固定在生物传感器尖端的（S201C）hGHBP结合的结果表明，K(d1)值为1.6×10⁻⁸ M。将该值作为上述函数中的常数，我们得到了计算的hGHR二聚化曲线与hGH浓度的关系。由于无法通过实验确定K(d2)值，因此通过改变K(d2)值模拟得到了曲线。当采用2.5×10⁻¹⁰ M作为K(d2)值时，模拟曲线模式与Ba/F3-hGHR细胞增殖的实验结果相符。总之，尽管20 kDa hGH与第一个hGHR结合的亲和力降至22 kDa hGH的十分之一，但与第二个hGHR结合的亲和力却增加了10倍，这表明在存在hGHBP的情况下，20 kDa hGH可能是一种有效的hGH异构体。
