Møldrup A, Allevato G, Dyrberg T, Nielsen J H, Billestrup N
Hagedorn Research Laboratory, Gentofte, Denmark.
J Biol Chem. 1991 Sep 15;266(26):17441-5.
Transfection of the insulin-producing rat islet tumor cell line RIN-5AH with a full length cDNA of the rat hepatic growth hormone (GH) receptor (GH-R1-638) augments the GH-responsive insulin synthesis in these cells. Using this functional system we analyzed the effect of COOH-terminal truncation of the GH receptor. Two mutated cDNAs encoding truncated GH receptors, GH-R1-294 and GH-R1-454, respectively, were generated by site-directed mutagenesis and transfected into the RIN cells. Both receptor mutants were expressed on the cell surface and displayed normal GH binding affinity. Whereas GH-R1-638 had a molecular mass of about 110 kDa, GH-R1-294 and GH-R1-454 showed molecular masses of 49 and 80 kDa, respectively. Cells expressing GH-R1-454 internalized GH to a similar extent as cells transfected with the full length receptor and the parent cell line, but GH-R1-294-expressing cells showed a markedly reduced capability of GH internalization. In contrast to cells transfected with GH-R1-638, none of the cell lines expressing truncated GH receptors exhibited any increase of the GH-stimulated insulin production. We conclude that domains within the COOH-terminal half of the cytoplasmic part of the GH receptor are required for transduction of the signal for GH-stimulated insulin synthesis, whereas cytoplasmic domains proximal to the transmembrane region are involved in receptor-mediated GH internalization.
用大鼠肝脏生长激素（GH）受体的全长cDNA（GH-R1-638）转染产生胰岛素的大鼠胰岛肿瘤细胞系RIN-5AH，可增强这些细胞中对GH应答的胰岛素合成。利用这个功能系统，我们分析了GH受体COOH末端截短的影响。通过定点诱变分别产生了两个编码截短的GH受体的突变cDNA，即GH-R1-294和GH-R1-454，并将其转染到RIN细胞中。两种受体突变体均在细胞表面表达，并表现出正常的GH结合亲和力。GH-R1-638的分子量约为110 kDa，而GH-R1-294和GH-R1-454的分子量分别为49 kDa和80 kDa。表达GH-R1-454的细胞内化GH的程度与用全长受体和原代细胞系转染的细胞相似，但表达GH-R1-294的细胞显示出明显降低的GH内化能力。与用GH-R1-638转染的细胞相反，表达截短的GH受体的细胞系均未表现出GH刺激的胰岛素产生增加。我们得出结论，GH受体胞质部分COOH末端一半内的结构域是GH刺激胰岛素合成信号转导所必需的，而跨膜区域近端的胞质结构域则参与受体介导的GH内化。
