A point mutation in the glucose-dependent insulinotropic peptide receptor confers constitutive activity.
A point mutation in the glucose-dependent insulinotropic peptide receptor confers constitutive activity.
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DOI：
10.1006/bbrc.1997.6231
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发表时间：
1997-03
影响因子：
3.1
通讯作者：
C. Tseng;L. Lin
中科院分区：
文献类型：
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作者：
C. Tseng;L. Lin
葡萄糖依赖性促胰岛素肽受体（GIP-R）是分泌素和甲状旁腺激素（PTH）家族的7种跨膜受体的成员。据报道，人PTH受体的第一胞内环和第二跨膜结构域之间的连接处的组氨酸和第六跨膜结构域中的苏氨酸的点突变与詹森型干骺端软骨发育不良中PTH受体的组成性激活相关。在这项研究中，我们探讨了GIP-R中的这种突变是否可能类似地诱导受体的组成性、配体非依赖性激活。通过定点诱变在GIP受体中进行单个氨基酸取代，并在转染的人胚肾细胞系（L293）中测量受体结合和cAMP水平。在第六跨膜结构域中的位置340处的苏氨酸突变为脯氨酸（T340 P）导致激动剂非依赖性组成型活性，并且与野生型GIP-R相比，表现出基础cAMP水平的四倍增加。T340 P突变体中cAMP水平的增加与转染质粒的量成正比，并与细胞表面受体的数量相对应。尽管其高的基础cAMP水平，T340 P突变体可以进一步刺激GIP，与野生型受体的最大cAMP生成。然而，在位置169处的氨基酸组氨酸变为精氨酸（H169 R），表现得像野生型受体，并且不具有组成型活性。这些结果说明，在位置340处苏氨酸至脯氨酸的点突变导致GIP受体的组成型活化，而不影响其对激动剂刺激的敏感性。
The glucose-dependent insulinotropic peptide receptor (GIP-R) is a member of the secretin and parathyroid hormone (PTH) family of seven transmembrane-spanning receptors. Point mutations of a histidine at the junction between the first intracellular loop and the second membrane-spanning domain and a threonine in the sixth membrane-spanning domain of the human PTH-receptor have been reported to be associated with constitutive activation of the PTH receptor in Jansen-type metaphyseal chondrodysplasia. In this study, we explored whether such mutations in the GIP-R might similarly induce constitutive, ligand-independent activation of the receptor. Single amino acid substitutions in the GIP receptor were made by site-directed mutagenesis and receptor binding and cAMP levels were measured in transfected human embryonal kidney cell line (L293). Mutation of the threonine at position 340 in the sixth transmembrane spanning domain to proline (T340P) led to agonist-independent constitutive activity and exhibited a four-fold increase in basal cAMP level as compared to the wild-type GIP-R. The increase in cAMP level in T340P mutant was proportional to the amount of transfected plasmid and corresponded to the receptor number on the cell surface. Despite its high basal cAMP level, the T340P mutant could be further stimulated by GIP, with maximal cAMP generation comparable to the wild-type receptor. The change of amino acid histidine at position 169 to arginine (H169R), however, behaved like the wild type receptor and did not possess constitutive activity. These results illustrate that a point mutation of threonine to proline at position 340 results in constitutive activation of the GIP receptor, without affecting its sensitivity to agonist stimulation.