Generation of a constitutively active mutant of human GPR48/LGR4, a G-protein-coupled receptor.
Generation of a constitutively active mutant of human GPR48/LGR4, a G-protein-coupled receptor.
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发表时间：
2006-03
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通讯作者：
Yun Gao;K. Kitagawa;M. Shimada;C. Uchida;T. Hattori;T. Oda;M. Kitagawa
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作者：
Yun Gao;K. Kitagawa;M. Shimada;C. Uchida;T. Hattori;T. Oda;M. Kitagawa
GPR48，也称为富含亮氨酸重复序列 (LRR) 的 G 蛋白偶联受体 4 (LGR4)，是 G 蛋白偶联受体 (GPCR) 蛋白家族的成员。然而，其生物学功能仍不清楚，因为其配体和信号转导途径均未确定，并且通常很难解决此类孤儿受体的功能。本研究的目的是产生人 GPR48 的组成型活性形式，其将形成配体独立的活性构象，并且可能以与配体结合后激活的 GPR48 类似的方式发挥作用。我们在人类 GPR48 cDNA 的跨膜结构域 V 和 VI 中引入了四个独立的突变。通过瞬时转染适当的表达质粒，在 HEK293 细胞中表达野生型和突变型 GPR48。由于促性腺激素的配体激活受体在结构上与 GPR48 相似，会刺激腺苷酸环化酶并增加细胞环 AMP，因此我们研究了 GPR48 转染的细胞是否表现出环 AMP 水平改变。转染野生型 GPR48 后，HEK293 细胞中的细胞环 AMP 水平以剂量依赖性方式增加。此外，转染 GPR48-T7551 突变体（其中苏氨酸-755 被异亮氨酸取代）可显着提高环 AMP 水平。来自组成型表达 GPR48-T7551 突变体的 HCT116 细胞的稳定转化子也显示出高环 AMP 水平。这些结果表明GPR48-T7551突变体是组成型活性突变体。该突变体可能有助于研究 GPR48 和 GPR48 介导的信号转导的生物学功能，即使未来特定配体仍然未知。
GPR48, also known as leucine-rich repeat (LRR)-containing G-protein-coupled receptor 4 (LGR4), is a member of the G-protein-coupled receptor (GPCR) family of proteins. However, its biological functions remain unclear, since neither its ligand nor signal transduction pathway have been identified, and it is usually difficult to solve the function of such orphan receptors. The aim of this study was to generate a constitutively active form of human GPR48, that would form a ligand-independent active conformation and may function in a similar manner to activated GPR48 following ligand binding. We introduced four independent mutations into transmembrane domains V and VI of a human GPR48 cDNA. The wild-type and mutant GPR48s were expressed in HEK293 cells by transient transfection of appropriate expression plasmids. Since ligand-activated receptors for gonadotropins, which are structurally similar to GPR48, stimulate adenylate cyclase and increase cellular cyclic AMP, we investigated, whether the GPR48-transfected cells showed altered cyclic AMP levels. The cellular cyclic AMP level in HEK293 cells was increased following transfection of wild-type GPR48 in a dose-dependent manner. Moreover, transfection of a GPR48-T7551 mutant, in which threonine-755 was replaced with isoleucine, dramatically increased the cyclic AMP level. Stable transformants derived from HCT116 cells that constitutively expressed the GPR48-T7551 mutant also showed high cyclic AMP levels. These results indicate that the GPR48-T7551 mutant is a constitutively active mutant. This mutant may be useful for studying the biological functions of GPR48 and GPR48-mediated signal transduction, even if the specific ligand remains unknown in the future.