Yin Dezhong, Shumay Elena, Wang Hsien-Yu, Malbon Craig C
Department of Pharmacology, Diabetes & Metabolic Diseases Research Center, School of Medicine, SUNY/Stony Brook, Stony Brook, NY 11794-8651, USA.
J Mol Signal. 2006 Nov 10;1:2. doi: 10.1186/1750-2187-1-2.
Mammalian receptors that couple to effectors via heterotrimeric G proteins (e.g., beta 2-adrenergic receptors) and receptors with intrinsic tyrosine kinase activity (e.g., insulin and IGF-I receptors) constitute the proximal points of two dominant cell signaling pathways. Receptors coupled to G proteins can be substrates for tyrosine kinases, integrating signals from both pathways. Yeast cells, in contrast, display G protein-coupled receptors (e.g., alpha-factor pheromone receptor Ste2) that have evolved in the absence of receptor tyrosine kinases, such as those found in higher organisms. We sought to understand the motifs in G protein-coupled receptors that act as substrates for receptor tyrosine kinases and the functional consequence of such phosphorylation on receptor biology. We expressed in human HEK 293 cells yeast wild-type Ste2 as well as a Ste2 chimera engineered with cytoplasmic domains of the beta2-adrenergic receptor and tested receptor sequestration in response to activation of the insulin receptor tyrosine kinase.
The yeast Ste2 was successfully expressed in HEK 293 cells. In response to alpha-factor, Ste2 signals to the mitogen-activated protein kinase pathway and internalizes. Wash out of agonist and addition of antagonist does not lead to Ste2 recycling to the cell membrane. Internalized Ste2 is not significantly degraded. Beta2-adrenergic receptors display internalization in response to agonist (isoproterenol), but rapidly recycle to the cell membrane following wash out of agonist and addition of antagonist. Beta2-adrenergic receptors display internalization in response to activation of insulin receptors (i.e., cross-regulation), whereas Ste2 does not. Substitution of the cytoplasmic domains of the beta2-adrenergic receptor for those of Ste2 creates a Ste2/beta2-adrenergic receptor chimera displaying insulin-stimulated internalization.
Chimera composed of yeast Ste2 into which domains of mammalian G protein-coupled receptors have been substituted, when expressed in animal cells, provide a unique tool for study of the regulation of G protein-coupled receptor trafficking by mammalian receptor tyrosine kinases and adaptor proteins.
通过异源三聚体G蛋白偶联效应器的哺乳动物受体（如β2 - 肾上腺素能受体）以及具有内在酪氨酸激酶活性的受体（如胰岛素和IGF - I受体）构成了两条主要细胞信号通路的近端节点。与G蛋白偶联的受体可以是酪氨酸激酶的底物，整合来自两条通路的信号。相比之下，酵母细胞展示了在没有受体酪氨酸激酶（如高等生物中发现的那些）的情况下进化而来的G蛋白偶联受体（如α - 因子信息素受体Ste2）。我们试图了解作为受体酪氨酸激酶底物的G蛋白偶联受体中的基序以及这种磷酸化对受体生物学的功能影响。我们在人HEK 293细胞中表达了酵母野生型Ste2以及用β2 - 肾上腺素能受体的胞质结构域工程改造的Ste2嵌合体，并测试了响应胰岛素受体酪氨酸激酶激活时受体的隔离情况。
酵母Ste2在HEK 293细胞中成功表达。响应α - 因子，Ste2向丝裂原活化蛋白激酶通路发出信号并内化。洗脱激动剂并添加拮抗剂不会导致Ste2再循环到细胞膜。内化的Ste2没有明显降解。β2 - 肾上腺素能受体响应激动剂（异丙肾上腺素）发生内化，但在洗脱激动剂并添加拮抗剂后迅速再循环到细胞膜。β2 - 肾上腺素能受体响应胰岛素受体的激活发生内化（即交叉调节），而Ste2则不会。用β2 - 肾上腺素能受体的胞质结构域替换Ste2的胞质结构域产生了一种显示胰岛素刺激内化的Ste2/β2 - 肾上腺素能受体嵌合体。
由酵母Ste2组成并替换了哺乳动物G蛋白偶联受体结构域的嵌合体，当在动物细胞中表达时，为研究哺乳动物受体酪氨酸激酶和衔接蛋白对G蛋白偶联受体运输的调节提供了一种独特的工具。
