Department of Veterinary Biosciences and Biochemistry, The Ohio State University, Columbus, OH 43210, USA.
Biochemistry. 2011 Jun 14;50(23):5333-44. doi: 10.1021/bi101931u. Epub 2011 May 17.
Hormone binding creates active receptor dimers for class 1 cytokine receptors; however, the detailed molecular mechanism by which these receptors are activated by their ligands is not well characterized, and it is unknown if these receptors share common mechanisms. A rotation model has been proposed for the activation of human erythropoietin receptor and human growth hormone receptor and is supported by evidence showing that additions of alanine at the junction of the transmembrane (TM) and intracellular (IC) domains and/or within the TM domain influenced receptor activities. This evidence suggests that alanine additions changed the relative orientations of the IC domains and their subsequent activation. We wished to determine if a similar mechanism was at play with human prolactin receptor (hPRLr). Up to four alanines were added between the TM and either the IC or extracellular (EC) domains to extend the TM helix and to rotate the IC or EC domains. Also, up to four glycines were placed between the TM and IC domains to provide increased flexibility between these two domains. Wild-type hPRLr or various mutant receptors were expressed in human embryonic kidney 293T cells that express endogenous Janus kinase 2. In the absence of human prolactin (hPRL), none of the alanine or glycine additions increased the level of receptor phosphorylation above that of wild-type hPRLr. In the presence of hPRL, both wild-type hPRLr and each of the mutant receptors were successfully phosphorylated. These data do not support a rotation mechanism for hPRLr activation or a requirement of a fixed spatial relationship between the TM and IC domains for hPRLr activation. In a second set of experiments, both wild-type hPRLr and either alanine- or glycine-extended receptors were coexpressed in 293T cells. In the absence of hPRL, there was no detectable phosphorylation of hPRLr. Such data do not support a piston movement between the hPRLr pair in their activation.
激素结合为 I 类细胞因子受体创建了活性受体二聚体；然而，这些受体被其配体激活的详细分子机制尚未很好地描述，并且不知道这些受体是否共享共同的机制。旋转模型已被提出用于激活人促红细胞生成素受体和人生长激素受体，并得到以下证据的支持：在跨膜 (TM) 和细胞内 (IC) 结构域以及/或 TM 结构域内连接处添加丙氨酸会影响受体活性。该证据表明，丙氨酸的添加改变了 IC 结构域的相对取向及其随后的激活。我们希望确定人催乳素受体 (hPRLr) 是否存在类似的机制。在 TM 与 IC 或细胞外 (EC) 结构域之间最多可以添加四个丙氨酸以延长 TM 螺旋并旋转 IC 或 EC 结构域。另外，在 TM 和 IC 结构域之间最多可以添加四个甘氨酸以提供这两个结构域之间的更大灵活性。野生型 hPRLr 或各种突变受体在表达内源性 Janus 激酶 2 的人胚肾 293T 细胞中表达。在没有人类催乳素 (hPRL) 的情况下，添加的丙氨酸或甘氨酸都不会使受体磷酸化水平高于野生型 hPRLr。在存在 hPRL 的情况下，野生型 hPRLr 和每种突变受体均成功磷酸化。这些数据不支持 hPRLr 激活的旋转机制，也不支持 TM 和 IC 结构域之间固定空间关系对于 hPRLr 激活的要求。在第二组实验中，野生型 hPRLr 和丙氨酸或甘氨酸扩展的受体都在 293T 细胞中共同表达。在没有 hPRL 的情况下，hPRLr 没有可检测到的磷酸化。这样的数据不支持 hPRLr 对在其激活过程中活塞运动。
