Gimpl Gerald, Wiegand Volker, Burger Katja, Fahrenholz Falk
Institute of Biochemistry, Johannes Gutenberg-University of Mainz, Becherweg 30, D-55099 Mainz, Germany.
Prog Brain Res. 2002;139:43-55. doi: 10.1016/s0079-6123(02)39006-x.
The function and physiological regulation of the oxytocin-receptor system is strongly steroid-dependent. This is, unexpectedly, only partially reflected by the promoter sequences in the oxytocin receptor and favors the idea that posttranscriptional mechanisms may also play a significant role for the physiological regulation of the oxytocin-receptor system. Our data indicate that cholesterol acts as an allosteric modulator of the oxytocin receptor and stabilizes both membrane-associated and solubilized OT receptors in a high-affinity state for agonists and antagonists. Moreover, high-affinity OT receptors are 2-fold enriched in cholesterol-rich plasma membrane domains in HEK293 fibroblasts stably expressing the human OT receptor. Biochemical data suggest a direct and cooperative molecular interaction of cholesterol molecules with OT receptors. To localize the cholesterol interacting domain of the oxytocin receptor the C-terminal part including the last two transmembrane domains have been exchanged by the corresponding sequences of the cholecystokinin type B receptor, which is functionally not dependent on cholesterol. Concerning its ligand-binding behavior this chimeric receptor protein showed the same dependence on cholesterol and its analogues as the wild type oxytocin receptor. From mutagenesis experiments and studies with receptor chimera between the OTR and cholecystokinin type B receptor, we conclude that a major part of the cholesterol interacting domain may be localized in the first part of the oxytocin receptor, possibly in a domain nearby the agonist binding site. Progesterone is considered to be essential to maintain the uterine quiescence. High concentrations of progesterone (> 10 microM) attenuate or block the signaling of several GPCRs, including the OT receptor via a fast, reversible and non-genomic pathway. Progesterone is known to inhibit both cholesterol biosynthesis and the intracellular trafficking of cholesterol. We therefore test the hypothesis that progesterone affects the signal transduction and subdomain localization of receptors via its influence on cholesterol trafficking. Since cholesterol-rich subdomains (rafts) are considered to be organization centers for cellular signal transduction, changes of the level or distribution of cholesterol may have profound effects on receptor-mediated signaling in general. Using fluorescence recovery after photobleaching (FRAP) measurements with GFP-tagged oxytocin receptors the influence of steroids on the mobility and distribution of the oxytocin receptor in the plasma membrane was analyzed. Progesterone had no effect on the lateral mobility of the oxytocin receptor, but it led to marked inhibition of cellular motility such as vesicle trafficking and movements of filopodia. Non-genomic effects of progesterone and estradiol with respect to receptor signaling as well as the influence of cholesterol on signal transduction will be discussed in more detail.
催产素受体系统的功能和生理调节强烈依赖于类固醇。出乎意料的是，这一点仅部分地反映在催产素受体的启动子序列中，这支持了转录后机制可能在催产素受体系统的生理调节中也发挥重要作用的观点。我们的数据表明，胆固醇作为催产素受体的变构调节剂，使膜相关的和可溶的催产素受体稳定在对激动剂和拮抗剂具有高亲和力的状态。此外，在稳定表达人催产素受体的HEK293成纤维细胞中，高亲和力的催产素受体在富含胆固醇的质膜结构域中富集了2倍。生化数据表明胆固醇分子与催产素受体之间存在直接的协同分子相互作用。为了定位催产素受体的胆固醇相互作用结构域，包含最后两个跨膜结构域的C末端部分已被胆囊收缩素B型受体的相应序列替换，该受体在功能上不依赖于胆固醇。关于其配体结合行为，这种嵌合受体蛋白对胆固醇及其类似物的依赖性与野生型催产素受体相同。从诱变实验以及催产素受体和胆囊收缩素B型受体之间的受体嵌合体研究中，我们得出结论，胆固醇相互作用结构域的主要部分可能位于催产素受体的第一部分，可能在激动剂结合位点附近的一个结构域中。孕酮被认为对维持子宫静息至关重要。高浓度的孕酮（>10 microM）通过快速、可逆和非基因组途径减弱或阻断包括催产素受体在内的几种GPCR的信号传导。已知孕酮会抑制胆固醇生物合成和胆固醇的细胞内运输。因此，我们检验了孕酮通过影响胆固醇运输来影响受体信号转导和亚结构域定位的假设。由于富含胆固醇的亚结构域（脂筏）被认为是细胞信号转导的组织中心，胆固醇水平或分布的变化可能总体上对受体介导的信号传导产生深远影响。使用带有GFP标签的催产素受体进行光漂白后荧光恢复（FRAP）测量，分析了类固醇对质膜中催产素受体的流动性和分布的影响。孕酮对催产素受体的侧向流动性没有影响，但它导致对细胞运动如囊泡运输和丝状伪足运动的显著抑制。将更详细地讨论孕酮和雌二醇关于受体信号传导的非基因组效应以及胆固醇对信号转导的影响。
