Ladds Graham, Davis Kevin, Hillhouse Edward W, Davey John
Department of Biological Sciences, University of Warwick, Coventry, UK.
Mol Microbiol. 2003 Feb;47(3):781-92. doi: 10.1046/j.1365-2958.2003.03336.x.
G protein-coupled receptors (GPCRs) help to regulate the physiology of all the major organ systems. They respond to a multitude of ligands and activate a range of effector proteins to bring about the appropriate cellular response. The choice of effector is largely determined by the interaction of individual GPCRs with different G proteins. Several factors influence this interaction, and a better understanding of the process may enable a more rational approach to identifying compounds that affect particular signalling pathways. A number of systems have been developed for the analysis of GPCRs. All provide useful information, but the genetic amenability and relative simplicity of yeast makes them a particularly attractive option for ligand identification and pharmaceutical screening. Many, but not all, GPCRs are functional in the budding yeast Saccharomyces cerevisiae, and we have developed reporter strains of the fission yeast Schizosaccharomyces pombe as an alternative host. To provide a more generic system for investigating GPCRs, we created a series of yeast-human Galpha-transplants, in which the last five residues at the C-terminus of the yeast Galpha-subunit are replaced with the corresponding residues from different human G proteins. These enable GPCRs to be coupled to the Sz. pombe signalling machinery so that stimulation with an appropriate ligand induces the expression of a signal-dependent lacZ reporter gene. We demonstrate the specificity of the system using corticotropin releasing factor (CRF) and CRF-related peptides on two CRF receptors. We find that different combinations of ligand and receptor activate different Galpha-transplants, and the specificity of the coupling is similar to that in mammalian systems. Thus, CRF signalled through the Gs- and Gi-transplants, consistent with its regulation of adenylate cyclase, and was more active against the CRF-R1A receptor than against the CRF-R2B receptor. In contrast, urocortin II and urocortin III were selective for the CRF-R2B receptors. Furthermore, urocortin, but not CRF, induced signalling through the CRF-R1A receptor and the Gq-transplant. This is the first time that human GPCRs have been coupled to the signalling pathway in Sz. pombe, and the strains described in this study will complement the other systems available for studying this important family of receptors.
G蛋白偶联受体（GPCRs）有助于调节所有主要器官系统的生理功能。它们对多种配体作出反应，并激活一系列效应蛋白以引发适当的细胞反应。效应器的选择在很大程度上取决于单个GPCR与不同G蛋白的相互作用。有几个因素会影响这种相互作用，更好地理解这一过程可能有助于采用更合理的方法来鉴定影响特定信号通路的化合物。已经开发了许多用于分析GPCR的系统。所有这些系统都提供了有用的信息，但酵母的遗传易处理性和相对简单性使其成为配体鉴定和药物筛选的特别有吸引力的选择。许多（但不是全部）GPCR在出芽酵母酿酒酵母中具有功能，我们已经开发了裂殖酵母粟酒裂殖酵母的报告菌株作为替代宿主。为了提供一个更通用的系统来研究GPCR，我们创建了一系列酵母-人Gα移植体，其中酵母Gα亚基C末端的最后五个残基被来自不同人类G蛋白的相应残基取代。这些使得GPCR能够与粟酒裂殖酵母的信号传导机制偶联，从而用适当的配体刺激诱导信号依赖性lacZ报告基因的表达。我们使用促肾上腺皮质激素释放因子（CRF）和与CRF相关的肽在两种CRF受体上证明了该系统的特异性。我们发现配体和受体的不同组合激活不同的Gα移植体，并且偶联的特异性与哺乳动物系统中的相似。因此，CRF通过Gs和Gi移植体发出信号，与其对腺苷酸环化酶的调节一致，并且对CRF-R1A受体的活性比对CRF-R2B受体的活性更高。相比之下，尿皮质素II和尿皮质素III对CRF-R2B受体具有选择性。此外，尿皮质素而非CRF通过CRF-R1A受体和Gq移植体诱导信号传导。这是人类GPCR首次与粟酒裂殖酵母中的信号通路偶联，本研究中描述的菌株将补充可用于研究这一重要受体家族的其他系统。
