Brazill D T, Lindsey D F, Bishop J D, Gomer R H
Howard Hughes Medical Institute, Department of Biochemistry and Cell Biology, MS-140, Rice University, Houston, Texas 77005-1892, USA.
J Biol Chem. 1998 Apr 3;273(14):8161-8. doi: 10.1074/jbc.273.14.8161.
When the unicellular eukaryote Dictyostelium discoideum starves, it senses the local density of other starving cells by simultaneously secreting and sensing a glycoprotein called conditioned medium factor (CMF). When the density of starving cells is high, the corresponding high density of CMF permits signal transduction through cAR1, the chemoattractant cAMP receptor. cAR1 activates a heterotrimeric G protein whose alpha-subunit is Galpha2. CMF regulates cAMP signal transduction in part by regulating the lifetime of the cAMP-stimulated Galpha2-GTP configuration. We find here that guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) inhibits the binding of CMF to membranes, suggesting that the putative CMF receptor is coupled to a G protein. Cells lacking Galpha1 (Galpha1 null) do not exhibit GTPgammaS inhibition of CMF binding and do not exhibit CMF regulation of cAMP signal transduction, suggesting that the putative CMF receptor interacts with Galpha1. Work by others has suggested that Galpha1 inhibits phospholipase C (PLC), yet when cells lacking either Galpha1 or PLC were starved at high cell densities (and thus in the presence of CMF), they developed normally and had normal cAMP signal transduction. We find that CMF activates PLC. Galpha1 null cells starved in the absence or presence of CMF behave in a manner similar to control cells starved in the presence of CMF in that they extend pseudopods, have an activated PLC, have a low cAMP-stimulated GTPase, permit cAMP signal transduction, and aggregate. Cells lacking Gbeta have a low PLC activity that cannot be stimulated by CMF. Cells lacking PLC exhibit IP3 levels and cAMP-stimulated GTP hydrolysis rates intermediate to what is observed in wild-type cells starved in the absence or in the presence of an optimal amount of CMF. We hypothesize that CMF binds to its receptor, releasing Gbetagamma from Galpha1. This activates PLC, which causes the Galpha2 GTPase to be inhibited, prolonging the lifetime of the cAMP-activated Galpha2-GTP configuration. This, in turn, allows cAR1-mediated cAMP signal transduction to take place.
当单细胞真核生物盘基网柄菌饥饿时，它通过同时分泌和感知一种名为条件培养基因子（CMF）的糖蛋白来检测其他饥饿细胞的局部密度。当饥饿细胞的密度较高时，相应高密度的CMF允许通过趋化因子cAMP受体cAR1进行信号转导。cAR1激活一种异源三聚体G蛋白，其α亚基是Gα2。CMF部分通过调节cAMP刺激的Gα2 - GTP构象的寿命来调节cAMP信号转导。我们在此发现，鸟苷5'-3 - O -（硫代）三磷酸（GTPγS）抑制CMF与膜的结合，这表明推定的CMF受体与一种G蛋白偶联。缺乏Gα1（Gα1缺失）的细胞不表现出GTPγS对CMF结合的抑制作用，也不表现出CMF对cAMP信号转导的调节作用，这表明推定的CMF受体与Gα1相互作用。其他人的研究表明Gα1抑制磷脂酶C（PLC），然而，当缺乏Gα1或PLC的细胞在高细胞密度下饥饿时（因此在CMF存在的情况下），它们正常发育且具有正常的cAMP信号转导。我们发现CMF激活PLC。在有无CMF存在的情况下饥饿的Gα1缺失细胞的行为方式与在有CMF存在的情况下饥饿的对照细胞相似，即它们伸出伪足，具有激活的PLC，具有低的cAMP刺激的GTP酶，允许cAMP信号转导并聚集。缺乏Gβ的细胞具有低的PLC活性，且不能被CMF刺激。缺乏PLC的细胞表现出的肌醇三磷酸（IP3）水平和cAMP刺激的GTP水解速率介于在无CMF或有最佳量CMF存在的情况下饥饿的野生型细胞所观察到的中间水平。我们假设CMF与其受体结合，使Gβγ从Gα1上释放。这激活PLC，导致Gα2 GTP酶被抑制，延长cAMP激活的Gα2 - GTP构象的寿命。反过来，这允许cAR1介导的cAMP信号转导发生。
