Xiang Yang, Rybin Vitalyi O, Steinberg Susan F, Kobilka Brian
Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Stanford Medical Center, Palo Alto, CA 94305, USA.
J Biol Chem. 2002 Sep 13;277(37):34280-6. doi: 10.1074/jbc.M201644200. Epub 2002 Jul 3.
There is a growing body of evidence that G protein-coupled receptors function in the context of plasma membrane signaling compartments. These compartments may facilitate interaction between receptors and specific downstream signaling components while restricting access to other signaling molecules. We recently reported that beta(1)- and beta(2)-adrenergic receptors (AR) regulate the intrinsic contraction rate in neonatal mouse myocytes through distinct signaling pathways. By studying neonatal myocytes isolated from beta(1)AR and beta(2)AR knockout mice, we found that stimulation of the beta(1)AR leads to a protein kinase A-dependent increase in the contraction rate. In contrast, stimulation of the beta(2)AR has a biphasic effect on the contraction rate. The biphasic effect includes an initial protein kinase A-independent increase in the contraction rate followed by a sustained decrease in the contraction rate that can be blocked by pertussis toxin. Here we present evidence that caveolar localization is required for physiologic signaling by the beta(2)AR but not the beta(1)AR in neonatal cardiac myocytes. Evidence for beta(2)AR localization to caveolae includes co-localization by confocal imaging, co-immunoprecipitation of the beta(2)AR and caveolin 3, and co-migration of the beta(2)AR with a caveolin-3-enriched membrane fraction. The beta(2)AR-stimulated increase in the myocyte contraction rate is increased by approximately 2-fold and markedly prolonged by filipin, an agent that disrupts lipid rafts such as caveolae and significantly reduces co-immunoprecipitation of beta(2)AR and caveolin 3 and co-migration of beta(2)AR and caveolin-3 enriched membranes. In contrast, filipin has no effect on beta(1)AR signaling. These observations suggest that beta(2)ARs are normally restricted to caveolae in myocyte membranes and that this localization is essential for physiologic signaling of this receptor subtype.
越来越多的证据表明，G蛋白偶联受体在质膜信号区室的背景下起作用。这些区室可能促进受体与特定下游信号成分之间的相互作用，同时限制与其他信号分子的接触。我们最近报道，β(1)-和β(2)-肾上腺素能受体（AR）通过不同的信号通路调节新生小鼠心肌细胞的内在收缩率。通过研究从β(1)AR和β(2)AR基因敲除小鼠分离的新生心肌细胞，我们发现刺激β(1)AR会导致蛋白激酶A依赖性的收缩率增加。相比之下，刺激β(2)AR对收缩率有双相作用。双相作用包括收缩率最初的蛋白激酶A非依赖性增加，随后是收缩率的持续下降，这可被百日咳毒素阻断。在此我们提供证据表明，在新生心肌细胞中，β(2)AR的生理信号传导需要小窝定位，而β(1)AR则不需要。β(2)AR定位于小窝的证据包括共聚焦成像的共定位、β(2)AR与小窝蛋白3的共免疫沉淀，以及β(2)AR与富含小窝蛋白3的膜组分的共迁移。破坏脂筏（如小窝）并显著减少β(2)AR与小窝蛋白3的共免疫沉淀以及β(2)AR与富含小窝蛋白3的膜的共迁移的试剂菲律宾菌素，使β(2)AR刺激的心肌细胞收缩率增加约2倍并显著延长。相比之下，菲律宾菌素对β(1)AR信号传导没有影响。这些观察结果表明，β(2)AR通常局限于心肌细胞膜中的小窝，并且这种定位对于该受体亚型的生理信号传导至关重要。
