Do biased agonists at the mu-opioid receptor induce different patterns of receptor phosphorylation?

Mu-阿片受体(MOPr)是一种蛋白质受体，存在于大脑和脊髓的某些神经细胞膜上。它与体内的内啡肽和脑啡肽等化学物质相互作用，导致神经细胞的电兴奋性降低。在整个生物体中，这会导致许多结果，其中最显著的是缓解疼痛，尽管MOPr还控制着许多其他过程，包括情绪和情绪、胃和肠道的分泌、呼吸频率和体温。因此，MOPr是一种非常重要的受体，因此多年来已经制造了许多能够与该受体相互作用的化合物(配体)。虽然这些配体中的许多在与MOPr相互作用时似乎产生了相似的作用，但我们最近发现，一些配体与受体相互作用，产生与其他配体显著不同的作用。这可以看作是一个配体强烈激活了一个特定的细胞内信号通路而不是另一个，而另一个配体可能更强烈地激活了后者。这种现象被称为“偏向激动症”，但迄今为止，产生偏向激动症的机制尚不清楚。我们认为，不同的配体能够与MOPr结合，使受体呈现不同的形状。这不仅使MOPr容易受到称为磷酸化的化学过程的影响，而且磷酸化可以在MOPr的不同部分发生，这取决于它与哪种配体相互作用。磷酸化是指将被称为磷酸基团的化学基团添加到蛋白质中，磷酸化通常会导致蛋白质以某种方式改变其功能，或改变它做某事的速度。MOPr的磷酸化导致它在某些方面变得不那么活跃，但在另一些方面变得更活跃。我们认为，不同的MOPr配体以不同的方式使其磷酸化，从而导致不同的细胞信号输出。这就是我们在这个提案中想要测量的--我们想要调查不同的配体是否以不同的方式磷酸化MOPr，以及这是否导致不同的信号转导，从而在生物体中产生不同的影响。从这项工作中获得的知识将使我们以一种全新的方式看待MOPr等受体，这意味着我们可以制造与相同受体(MOPr)结合的配体，但可能会在整个生物体中产生不同的影响。此外，这种现象很可能发生在许多其他类型的相关受体(哺乳动物基因组中的&gt；800)，因此MOPr可以作为一个很好的模型系统来检验我们的假设。因此，这项工作的主要目的是：(1)当MOPr与不同的配体相互作用时，准确地识别MOPr的哪些部分被磷酸化(2)利用这一知识制造识别磷酸化的MOPr的抗体，并利用它们来观察不同的配体如何在受体上产生不同的磷酸化事件(3)使用细胞成像技术研究MOPr的磷酸化程度和类型是否与MOPr位于神经细胞中的位置之间存在相关性(4)研究大脑中的MOPr是否以及如何被磷酸化(5)识别偏向MOPr的配体可能产生的不同细胞信号

The mu-opioid receptor (MOPr) is a protein receptor found in the membrane of certain nerve cells in the brain and spinal cord. It interacts with (i.e. is receptive to) chemicals such as endorphins and enkephalins in the body causing a reduction in the electrical excitability of the nerve cells. In the whole organism, this leads to a number of outcomes, most notably the relief of pain, although MOPr also controls a number of other processes including mood and emotion, secretions in the stomach and intestine, the rate of breathing, and body temperature. The MOPr is therefore a very important receptor and consequently over the years many chemical compounds (ligands) have been made that can interact with this receptor. Although many of these ligands seem to produce similar effects when they interact with MOPr, we have recently found that some ligands interact with the receptor to produce significantly different effects from others. This can be seen as one ligand strongly activating one particular intracellular signaling pathway rather than another, whereas another ligand might activate the latter pathway more strongly. This phenomenon has been called 'biased agonism', but as yet the mechanisms involved in producing biased agonism are not known. We think that what happens is that the different ligands are able to bind to the MOPr and make the receptor take up different shapes. This not only makes the MOPr susceptible to a chemical process called phosphorylation, but the phosphorylation can take place on different parts of the MOPr, depending upon which ligand it is interacting with. Phosphorylation is where chemical groups called phosphate groups are added to a protein, and phosphorylation normally leads to the protein changing its function in some way, or the rate at which it does something. Phosphorylation of MOPr leads to it becoming less active in some regards, but more active in others. We think that different MOPr ligands make it become phosphorylated in different ways and this leads to distinct cell signaling outputs. This is what we want to measure in this proposal - we want to investigate whether different ligands phosphorylate MOPr in different ways, and whether this leads to different signaling and hence different effects in the organism. Knowledge gained from this work will allow us regard receptors such as MOPr in a completely new way, meaning that we could make ligands that bind to the same receptor (MOPr), but can lead to different effects in the whole organism. In addition, this phemomenon is likely to occur for many of the other types of related receptor (>800 in the mammalian genome) and so the MOPr can be used as an excellent model system to test out our hypothesis. The major aims of this work are therefore to: (1) identify exactly which parts of MOPr are phosphorylated when it interacts with different ligands (2) use this knowledge to make antibodies that will recognize the phosphorylated MOPr and use them to see how different ligands might produce different phosphorylation events on the receptor (3) investigate using cell imaging technology whether there is a correlation between the amount and type of phosphorylation of MOPr and where the MOPr is located in a nerve cell (4) investigate whether and how MOPr in brain is phosphorylated (5) identify the different cellular signals that biased ligands at MOPr might produce