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

阿片受体（mu-opioid receptor, MOPr）是一种蛋白质受体，存在于大脑和脊髓某些神经细胞的膜中。它与体内的内啡肽和脑啡肽等化学物质相互作用（即接受），导致神经细胞的电兴奋性降低。在整个机体中，这导致了许多结果，最明显的是疼痛的缓解，尽管MOPr也控制着许多其他过程，包括情绪和情感，胃和肠的分泌物，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，许多化合物（配体）已被制成可以与该受体相互作用。虽然许多这些配体在与MOPr相互作用时似乎产生相似的作用，但我们最近发现一些配体与受体相互作用时产生的作用与其他配体明显不同。这可以看作是一种配体强烈激活一种特定的细胞内信号通路而不是另一种，而另一种配体可能更强烈地激活后一种途径。这种现象被称为“偏颇激动作用”，但迄今为止，产生偏颇激动作用的机制尚不清楚。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，而且磷酸化可以发生在MOPr的不同部分，这取决于它与哪个配体相互作用。磷酸化是指在蛋白质上加入磷酸基团，磷酸化通常会导致蛋白质以某种方式改变其功能，或者改变其做某事的速度。MOPr的磷酸化导致它在某些方面变得不那么活跃，但在其他方面变得更活跃。我们认为不同的MOPr配体使其以不同的方式磷酸化，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——我们想要研究不同的配体是否以不同的方式磷酸化MOPr，以及这是否会导致不同的信号传导，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，但可以在整个生物体中产生不同的作用。此外，这种现象也可能发生在许多其他类型的相关受体（bbb800在哺乳动物基因组中），因此MOPr可以作为一个很好的模型系统来验证我们的假设。因此，这项工作的主要目标是：(1)确定哪些部分MOPr磷酸化当它与不同的配体(2)使用这些知识使抗体能识别磷酸化MOPr和使用它们来看看不同的配体可能产生不同的磷酸化受体(3)上的事件调查使用细胞成像技术之间是否有相关性的数量和类型的磷酸化MOPr和MOPr所在地神经细胞(4)调查是否和大脑中的MOPr是如何被磷酸化的？(5)识别MOPr的偏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