Transgenic Targeting of Vascular G Protein Signaling

描述(申请人提供)：以激动剂依赖的方式，G蛋白偶联受体激酶(GRKs)磷酸化7个跨膜受体(7TMR)，导致经典的同源脱敏。有7个GRK(GRK1-7)，它们似乎有特定的7TMR底物。GRK磷酸化的7TMR通过arrestin结合而被抑制与其正常的异三聚体G蛋白结合伙伴。重要的是，最近人们认识到，尽管经典信号可能被磷酸化减敏和/或下调，但现在可能发现由受体磷酸化刺激的替代信号通路。例如，很大一部分血管紧张素II或加压素受体的GRK磷酸化是由GRK2介导的，并导致arrestin的募集和内化。然而，最近的研究表明，血管紧张素II或加压素刺激后Arrestin介导的ERK激活需要GRK5的磷酸化。此外，21-肾上腺素能受体(21AR)通常不与腺酰环化酶抑制的异三聚体G蛋白GI偶联，然而，培养细胞中的数据表明，GRK5使21AR的磷酸化可以允许GI偶联，这是由于GRK5的作用导致21AR的羧尾与PSD-95之间的解离。当22AR被蛋白激酶A(PKA)磷酸化时，GS-GI开关也被显示出来。在血管平滑肌(VSM)中，由于Gs介导血管扩张，而GI激活导致血管收缩，2AR-GI偶联将产生深刻的生理效应。因此，了解7TMR信号在磷酸化后的变化对于更好地了解信号在疾病状态下的变化至关重要。事实上，在高血压的动物模型中，GI与高血压的发生和发展有关。此外，GRK5的表达和活性在高血压中升高，我们发现仅GRK5的VSM过度表达就足以导致高血压。重要的是，我们有初步数据表明，GRK5可以诱导VSM中21AR的Gs到GI的转换，这可能是当GRK5水平增加时导致高血压的至少一个机制。我们的中心假设是，GRK5修饰的“非经典”7TMR信号促进了其他信号通路的选择，使GI介导的信号成为高血压发展和/或进展中VSM血管收缩加剧的关键成分。针对这一假说的具体目的包括：1.阐明G蛋白偏好转换通过增加GRK5表达所起的作用。2.阐明GRK5缺失对替代信号通路和高血压发生发展的影响。3.确定VSM GI介导的信号在正常和病理生理环境下BP的建立中的作用。公共卫生相关性：在高血压等疾病状态下，许多蛋白质的表达水平会发生变化。重要的是确定这些蛋白质，特别是具有酶活性的蛋白质，如GRK5，如何扰乱正常的信号模式，以制定更有效地治疗和/或预防心血管疾病的策略。这些实验的数据也应该增加我们对21AR信号在高血压环境中的理解，并有助于开发新的抗高血压治疗策略。

DESCRIPTION (provided by applicant): In an agonist-dependent manner, G protein-coupled receptor kinases (GRKs) phosphorylate 7 transmembrane spanning receptors (7TMRs) leading to classical homologous desensitization. There are 7 GRKs (GRK1-7) and they appear to have specific 7TMR substrates. GRK-phosphorylated 7TMRs are inhibited from association with their normal heterotrimeric G protein binding partner via arrestin binding. Importantly, it has recently been appreciated that although classical signaling might be desensitized and/or downregulated with phosphorylation, alternative signaling pathways may now be uncovered that are stimulated by receptor phosphorylation. For example, a large portion of GRK phosphorylation of either angiotensin II or vasopressin receptor is GRK2 mediated and leads to arrestin recruitment and internalization. However, it has recently been shown that arrestin-mediated ERK activation subsequent to either angiotensin II or vasopressin stimulation requires GRK5 phosphorylation. In addition, 21-adrenergic receptors (21ARs) generally do not couple to the adenylyl cyclase inhibitory heterotrimeric G protein, Gi, however, data in cultured cells indicate that phosphorylation of 21ARs by GRK5 can allow for Gi-coupling due to GRK5 actions causing a dissociation between the carboxyl-tail of 21ARs and PSD-95. Gs-Gi switching has also been shown for the 22AR when this receptor is phosphorylated by protein kinase A (PKA). In vascular smooth muscle (VSM), 2AR-Gi coupling would have profound physiological effects since Gs mediates vasodilation whereas Gi activation leads to vasoconstriction. Therefore, understanding changes in 7TMR signaling subsequent to phosphorylation is critical to gain a better insight into how signaling may change during disease states. In fact, Gi has been implicated in the development and progression of hypertension in animal models of the disease. In addition, GRK5 expression and activity is elevated in hypertension and we have found that VSM overexpression of GRK5 alone is sufficient to cause hypertension. Importantly, we have preliminary data to suggest that GRK5 can induce Gs to Gi switching of 21ARs in VSM and that this is likely at least one mechanism underlying hypertension when GRK5 levels are increased. Our Central Hypothesis is that "non-classical" 7TMR signaling as modified by GRK5 facilitates alternative signaling pathways such that Gi-mediated signaling becomes a critical component in the exacerbation of VSM vasoconstriction in the development and/or progression of hypertension. The Specific Aims addressing this hypothesis include: 1. To elucidate the role of G protein preference switching through increased GRK5 expression. 2. To delineate how the lack of GRK5 affects alternative signaling pathways and the development of high blood pressure (BP). 3. To determine the role of VSM Gi-mediated signaling in the establishment of BP under normal and pathophysiological settings. PUBLIC HEALTH RELEVANCE: There are changes in expression levels of numerous proteins during disease states such as hypertension. It is important to determine how these proteins, and particularly proteins with enzymatic activity such as GRK5, disrupt normal signaling patterns to develop strategies to more effectively treat and/or prevent cardiovascular disease. Data from these experiments should also increase our understanding of 21AR signaling in the setting of high blood pressure and help in the development of novel antihypertensive therapeutic strategies.