SIGNALING PATHWAYS OF ARF GTPASES AND ARF GAPS

G蛋白偶联受体以激动剂依赖的方式激活异三聚体G蛋白。这些激活受体的G蛋白偶联受体激酶(GRK)的磷酸化促进了arrestin蛋白的结合，从而阻止了G蛋白的进一步激活，导致激活的受体与G蛋白解偶联。这种调节机制的一个值得注意的特征是，像G蛋白本身一样，GRK与激动剂占据的受体结合并被激活。GRKs的这种膜募集可能起到直接的信号作用，GRKs作为适配器将相关蛋白带到膜上，以响应受体的激活。GIT1是一种新的GRK相关蛋白，在HEK293细胞中过表达导致激动剂依赖的β2肾上腺素能受体cAMP信号、磷酸化和隔离发生显著变化。Git蛋白是ARF小GTP结合蛋白的GTP酶激活蛋白家族，当GIT1的ARF GAP活性被阻断时，GIT1过度表达的细胞效应被阻断。GIT1改变了G蛋白偶联受体的功能，而G蛋白偶联受体似乎是通过被笼罩的凹坑内化的，但不改变使用其他内化机制的受体的功能。这些观察表明，ARF和ARF GAP蛋白的Git家族在调节受体进出质膜的运输方面发挥着重要作用，从而影响受体信号转导。Git蛋白还与几个不同的信号和支架蛋白在复合体中相互作用，这些蛋白可能通过rac1和CDc42小GTP结合蛋白以及ARF将Git功能与细胞骨架的调节联系起来。GIT12还与GRKs相互作用，这进一步表明G蛋白偶联受体可能利用这些相互作用来影响各种小的GTP结合蛋白及其后续效应物的活性。这项研究的主要假设是G蛋白偶联受体可以通过GRKs和GRK相关蛋白(如GIT1)发出信号。这个项目的主要目的有两个：确定Git和ARF蛋白在调节G蛋白偶联受体信号转导和调节G蛋白偶联受体细胞定位中的作用。拟议的实验采用生化和细胞生物学技术相结合的方法来确定Git蛋白和Git相关蛋白作为异源三聚体G蛋白偶联受体信号和功能调节因子的功能意义。

G protein-couple receptors activate heterotrimeric G proteins in an agonist-dependent manner. G protein-coupled receptor kinase (GRK) phosphorylation of these activated receptors facilitates arrestin protein binding, which prevents further G protein activation and results in uncoupling of activated receptors from G proteins. A noteworthy feature of this regulatory mechanism is that, like the G proteins themselves, GRKs bind to and are activated by agonist-occupied receptors. This membrane recruitment of GRKs may serve a direct signaling role in which GRKs act as adaptors to bring associated proteins to the membrane in response to receptor activation. Over-expression in HEK293 cells of GIT1, a novel GRK-associated protein, leads to significant alterations in agonist-dependent beta2-adrenergic receptor cAMP signaling, phosphorylation and sequestration. GIT proteins are a family of GTPase- activating proteins (GAPs) for ARF small GTP-binding proteins, and the cellular effects of GIT1 over-expression are blocked when the ARF GAP activity of GIT1 is ablated. GIT1 alters the function of G protein-coupled receptors that appear to internalize via clathrin-coated pits, but not of receptors that use other internalization mechanisms. These observations imply that ARF and the GIT family of ARF GAP proteins play an important role in regulation of receptor trafficking to and from the plasma membrane, and thereby influence receptor signaling. GIT proteins also interact in a complex with several distinct signaling and scaffolding proteins that may link GIT function to regulation of the cytoskeleton via the rac1 and cdc42 small GTP-binding proteins, as well as ARF. That GIT12 also interacts with GRKs further suggests that G protein-coupled receptors may utilize these interactions to influence the activity of various small GTP-binding proteins and their subsequent effectors. The main postulate underlying this study is that G protein-coupled receptors can signal via GRKs and GRK-associated proteins, such as GIT1. The major aims of this project are two-fold: to determine the role of GIT and ARF proteins in regulating G protein-coupled receptor signaling and in regulating G protein-coupled receptor cellular localization. The proposed experiments employ a combination of biochemical and cell biological techniques to define the functional significance of GIT proteins and GIT- associated proteins as regulators of heterotrimeric G protein coupled receptor signaling and function.