SIGNALING PATHWAYS OF ARF GTPASES AND ARF GAPS

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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.