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.
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.
