No description available.

No description available.

No description available.

No description available.

Regulators of G-protein signaling (RGS) modulate G-protein coupled receptor (GPCR) activity in vascular smooth muscle cells (VSMCs). One such protein, RGS5, has been shown to have selective expression in VSMCs and pericytes, and can inhibit signaling from Gαq and Gαi subunits. Using an RGS5 knockout model, we assessed the functional effect of RGS5 in the constriction and dilation of resistance arterioles. Furthermore, we examined the intracellular lipid interaction of RGS proteins to identify the determinants regulating the biologic function of RGS5. Surprisingly, loss of RGS5 function in mesenteric arterioles had no effect on constriction and dilation of resistance arterioles. Cultured VSMCs showed increased basal ERK1/2 phosphorylation and increased VASP signaling in response to SNP treatment in RGS5KO VSMCs as compared to wild type controls, with no effect on cell proliferation. These data suggest RGS5 may integrate multiple intracellular pathways with competing effects on VSMC contraction.

Regulators of G-protein signaling (RGS) modulate G-protein coupled receptor (GPCR) activity in vascular smooth muscle cells (VSMCs). One such protein, RGS5, has been shown to have selective expression in VSMCs and pericytes, and can inhibit signaling from Gαq and Gαi subunits. Using an RGS5 knockout model, we assessed the functional effect of RGS5 in the constriction and dilation of resistance arterioles. Furthermore, we examined the intracellular lipid interaction of RGS proteins to identify the determinants regulating the biologic function of RGS5. Surprisingly, loss of RGS5 function in mesenteric arterioles had no effect on constriction and dilation of resistance arterioles. Cultured VSMCs showed increased basal ERK1/2 phosphorylation and increased VASP signaling in response to SNP treatment in RGS5KO VSMCs as compared to wild type controls, with no effect on cell proliferation. These data suggest RGS5 may integrate multiple intracellular pathways with competing effects on VSMC contraction.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 6 uppsatser. Med sammanfattning på svenska.

G protein-coupled receptors (GPCRs) activate heterotrimeric G proteins (Gαβγ), which together form one of the most important signaling axes found in the cell. Because GPCRs are very common targets for therapeutic drugs, the mechanisms underlying their regulation are of high biomedical importance. Downstream of GPCR activation, there are many cytoplasmic proteins that regulate the activity of G proteins, providing an opportunity for therapeutic intervention. The neuronal protein GINIP binds directly to Gαi and is believed to play a role in modulating GPCR-mediated neurotransmission, an exquisitely-balanced process whereby dysregulation leads to neurological disorders including chronic pain and epilepsy. However, the molecular and structural determinants of GINIP underlying and required for proper regulation of G protein signaling downstream of GPCRs are unclear. In the studies presented here, we dissect the molecular and structural basis by which GINIP regulates G proteins after receptor activation and contributes to the fine-tuning of neurotransmitter responses in the nervous system. First, we revealed a new paradigm of G protein regulation by GINIP whereby it biases GPCR responses favoring Gβγ-mediated signaling to the detriment of Gα-mediated signaling. Second, we demonstrated that GINIP uses specific residues in the first loop of the PHD domain to physically engage Gαi by adopting a binding mode similar to that of G protein effectors like adenylyl cyclase, which is in turn required for the subsequent modulation of G protein signaling. Together, these insights advance our understanding of how GPCR signaling is fine-tuned by GINIP to set the tone of neurotransmission. Characterizing this layer of G protein regulation after receptor activation is crucial for developing novel therapeutic approaches to target diseases that arise from dysregulated GPCR signaling.

No description available.

G-protein coupled receptors (GPCRs) are integral membrane proteins which represent primary cellular targets for intracellular signalling. Many of these receptors are altered in disease states and hence are the target for over 50% of marketed drugs. Despite their physiological importance, high-throughput, cell-free assays which measure functional or signalling activity are only recently being investigated. The current approach by the pharmaceutical industry to initially screen compounds for functionality is to use heterologous cell-based assay formats. The aim of this work was to reconstitute a cell-free GPCR signalling system on an appropriate platform (surface) as a prototype for future rapid drug screening and other applications. The proof-of-concept approach involved using the �2A-adrenergic receptor (�2A-AR) containing cell membrane preparations as the model GPCR, reconstituted with a set of heterotrimeric G-proteins; G�i1 and �1�2 (the signal transducing complex being termed a �transductosome�). However, other receptors and G-proteins were also investigated. Receptors were initially obtained from natural (tissue) sources, however in the later stages they were expressed in a heterologous system (insect or mammalian expression system). G-proteins were expressed in Spodoptera frugiperida (Sf9) insect cells using the baculovirus expression system. Receptor expression was verified by radioligand binding assays and endogenous G-proteins were removed from membrane preparations using the chaotropic agent urea to allow for reconstitution with purified G-proteins. Signal transduction through the transductosome was measured using the [35S]GTP�S binding assay. Receptor activated [35S]GTP�S binding was used to determine functional reconstitution and to validate that the system was working in the normal physiological manner both on and off a surface (with surface attachment being via histidine attachment on the G�i1 (6xHIS) subunit). Using the captured (surface-attached) transductosomes, the IC50 values for Rauwolscine, Yohimbine (potent �2-AR antagonists), Prazosin (potent �1- AR antagonist) and Propranolol (�-AR antagonist) displayed the appropriate rank order for this class of receptor. This cell-free, surface-attached signalling complex prototype may have use in the future development of drug screening and discovery assay technologies as well as other applications as an alternative to cell-based assays which are not readily amendable to miniaturisation, long term storage and therefore stable robust microarray formats.