Identification of the endophilins (SH3p4/p8/p13) as novel binding partners for the beta1-adrenergic receptor.

Several G-protein coupled receptors, such as the beta1-adrenergic receptor (beta1-AR), contain polyproline motifs within their intracellular domains. Such motifs in other proteins are known to mediate protein-protein interactions such as with Src homology (SH)3 domains. Accordingly, we used the proline-rich third intracellular loop of the beta1-AR either as a glutathione S-transferase fusion protein in biochemical "pull-down" assays or as bait in the yeast two-hybrid system to search for interacting proteins. Both approaches identified SH3p4/p8/p13 (also referred to as endophilin 1/2/3), a SH3 domain-containing protein family, as binding partners for the beta1-AR. In vitro and in human embryonic kidney (HEK) 293 cells, SH3p4 specifically binds to the third intracellular loop of the beta1-AR but not to that of the beta2-AR. Moreover, this interaction is mediated by the C-terminal SH3 domain of SH3p4. Functionally, overexpression of SH3p4 promotes agonist-induced internalization and modestly decreases the Gs coupling efficacy of beta1-ARs in HEK293 cells while having no effect on beta2-ARs. Thus, our studies demonstrate a role of the SH3p4/p8/p13 protein family in beta1-AR signaling and suggest that interaction between proline-rich motifs and SH3-containing proteins may represent a previously underappreciated aspect of G-protein coupled receptor signaling.

Receptor and G betagamma isoform-specific interactions with G protein-coupled receptor kinases.

The G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate and desensitize agonist-occupied GPCRs. GRK2-mediated receptor phosphorylation is preceded by the agonist-dependent membrane association of this enzyme. Previous in vitro studies with purified proteins have suggested that this translocation may be mediated by the recruitment of GRK2 to the plasma membrane by its interaction with the free betagamma subunits of heterotrimeric G proteins (G betagamma). Here we demonstrate that this mechanism operates in intact cells and that specificity is imparted by the selective interaction of discrete pools of G betagamma with receptors and GRKs. Treatment of Cos-7 cells transiently overexpressing GRK2 with a beta-receptor agonist promotes a 3-fold increase in plasma membrane-associated GRK2. This translocation of GRK2 is inhibited by the carboxyl terminus of GRK2, a known G betagamma sequestrant. Furthermore, in cells overexpressing both GRK2 and G beta1 gamma2, activation of lysophosphatidic acid receptors leads to the rapid and transient formation of a GRK/G betagamma complex. That G betagamma specificity exists at the level of the GPCR and the GRK is indicated by the observation that a GRK2/G betagamma complex is formed after agonist occupancy of the lysophosphatidic acid and beta-adrenergic but not thrombin receptors. In contrast to GRK2, GRK3 forms a G betagamma complex after stimulation of all three GPCRs. This G betagamma binding specificity of the GRKs is also reflected at the level of the purified proteins. Thus the GRK2 carboxyl terminus binds G beta1 and G beta2 but not G beta3, while the GRK3 fusion protein binds all three G beta isoforms. This study provides a direct demonstration of a role for G betagamma in mediating the agonist-stimulated translocation of GRK2 and GRK3 in an intact cellular system and demonstrates isoform specificity in the interaction of these components.

Impacts of Mountaintop Removal Coal Mining on the Mud River, West Virginia: Selenium Accumulation, Trophic Transfer, and Toxicity in Fish

Selenium (Se) is a micronutrient necessary for the function of a variety of important enzymes; Se also exhibits a narrow range in concentrations between essentiality and toxicity. Oviparous vertebrates such as birds and fish are especially sensitive to Se toxicity, which causes reproductive impairment and defects in embryo development. Selenium occurs naturally in the Earth's crust, but it can be mobilized by a variety of anthropogenic activities, including agricultural practices, coal burning, and mining.

Mountaintop removal/valley fill (MTR/VF) coal mining is a form of surface mining found throughout central Appalachia in the United States that involves blasting off the tops of mountains to access underlying coal seams. Spoil rock from the mountain is placed into adjacent valleys, forming valley fills, which bury stream headwaters and negatively impact surface water quality. This research focused on the biological impacts of Se leached from MTR/VF coal mining operations located around the Mud River, West Virginia.

In order to assess the status of Se in a lotic (flowing) system such as the Mud River, surface water, insects, and fish samples including creek chub (Semotilus atromaculatus) and green sunfish (Lepomis cyanellus) were collected from a mining impacted site as well as from a reference site not impacted by mining. Analysis of samples from the mined site showed increased conductivity and Se in the surface waters compared to the reference site in addition to increased concentrations of Se in insects and fish. Histological analysis of mined site fish gills showed a lack of normal parasites, suggesting parasite populations may be disrupted due to poor water quality. X-ray absorption near edge spectroscopy techniques were used to determine the speciation of Se in insect and creek chub samples. Insects contained approximately 40-50% inorganic Se (selenate and selenite) and 50-60% organic Se (Se-methionine and Se-cystine) while fish tissues contained lower proportions of inorganic Se than insects, instead having higher proportions of organic Se in the forms of methyl-Se-cysteine, Se-cystine, and Se-methionine.

Otoliths, calcified inner ear structures, were also collected from Mud River creek chubs and green sunfish and analyzed for Se content using laser ablation inductively couple mass spectrometry (LA-ICP-MS). Significant differences were found between the two species of fish, based on the concentrations of otolith Se. Green sunfish otoliths from all sites contained background or low concentrations of otolith Se (< 1 µg/g) that were not significantly different between mined and unmined sites. In contrast creek chub otoliths from the historically mined site contained much higher (≥ 5 µg/g, up to approximately 68 µg/g) concentrations of Se than for the same species in the unmined site or for the green sunfish. Otolith Se concentrations were related to muscle Se concentrations for creek chubs (R2 = 0.54, p = 0.0002 for the last 20% of the otolith Se versus muscle Se) while no relationship was observed for green sunfish.

Additional experiments using biofilms grown in the Mud River showed increased Se in mined site biofilms compared to the reference site. When we fed fathead minnows (Pimephales promelas) on these biofilms in the laboratory they accumulated higher concentrations of Se in liver and ovary tissues compared to fathead minnows fed on reference site biofilms. No differences in Se accumulation were found in muscle from either treatment group. Biofilms were also centrifuged and separated into filamentous green algae and the remaining diatom fraction. The majority of Se was found in the diatom fraction with only about 1/3rd of total biofilm Se concentration present in the filamentous green algae fraction

Finally, zebrafish (Danio rerio) embryos were exposed to aqueous Se in the form of selenate, selenite, and L-selenomethionine in an attempt to determine if oxidative stress plays a role in selenium embryo toxicity. Selenate and selenite exposure did not induce embryo deformities (lordosis and craniofacial malformation). L-selenomethionine, however, induced significantly higher deformity rates at 100 µg/L compared to controls. Antioxidant rescue of L-selenomethionime induced deformities was attempted in embryos using N-acetylcysteine (NAC). Pretreatment with NAC significantly reduced deformities in the zebrafish embryos secondarily treated with L-selenomethionine, suggesting that oxidative stress may play a role in Se toxicity. Selenite exposure also induced a 6.6-fold increase in glutathione-S-transferase pi class 2 gene expression, which is involved in xenobiotic transformation. No changes in gene expression were observed for selenate or L-selenomethionine-exposed embryos.

The findings in this dissertation contribute to the understanding of how Se bioaccumulates in a lotic system and is transferred through a simulated foodweb in addition to further exploring oxidative stress as a potential mechanism for Se-induced embryo toxicity. Future studies should continue to pursue the role of oxidative stress and other mechanisms in Se toxicity and the biotransformation of Se in aquatic ecosystems.