Over the past two decades, a vast body of research has demonstrated the importance of G protein-coupled receptor kinase 2 (GRK2) in the physiology and pathophysiology of the heart. Adrenergic receptors are the primary target for GRK2 activity in the heart; phosphorylation by GRK2 leads to desensitization of these receptors. As such, levels of GRK2 activity in the heart directly correlate with cardiac contractile function. Furthermore, increased expression of GRK2 following cardiac insult exacerbates injury and speeds progression to heart failure. In this dissertation we turned our attention towards two novel aspects of GRK2 biology. Firstly, despite the importance of this GRK2 activity in both the physiology and pathophysiology of the heart, relatively little is known about the role of GRK2 in skeletal muscle function and disease. In the first study of this dissertation, we generated a novel skeletal muscle specific GRK2 knockout (KO) mouse (MLC-Cre:GRK2fl/fl) to gain a better understanding of the role of GRK2 in skeletal muscle physiology. In isolated muscle mechanics testing, GRK2 ablation caused a significant decrease in the specific force of contraction of the fast-twitch extensor digitorum longus muscle, yet had no effect on the slow-twitch soleus muscle. Despite these effects in isolated muscle, exercise capacity was not altered in MLC-Cre:GRK2fl/fl mice compared to wild-type controls. Skeletal muscle hypertrophy stimulated by clenbuterol, a β2-adrenergic receptor agonist, was significantly enhanced in MLC-Cre:GRK2fl/fl mice; mechanistically, this seems to be due to increased clenbuterol-stimulated pro-hypertrophic Akt signaling in the GRK2 KO skeletal muscle. In summary, this study provides the first insights into the role of GRK2 in skeletal muscle physiology, and points to a role for GRK2 as a modulator of contractile properties in skeletal muscle as well as β2-adrenergic receptor-induced hypertrophy. In the second part of this dissertation, we report surprising novel metabolic phenotypes that arise from modulating GRK2 activity exclusively in the heart. We show that transgenic βARKct (TgβARKct) mice (cardiac specific expression of a GRK2 inhibitory peptide) are more susceptible to high-fat diet (HFD) induced obesity. TgβARKct mice exhibit marked increase in adiposity on HFD relative to control animals. Conversely transgenic GRK2 mice (TgGRK2) mice (cardiac specific overexpression of GRK2) show resistance to weight gain on a HFD and decrease in adipose tissue mass relative to control animals. Furthermore, conditioned media from βARKct expressing neonatal rat ventricular myocytes enhances adipocyte differentiation in vitro. These results suggest that the heart produces a secreted factor to control whole body metabolism, and that GRK2 is a regulator of this mechanism. / Biomedical Sciences

Rationale: Pathological remodeling of the heart is a hallmark of chronic heart failure (HF) and these structural changes further perpetuate the disease. Cardiac fibroblasts are the critical cell type that is responsible for maintaining the structural integrity of the heart. Stress conditions, such as a myocardial infarction (MI), can activate quiescent fibroblasts into synthetic and contractile myofibroblasts. G protein-coupled receptor (GPCR) kinase (GRK) 5 is an important mediator of cardiovascular homeostasis through dampening of GPCR signaling, and is expressed in the heart and upregulated in human HF. Of note, GRK5 has been demonstrated to translocate to the nucleus in cardiomyocytes in a calcium- calmodulin (Ca2+-CAM)-dependent manner, promoting hypertrophic gene transcription through activation of NFAT. Interestingly, NFAT is also involved in fibroblast activation. GRK5 is highly expressed and active in cardiac fibroblasts (CFs), however its pathophysiological role in these crucial cardiac cells is unknown. Objective: The aim of this study is to elucidate the role of GRK5 in the activation of cardiac fibroblasts in vitro and cardiac fibrosis after injury in vivo. Methods and Results: We demonstrate using adult cardiac fibroblasts that genetic deletion of GRK5 inhibits Angiotensin II (AngII) mediated fibroblast activation. Fibroblast-specific deletion of GRK5 in mice decreased fibrosis and cardiac hypertrophy after chronic AngII infusion compared to non-transgenic littermate controls (NLCs). Fibroblast-specific deletion of GRK5 was also protective in mice after ischemic injury as they presented with preserved systolic function, decreased fibrosis, and decreased hypertrophy compared to NLCs. Mechanistically, we show that nuclear translocation of GRK5 is involved in fibroblast activation. Conclusions: We present novel data demonstrating that GRK5 is a regulator of fibroblast activation in vitro and cardiac fibrosis in vivo. This adds to previously published data which demonstrates the potential beneficial effects of GRK5 inhibition in the context of cardiac disease. / Biomedical Sciences

Les récepteurs couplés aux protéines G (RCPG) peuvent activer des voies de signalisation indépendantes des protéines G. Cependant, la régulation de ces voies, et en particulier leur désensibilisation, est peu connue. Le récepteur de la sérotonine de type 4 (R-5-HT4) est un RCPG exprimé dans le cerveau et les organes périphériques. Il est impliqué dans des fonctions physiologiques importantes comme la mémoire, l'apprentissage, la prise de nourriture, le contrôle respiratoire et la mobilité gastro-intestinale. Le R-5-HT4 est couplé à la protéine Gs. De plus, il active la voie Src/ERK1/2, indépendamment des protéines G et des β-arrestines.Nous avons montré que GRK5, physiquement associé à la région C-terminale (C-ter) du R-5-HT4 inhibait la voie Src/ERK1/2 couplée au récepteur, mais pas la voie Gs. Ce résultat a été observé dans la lignée de cellules HEK-293 mais aussi dans des neurones de collicules en culture. Cette inhibition nécessite deux séquences d'évènements : l'association de la β-arrestine1 à une région riche en sérines et thréonines, localisée dans le domaine C-ter du récepteur et la phosphorylation par GRK5, de la β-arrestine1 (en sérine 412) liée au récepteur. La β-arrestine1 phosphorylée empêche l'activation de Src, constitutivement liée au récepteur, nécessaire à l'activation d'ERK1/2. Ceci constitue la première démonstration que la phosphorylation d'une β-arrestine par une GRK régule la signalisation indépendante des protéines G. En plus de ces résultats, nous avons démontré que l'activation d'ERK1/2 par le R-5-HT4, indépendante des β-arrestines, implique la libération d'un ligand induite par une métalloprotéase, conduisant à la transactivation d'un autre récepteur. Par une approche protéomique, nous avons également identifiés plusieurs partenaires potentiels du R-5-HT4. L'étude de ces partenaires pourrait apporter un éclairage supplémentaire sur les voies de signalisation du récepteur et leur régulation. / G protein-coupled receptors (GPCRs) have been found to trigger G protein-independent signalling. However, the regulation of G protein-independent pathways, especially their desensitization, is poorly characterized.The 5-Hydroxytryptamine 4 receptor (5-HT4R) is a GPCR widely expressed in the brain and at the periphery. It is implicated in important physiological functions such as memory, cognition, feeding, respiratory control and gastrointestinal motility. 5-HT4R couples to the Gs/cAMP/PKA pathway. Moreover, this receptor can activate a Src/ERK pathway independently of both G proteins and β-arrestins.Here, we show that the G protein-independent 5-HT4R-operated Src/ERK pathway, but not the Gs pathway, is inhibited by GPCR kinase 5 (GRK5), physically associated with the proximal region of receptor C-terminus, in both HEK-293 cells and colliculi neurons. This inhibition requires two sequences of events: the association of β-arrestin1 to a phosphorylated serine/threonine cluster located within the receptor C-terminal domain and the phosphorylation by GRK5 of β-arrestin1 (at Ser 412) bound to the receptor. Phosphorylated β-arrestin1 prevents in turn activation of Src constitutively bound to 5-HT4R, a necessary step in receptor-stimulated ERK signalling. This is the first demonstration that β-arrestin phosphorylation by a GRK regulates G protein-independent signalling.In addition to these results, we also demonstrated that the β-arrestin-independent activation of ERK1/2 by the 5-HT4R involves a metalloprotease-dependant ectodomain shedding and transactivation of another receptor. By a proteomic approach, we also identified several potential partners of the 5-HT4R. Study of these proteins may provide a better understanding of 5-HT4R signalling and his regulation.

ENZYMATIC REGULATION OF OPIOID ANTINOCICEPTION AND TOLERANCE By Lynn C. Hull, Ph.D. A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University. Virginia Commonwealth University, 2009 Director: William L. Dewey, Ph.D. Department of Pharmacology and Toxicology The involvement of kinases in opioid actions has long been established. The acute actions of opioids, through the Gi/Go G-proteins, cause the inhibition of adenylyl cyclase and therefore a decrease in protein kinase A (PKA) activation. Additionally, acute opioid administration may cause the G-protein to activate the phospholipase C (PLC)-mediated cascade leading to the activation of protein kinase C (PKC). The phosphorylation of the MOR which can lead to both desensitization by uncoupling of the G-protein coupled receptors (GPCRs) from the G-proteins and to internalization by recruitment of β-arrestins has long been identified as a key process in tolerance. Phosphorylation by PKA and PKC leads primarily to uncoupling of the receptor from the G-proteins. Phosphorylation of the receptor by G-protein coupled receptor kinase (GRK) leads to the recruitment of β-arrestins and internalization of the receptor. Many in vitro studies have come to the conclusion that GRK induced internalization plays a more central role in the tolerance to high efficacy opioids and a lesser role in low- and moderate-efficacy opioid tolerance. In fact it has been hypothesized that morphine, a moderate-efficacy opioid, causes no internalization at all, while the desensitization of the receptor via phosphorylation by PKA and PKC plays a more central role in low- and moderate-efficacy opioid tolerance. We sought to test these in vitro findings in an in vivo model of opioid tolerance. Animals were made tolerant to one of a number of opioids of varying efficacy (low-efficacy meperidine, moderate-efficacy morphine and fentanyl, and high-efficacy [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO)) over an 8 hour period and then were administered one of the kinases’ inhibitors. Tolerance reversal was determined by challenging these mice with the same opioids to which they were tolerant. Calcium is known to play an important role in the acute antinociceptive actions of opioids as well as in opioid tolerance. Therefore it is important to determine how opioids are affecting the regulation of intracellular calcium. Our laboratory has previously shown that Calcium Induced Calcium Release (CICR), the ryanodine receptor and intracellular microsomal Ca2+ pools all play a role in opioids’ actions. It is also well known that mammalian ADP-ribosyl cyclase, CD38’s, product cADPR acts on the ryanodine receptor to cause Ca2+ release into the intracellular space. We chemically and genetically altered CD38 and then tested the acute effect of morphine as well as what effect these treatments had on morphine tolerance to determine what role if any, that CD38 may play in the acute actions of morphine antinociception as well as in morphine tolerance. Together, studies focusing on the role of an ADP-ribosyl cyclase, CD38, and 3 separate kinases, PKA, PKC and GRK, in opioids’ actions were performed in order to better understand the roles of these enzymes’ pathways in the actions of opioid-induced antinociception and subsequent development of tolerance. It is hoped that the results herein add useful knowledge to the general understanding of this drug class, and will one day be of use in the development of future analgesics and in the clinical treatment of pain and reduction in tolerance.

<p>Chemokine receptors are members of the seven transmembrane receptor (7TMR) superfamily and are regulated by the G-protein coupled Receptor Kinase (GRK)/ b-arrestin system. CCL19 and CCL21 are endogenous agonists for the chemokine receptor CCR7. They are known to be equipotent in promoting Gi/o mediated calcium mobilization, chemotaxis and inhibition of adenylyl cyclase activity. Here we test the hypothesis that these ligands are biased agonists that differentially activate the G-protein coupled Receptor Kinase (GRK)/ b-arrestin system.</p><p>In order to test whether these ligands have distinct activity, murine T lymphocytes were used to compare the effects of CCL19 and CCL21 activation of CCR7 at endogenous expression levels. While each ligand stimulates similar chemotactic responses, we also find that CCR7 ligands lead to differential signaling. For instance, CCL19 is markedly more efficacious than CCL21 for the activation of ERK and JNK, but not AKT in these cells. Furthermore, ERK activation and chemotaxis are maintained as separate pathways, also distinguishable by their dependency upon PKC and PI3 kinase, respectively. Thus, CCL19 and CCL21 stimulate equal activation of PI3 kinase, AKT, and chemotaxis, but are in fact biased agonists leading to differential activation of MAP kinase in murine T lymphocytes. </p><p>To determine the mechanism of CCR7 ligand bias, we used HEK-293 cells expressing CCR7 to compare the proximate signaling events following CCL19 and CCL21 activation. We found striking differences in the activation of the GRK/ b-arrestin system. CCL19 leads to robust CCR7 phosphorylation and b-arrestin2 recruitment catalyzed by both GRK3 and GRK6 while CCL21 activates GRK6 alone. This differential GRK activation leads to distinct functional consequences. Only CCL19 leads to the recruitment of b-arrestin2-GFP into endocytic vesicles and classical receptor desensitization. In contrast, each agonist is fully capable of signaling to MAP kinase through b-arrestin2 in a GRK6 dependent fashion. </p><p>Therefore, CCR7 and its ligands represent a natural example of ligand bias whose mechanism involves differential GRK isoform utilization by CCL19 and CCL21 despite similar G-protein signaling. This study suggests that the GRK signatures of 7TMRs can determine the function of discrete pools of b-arrestin and thus guide its cellular effects.</p> / Dissertation

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In this essay the author has chosen to analyze the similarities and differences between some of the northern Europe’s late Mesolithic and Neolithic cultures. The research is of a processual standpoint and the information is mainly gathered from secondary sources as well as ethnological studies. The material collected has been analyzed in both a processual and a post-processual manner to most accurately study the foundations of the three primary theories describing the Pitted Ware Culture’s origin. During the course of the study the author also found a possible fourth alternative, and the possibility of exogamy as a factor in the emerge of the Pitted Ware Culture has also been questioned. This work has prompted for a rigorous collection of information to be able to properly present all theories strong and weak points, without bias for any theory. The conclusion of these studies is that it is very hard to pinpoint exactly what it is that has given the Pitted Ware Culture its typical cultural traits, although a possible sequence of events has been presented in this text.

mRNA localisation is a widely used mechanism for achieving temporal-spatial restriction of protein expression and is essential during development to establish cell polarity. mRNA localisation is particularly well studied in the Drosophila egg chamber where gurken mRNA is localised to the dorsal-anterior corner of the oocyte in a Dynein-dependent process that establishes the anterior-posterior and dorsal-ventral axes of the future embryo. An RNA stem-loop called the gurken localisation signal is necessary and sufficient to drive gurken localisation through interactions with a specific complement of protein factors. However, the exact RNA sequence and structural features required to promote each stage of gurken localisation are unknown. Using a live-cell injection assay I have dissected regions of the mRNA signal that are responsible for driving gurken transport and anchoring through their association with Egalitarian, Me31B and Squid proteins. I show the structure of an AU-rich stem and a purine stack are essential for gurken transport, and demonstrate that the size of the internal loop between these stems is important. These features of the localisation signal are essential for recruitment of Egalitarian, which links the mRNA to the Dynein transport machinery. I also show that these mRNA sequence and structural elements are present in several other Dynein-transported mRNAs. The bulge at the distal end of the gurken localisation signal is important for anchoring grk at the dorsal-anterior of the oocyte, possibly through Squid binding, and the proximal third of the signal is essential for recruitment of the translation component Me31B. These studies indicate that the role of the gurken localisation signal in controlling gurken transport, anchoring and translation can be mapped to distinct regions of the signal and provide insights into how the signal carries out these numerous functions at a molecular level. Determining the molecular interactions involved in mRNA localisation improves our understanding of how specificity is generated to direct different mRNAs to distinct regions of the cell to restrict protein expression.