The cellular degradation of the low density lipoprotein (LDL) receptor, and its ligand, LDL, were investigated in order to clarify certain mechanistic aspects of these important processes. Long-term lymphoblastoid cell lines and cultured human skin fibroblasts were used to examine the fate of ¹²⁵I-LDL subsequent to its uptake via receptor-mediated endocytosis. In both cases, binding activity was saturable, depended on the presence of calcium ions in the medium, and was calculated to have an equilibrium dissociation constant at 4ᵒC of 2 μg ¹²⁵I-LDL/ml. No high-affinity binding was detected when the ligand was modified by acetylation. After incubating the monolayers at 37°C LDL/LDL receptor complexes were internalized, and the receptors were recycled back to the surface within about 10 minutes. Apolipo-protein B in the LDL particles was largely degraded to the amino acid level: chloroquine, a lysosomotropic agent, inhibited the formation of the ¹²⁵I-LDL degradation products. Cells obtained from a number of heterozygous and homozygous familial hypercholesterolemic patients, as expected, bound markedly reduced amounts of ligand. The half-life of ¹²⁵I-LDL was measured after it had been introduced into cultured fibroblasts by one of the following processes: (i) uptake via receptor-mediated endocytosis in human skin fibroblasts with normal LDL receptors, or (ii) incorporation via scrape-loading into fibroblasts defective in LDL receptor content. The half-lives obtained were about 1 hour and 50 hours, respectively, indicating that efficient degradation of LDL occurred only when it was deIivered to lysosomes via receptor-mediated endocytosis.

Immunohistochemical studies were conducted to assess the subunits of ionotropic and metabotropic glutamate receptor present in the rostral ventrolateral medulla (RVLM) of the rat. Double labeling the medullary sections with polyclonal GluR1, GluR2/3, GluR4, NMDAR1, NMDAR2A/B, mGluR1α, and mGluR2/3 antiserum and monoclonal tyrosine hydroxylase (TH) antiserum revealed nearly all TH immunoreactive (irTH) cells and many TH-negative neurons were immunoreactive to GluR2/3 (irGluR2/3), NMDAR1 (irNMDAR1), and NMDAR2A/B (irNMDAR2A/B). A few RVLM neurons were immunoreactive to GluR1 (irGluR1) and GluR4 (irGluR4), but they were generally TH-negative. Immunoreactivity to mGluR1α (irmGluR1α) appeared to be localized exclusively to fiber-like elements in the RVLM area. Our results show that neurons in the RVLM, including irTH, are endowed mainly with GluR2/3 and NMDAR1 or NMDAR2A/B ionotropic receptor subunits, and that irmGluR1α splice variant appears to be located on nerve fibers ramifying within the RVLM. Moreover, TH-negative neurons in the RVLM appear to bear similar subunits of ionotropic glutamate receptors.

The objective of the study was to determine if chronic interruption of all extrinsic nerve inputs to the heart alters cholinergic-mediated responses within the intrinsic cardiac nervous system (ICN). Extracardiac nerve inputs to the ICN were surgically interrupted (ICN decentralized). Three weeks later, the intrinsic cardiac right atrial ganglionated plexus (RAGP) was removed and intrinsic cardiac neuronal responses were evaluated electrophysiologically. Cholinergic receptor abundance was evaluated using autoradiography. In sham controls and chronic decentralized ICN ganglia, neuronal postsynaptic responses were mediated by acetylcholine, acting at nicotinic and muscarinic receptors. Muscarine- but not nicotine-mediated synaptic responses that were enhanced after chronic ICN decentralization. After chronic decentralization, muscarine facilitation of orthodromic neuronal activation increased. Receptor autoradiography demonstrated that nicotinic and muscarinic receptor density associated with the RAGP was unaffected by decentralization and that muscarinic receptors were tenfold more abundant than nicotinic receptors in the right atrial ganglia in each group. After chronic decentralization of the ICN, intrinsic cardiac neurons remain viable and responsive to cholinergic synaptic inputs. Enhanced muscarinic responsiveness of intrinsic cardiac neurons occurs without changes in receptor abundance.

The CXC chemokine receptor 4 (CXCR4) and the atypical chemokine receptor 3 (ACKR3) are seven transmembrane receptors that are involved in numerous pathologies, including several types of cancers. Both receptors bind the same chemokine, CXCL12, leading to significantly different outcomes. While CXCR4 activation generally leads to canonical GPCR signaling, involving Gi proteins and β‐arrestins, ACKR3, which is predominantly found in intracellular vesicles, has been shown to signal via β‐arrestin‐dependent signaling pathways. Understanding the dynamics and kinetics of their activation in response to their ligands is of importance to understand how signaling proceeds via these two receptors. In this thesis, different Förster resonance energy transfer (FRET)‐based approaches have been combined to individually investigate the early events of their signaling cascades. In order to investigate receptor activation, intramolecular FRET sensors for CXCR4 and ACKR3 were developed by using the pair of fluorophores cyan fluorescence protein and fluorescence arsenical hairpin binder. The sensors, which exhibited similar functional properties to their wild‐type counterparts, allowed to monitor their ligand-induced conformational changes and represent the first RET‐based receptor sensors in the field of chemokine receptors. Additional FRET‐based settings were also established to investigate the coupling of receptors with G proteins, rearrangements within dimers, as well as G protein activation. On one hand, CXCR4 showed a complex activation mechanism in response to CXCL12 that involved rearrangements in the transmembrane domain of the receptor followed by rearrangements between the receptor and the G protein as well as rearrangements between CXCR4 protomers, suggesting a role of homodimers in the activation course of this receptor. This was followed by a prolonged activation of Gi proteins, but not Gq activation, via the axis CXCL12/CXCR4. In contrast, the structural rearrangements at each step of the signaling cascade in response to macrophage migration inhibitory factor (MIF) were dynamically and kinetically different and no Gi protein activation via this axis was detected. These findings suggest distinct mechanisms of action of CXCL12 and MIF on CXCR4 and provide evidence for a new type of sequential signaling events of a GPCR. Importantly, evidence in this work revealed that CXCR4 exhibits some degree of constitutive activity, a potentially important feature for drug development. On the other hand, by cotransfecting the ACKR3 sensor with K44A dynamin, it was possible to increase its presence in the plasma membrane and measure the ligand‐induced activation of this receptor. Different kinetics of ACKR3 activation were observed in response to CXCL12 and three other agonists by means of using the receptor sensor developed in this thesis, showing that it is a valuable tool to study the activation of this atypical receptor and pharmacologically characterize ligands. No CXCL12‐induced G protein activation via ACKR3 was observed even when the receptor was re-localized to the plasma membrane by means of using the mutant dynamin. Altogether, this thesis work provides the temporal resolution of signaling patterns of two chemokine receptors for the first time as well as valuable tools that can be applied to characterize their activation in response to pharmacologically relevant ligands. / Der CXC Chemokin‐Rezeptor 4 (CXCR4) und der atypische Chemokin‐Rezeptor 3 (ACKR3) sind heptatransmembranäre Rezeptoren, die in zahlreichen Krankheitsbildern eine Rolle spielen, wie in einigen Krebsarten. Beide Rezeptoren werden zwar von dem gleichen Chemokin CXCL12 aktiviert, allerdings mit unterschiedlichen Signalweiterleitungsmustern. Die Aktivierung von CXCR4 führt zu kanonischer GPCR Signaltransduktion über Gi‐Proteine und β‐Arrestine. Die Signalweiterleitung des Rezeptors ACKR3 hingegen, welcher hauptsächlich in intrazellulären Vesikeln vorliegt, erfolgt über ß‐Arrestinabhängige Signalwege. Es ist von großer Wichtigkeit die Dynamik und Kinetik dieser beiden Rezeptoren hinsichtlich der Aktivierung durch ihre Liganden und der Signalweiterleitung zu verstehen. In dieser Arbeit wurden verschiedene Förster‐Resonanzenergietransfer (FRET) Anwendungen kombiniert, um die frühen Phasen der Signal‐Kaskade von CXCR4 und ACKR3 zu untersuchen. Zur genaueren Aufklärung der Rezeptoraktivierung wurden intramolekulare FRET‐Sensoren entwickelt, hierzu wurden die Fluorophore Cyan‐fluoreszierendes Protein und engl. fluorescence arsenical hairpin binder verwendet. Die generierten Sensoren zeigten ähnliche funktionelle Eigenschaften wie die unveränderten Rezeptoren. Liganden‐induzierte Änderungen der Rezeptorkonformation können mittels dieser Sensoren beobachtet werden und stellen die ersten RET‐basierten Sensoren auf dem Forschungsgebiet der Chemokin‐Rezeptoren dar. Weitere FRET‐basierte Methoden wurden zur Untersuchung von Interaktionen zwischen Rezeptor und G‐Protein, Neuanordnung von Dimeren, sowie der G‐Protein Aktivierung eingesetzt und für beide Chemokin‐Rezeptoren etabliert. CXCR4 zeigte einen komplexen Aktivierungsmechanismus nach Stimulation durch CXCL12, bei welchem zunächst eine Neuordnung der Rezeptor‐Transmembrandomäne gefolgt von Neuordnungen zwischen Rezeptor und G‐Protein und zuletzt eine Neuordnung zwischen CXCR4 Protomeren erfolgte. Dies impliziert, dass im Aktivierungsprozess des Rezeptors Homodimere eine Rolle spielen. Zudem wurde eine verlängerte Gi ‐Protein Aktivierung gegenüber der Gq‐Protein Aktivierung bei CXCL12 stimuliertem CXCR4 beobachtet. Hingegen zeigte eine Stimulierung mit dem Macrophage Migration Inhibitory Factor (MIF) bei jedem Schritt der frühen Singal‐Kaskade veränderte Dynamiken und Kinetiken im Vergleich zu CXCL12. Darüber hinaus konnte keine Gi ‐Protein Aktivierung festgestellt werden. Dieser Befund zeigt individuelle Mechanismen für MIF und CXCL12 am CXCR4‐Rezeptor und liefert Belege für eine neuer Art von sequenziellen Signalweiterleitungen an GPCRs. Eine wichtige Beobachtung dieser Arbeit für eine potentielle Medikamentenentwicklung ist das CXCR4 ligandenunabhängige Aktivität zeigt. Um die Aktivierung des ACKR3 Sensors messen zu können wurde durch eine Co‐Transfektion mit K44A Dynamin eine höhere Membranständigkeit erreicht. CXCL12 und drei weiteren Agonisten zeigten am hier entwickelten ACKR3‐Sensor unterscheidbare Kinetiken. Mit diesem wertvollen Werkzeug können Liganden an diesem atypischen Rezeptor pharmakologisch charakterisiert werden. Es konnte keine CXCL12‐induzierte G‐Protein Aktivierung gemessen werden, trotz der stärkeren Präsenz an der Plasmamembran mit Hilfe der Dynamin‐Mutante. In Summe liefert diese Arbeit zum ersten Mal eine zeitliche Auflösung von Signalweiterleitungsmustern von zwei Chemokin‐Rezeptoren sowie wertvolle Werkzeuge zur Charakterisierung der frühen Phase der Signal‐Kaskade durch andere pharmakologisch relevanten Liganden.

Phosducin (Pdc) is a 28 kDa binding partner for the G protein beta gamma subunit dimer (G-beta-gamma) found abundantly in the photoreceptor cells of the retina and pineal gland. In the retina, light-dependent changes in cAMP and Ca2+ control the phosphorylation of Pdc at serine 73 and 54, respectively, which in turn controls the binding of Pdc to G protein beta gamma subunit dimer . G protein beta gamma subunit dimer binding has been proposed to facilitate light-driven transport of G protein beta gamma subunit dimer from the site of phototransduction in the outer segment of the photoreceptor cell to the inner segment, thereby decreasing light sensitivity and contributing to the process of light adaptation. Dopamine and melatonin are neuromodulators whose concentrations in the retina vary reciprocally during the circadian cycle, with dopamine high during the day and melatonin high during the night. Together, they control numerous aspects of light and dark adaptation in the retina. In this study, we have investigated the possible roles of dopamine and melatonin in regulating Pdc phosphorylation. Using phosphorylation-site specific antibodies to serines 54 and 73, we show that dopamine decreases the phosphorylation of both sites. This decrease is blocked by D4 receptor antagonists and pertussis toxin, indicating that dopamine causes a decrease in photoreceptor cell cAMP and Ca2+ concentration via the D4 receptor coupled to the Gi protein. Conversely, melatonin increases the phosphorylation of both S54 and S73, most likely via the inhibition of dopamine synthesis. These results demonstrate that dopamine and melatonin control the phosphorylation state of phosducin by changing the concentration of cAMP and Ca2+ in photoreceptor cells, and they suggest that dopamine and melatonin may contribute to the light-induced movement of the photoreceptor G protein by regulating Pdc phosphorylation.

Calcium-influx through ionotropic glutamate receptors expressed on non-excitable cells, such as CNS glia, may regulate important cell events via intracellular signaling mechanisms. Oligodendrocytes and oligodendrocyte progenitors (OPCs), two glial populations supporting CNS myelination and myelin repair, express AMPA and NMDA receptors. Although calcium-influx through these receptors is thought to cause glutamate excitotoxicity to oligodendrocytes in CNS injuries, more recent studies suggest that AMPA or NMDA receptor-mediated synaptic transmission between neurons and OPCs plays a positive role in neuronal activity-dependent oligodendrocyte development and regeneration. Given the opposing roles of glutamate receptors in oligodendrocyte death and repair, the clinical relevance of these receptors in white matter injuries remain unclear. Another major challenge for exploring the role of these receptors in white matter injuries is that OPCs and neurons express a similar complement of AMPA and NMDA receptor subunits, which has complicated the interpretation of pharmacological manipulations and global genetic deletion approaches. To define the cell autonomous role of AMPA and NMDA receptor-mediated calcium signaling in oligodendroglia, I abolished the calcium influx through glutamate receptors using two different genetic approaches, and examined their impacts on oligodendrocyte development, injury-induced cell death, and regeneration. First, I employed a new mouse line which allows overexpression of GluA2, the calcium-impermeable AMPA receptor subunit, in a Cre activity-dependent manner. After crossing these mice with OPC- or oligodendrocyte-lineage-specific Cre mice, I applied hypoxic-ischemic injury to these multiple transgenic mice. Surprisingly, even though AMPA receptor-mediated calcium influx was blocked in OPCs, oligodendrogenesis or myelin integrity was not affected. However, GluA2 overexpression significantly promoted oligodendrocyte regeneration and OPC proliferation after injury, while the same manipulation in oligodendrocytes did not protect them from the initial cell loss. Moreover, GluA2 overexpression also stimulated transcriptional activities linked to myelinogenesis, even without injury. Second, I used conditional knockout mice for Grin1, the gene encoding an essential subunit of NMDA receptor complexes. As with GluA2 overexpressing mice, the removal of NMDA receptors from OPCs or all oligodendroglia did not significantly change normal oligodendrocyte development. However, the ablation of NMDA receptor in OPCs exacerbated oligodendrocyte loss by impairing new oligodendrogenesis in hypoxic-ischemic injury. These results suggest that neither AMPA receptors nor NMDA receptors mediate glutamate excitotoxicity in oligodendrocytes in neonatal hypoxic-ischemic injury. Instead, these receptors play distinct roles in post-injury oligodendrocyte development: AMPA receptor-mediated calcium suppresses oligodendrocyte regeneration, and NMDA receptor signaling supports oligodendrocyte regeneration after injury. / Biomedical Sciences

The innate immune system houses cellular signaling proteins called pattern recognition receptors (PRRs) that are responsible for recognizing highly-conserved molecular patterns associated with pathogens or damage to elicit an immune response. However, NLRX1 is a unique PRR in the NOD-like receptor (NLR) family that instead functions to attenuate pro-inflammatory pathways that are activated by other PRRs, such as NF-κB and type-1 interferon signaling which both have implications in cancer. NLRX1 can regulate additional cancer-associated pathways, such as MAPK and AKT, and cancer-associated functions like metabolism and reactive oxygen species (ROS) production. Interestingly, depending on the type and subtype of cancer, NLRX1 can either be tumor promoting or tumor suppressing. Here, we investigate the role of NLRX1 in two deadly cancers: triple-negative breast cancer (TNBC) and pancreatic cancer. In a murine mammary tumor model that highly mimics TNBC, we discovered that NLRX1 is protective against disease burden in vivo when NLRX1 is expressed in healthy host cells. NLRX1 exerts its protection through limiting the recruitment of eosinophils to the tumor, suppressing epithelial-mesenchymal transition (EMT), and attenuating the formation of the metastatic niche. Conversely, when NLRX1 is instead expressed by the mammary tumor cells, NLRX1 promotes cancer-associated characteristics in vitro and disease burden in vivo by promoting EMT. This indicates that the role of NLRX1 in TNBC is highly dependent on cellular context. Conversely, in murine pancreatic cancer cells, we found that NLRX1 expression by the tumor cells is protective against cancer-associated characteristics in vitro, and that this is likely driven by NF-κB, MAPK, AKT, and inflammasome signaling with a potential to also limit immune evasion. Together, this research indicates that the role of NLRX1 can be highly variable based on the cell and tumor type and identifies the underlying mechanisms through which NLRX1 functions in these two cancer models. This is critical information for drug development initiatives so therapies can be developed that target NLRX1 in the appropriate cell type and in the appropriate disease. / Doctor of Philosophy / Inflammation, which is characterized by redness, heat, pain, swelling, and sometimes loss of function, is a critical way in which our bodies fight infections and repair tissue damage. However, chronic inflammation occurs when our bodies are unable to turn inflammation off and can result in cancerous mutations. Therefore, the successful resolution of inflammation is critical to maintaining inflammatory balance and has previously been dubbed the "Goldilocks Conundrum". The immune system houses a class of cellular signaling proteins called pattern recognition receptors (PRRs), which often function to turn inflammation on. However, a unique PRR in the NOD-like receptor (NLR) family called "NLRX1" functions to turn inflammation off and therefore plays an important role in preventing damaging chronic inflammation. NLRX1 has historically been studied in the context of infectious diseases, but because NLRX1 is involved in inflammation and because inflammation is a critical factor of cancer, its role as a tumor suppressor or tumor promoter has recently become an area of interest. NLRX1 has also been found to regulate biological pathways beyond inflammation that are also important for cancer initiation and progression. Interestingly, depending on the type and subtype of cancer, NLRX1 can either be tumor promoting or tumor suppressing. Here, we investigate the role of NLRX1 in two deadly cancers: triple-negative breast cancer (TNBC) and pancreatic cancer. In a mouse mammary tumor model that highly mimics TNBC, we discovered that NLRX1 is protective against disease burden when NLRX1 is expressed in healthy, non-tumor cells. NLRX1 exerts its protection through impacting the immune cells recruited to the tumor, limiting the ability of the tumor cells to leave the original tumor and spread throughout the body in the process known as metastasis, and suppressing the formation of a favorable tumor metastasis environment in the lung. Conversely, when NLRX1 is instead expressed by the mammary tumor cells, NLRX1 promotes disease burden by helping tumor cells leave the original tumor and spread throughout the body. This indicates that the role of NLRX1 in TNBC is highly dependent on cellular context, including if the cell is healthy or cancerous. Conversely, in mouse pancreatic cancer cells, we found that NLRX1 expression by the tumor cells is protective against cancer-associated characteristics. Together, this research indicates that the role of NLRX1 can be highly variable based on the cell and tumor type. This is critical information for drug development initiatives so therapies can be developed that turn NLRX1 on or off in the appropriate cell type and in the appropriate disease.

A study was conducted to determine the effects of trenbolone acetate (TBA) and estradiol-17B (E2) implantation on the characteristics of the glucocorticoid and E2 receptor, skeletal muscle protein turnover and tissue growth. Twenty-four lambs were utilized. Trenbolone acetate did not ,affect (P>.10) degradation rates in the semitendinosus (ST) and triceps brachii (TB) muscles, the production of cortisol, adrenal weights and cytosolic glucocorticoid binding capacity (Bmax). Trenbolone acetate decreased synthesis rate of muscle protein (P<.Ol), the percent of [3H] dexamethasone binding in the nuclear fraction, Bmax and the disociation constant (Kd) of the cytosolic E2 receptor, only in the TB muscle. Deoxyribonucleic acid (DNA) of the TB was increased (P<.05) with TBA. Pituitary weights were decreased (P<.005) with TBA and increased (P<.Ol) with E2. Estradiol decreased (P<.05) Bmax of the cytosolic E2 receptor in the ST and decreased (P<.05) Bmax of the nuclear E2 receptor in the TB muscle. The TB muscle had greater (P<.05) synthesis rates than the ST and the protein:RNA ratio was decreased (P<.05) in the TB. The TB muscle had greater (P<.005) Bmax for the cytosolic glucocorticoid receptor. / Master of Science

Avian intestinal cytosoluble receptors for 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) were subjected to limited trypsin digestion, endogenous proteolytic action, as well as carboxypeptidase treatment, and the physical and functional properties of the resulting discrete polypeptide fragments were identified and contrasted with the native 1,25(OH)₂D₃ receptor. Resultant fragments were followed by tracing either radioactive 1,25(OH)₂D₃ or by probing with anti-receptor monoclonal antibodies. Two differentially trypsin-sensitive effects on the 1,25(OH)₂D₃ receptor were noted when fragments were detected by their ability to bind 1,25(OH)₂[³H]D₃. Two hormone-bound fragments of 40 and 30 kDa were formed; neither bound to DNA-cellulose nor anti-receptor monoclonal antibodies. Immunoblot technology was used to show the disappearance of the 60 kDa receptor with increasing trypsin concentrations, paralleling the appearance of an immunoreactive 20 kDa fragment. The 20 kDa fragment did not bind hormone but was capable of interacting with DNA-cellulose in a fashion identical to that of the 60 kDa receptor. This fragment is likely the complementary fragment to the hormone-bound fragment of 40 kDa that is described above. In contrast to the exogeneous effect of trypsin, incubation of chick intestinal cytosol resulted in the time-dependent formation of an endogenous protease-derived fragment of 45 kDa. This species retained the hormone-binding site and the antibody determinant, but was devoid of DNA-binding activity. Moreover, it did not generate the trypsin-dependent 20 kDa fragment and therefore was derived from the opposite end of the receptor molecule. Carboxypeptidase treatment of the 1,25(OH)₂D₃ receptor produces a 56 kDa fragment which does not retain hormone, but which does bind to DNA-cellulose and monoclonal antibody. These combined data from various limited enzymatic cleavage studies of the receptor have facilitated the construction of a schematic model of the chick receptor in which the immunoreactive epitope is located between the N-terminal DNA-binding domain and the C-terminal hormone-binding domain. This map for the 1,25(OH)₂D₃ receptor protein is consistent with the general structure of steroid and thyroid hormone receptors and places the vitamin D hormone receptor in a class of macromolecules that are postulated to bind enhancer regions of responsive DNA and thereby control target gene transcription.

No description available.