Effects of an F238L point mutation on intracellular trafficking and signaling of the cannabinoid type 1 receptor

The CB1 receptor is involved in many physiological functions, such as learning, memory and regulation of emotion. Furthermore, it plays a role in various pathological conditions, e.g. psychiatric and neurodegenerative. The CB1 receptors availability and its polarization towards the axon and the presynapse is amongst others regulated by endocytosis. It has been further suggested that endocytosis and polarization of the CB1 receptor positively correlate with the receptors activity. In this work, the effect of a F238L mutation in the fourth transmembrane domain of the CB1 receptor on its trafficking and signaling was examined. The CB1F238L receptor showed a reduced surface expression in HEK293 cells, which could be rescued by inverse agonist treatment and was found to be caused by an increased basal endocytosis via lipid rafts/caveolae. In line with this finding, an increased lipid raft allocation of the mutant CB1 receptor was observed. In accordance with the role of endocytosis in receptor polarization, the CB1F238L receptor showed increased axonal polarization, when ectopically expressed in primary hippocampal neurons. However, although the CB1F238L receptor showed increased endocytosis and polarization, its basal activity was found to be reduced. It was concluded that there might be other mechanisms for the regulation of CB1 receptor polarization, additionally to the receptors activity. The CB1 receptor is known to form heteromers with other GPCRs. Previous work by others showed the heteromerization of the CB1 receptor and the D2 receptor by co-immunoprecipitation and FRET. Additionally, activation of the CB1 receptor led to a reduction in the affinity of the D2 receptor for dopamine. In the present work, the co-immunoprecipitation of the two receptors was reproduced. Furthermore, it was shown that the mere co-expression of the CB1 receptor affects dopamine binding to the D2 receptor. In previous experiments, the CB1F238L mutation in rats was shown to lead to an increased binding potential of a D2 receptor PET radiotracer. In general, the binding potential of D2 radiotracers has been proposed to depend on D2 receptor expression and the subcellular localization of the receptor. However, previous experiments did not reveal changes in D2 receptor mRNA levels in the CB1F238L receptor mutant rat. Thus, further experiments were performed in order to determine the protein level of the D2 receptor in the striatum of the CB1F238L mutant rat. However, receptor autoradiography and western blot experiments yielded contradictory data. As the F238L mutation in the CB1 receptor affects the receptors trafficking, and as receptors can co-traffick in heteromers, it was then hypothesized that differential co-trafficking of the D2 receptor with the CB1F238L receptor could account for the differences observed in the PET ratiotracer binding potential. To test this hypothesis, HEK293 cells were generated which express the D2 receptor either alone or together with the CB1wt receptor or the CB1F238L receptor.Der CB1 Rezeptor ist an vielen physiologischen Prozessen beteiligt, wie beispielweise beim Lernen und Erinnern oder bei der Regulation von Emotionen. Dementsprechend ist dieser Rezeptor ebenfalls in pathologische Prozesse bei psychiatrischen oder neurodegenerativen Erkrankungen involviert. Die Verfügbarkeit des CB1 Rezeptors und dessen Transport zur Präsynapse wird unter anderem durch Endozytose reguliert. Es gibt Hinweise darauf, dass die Endozytose des CB1 Rezeptors wiederum in einem positiven Zusammenhang mit seiner Aktivität steht. In dieser Arbeit wurde der Effekt der F238L Mutation in der vierten Transmembrandomäne des CB1 Rezeptors auf dessen subzellulären Transport und dessen Signaltransduktion untersucht. Es wurde gezeigt, dass der CB1F238L Rezeptor eine verringerte Oberflächenexpression aufweist, was auf verstärkte „lipid raft/caveolae“ vermittelte Endozytose zurückgeführt werden konnte. Dementsprechend wurde eine verstärkte Anreicherung des CB1F238L Rezeptors in „lipid raft“ Präparationen beobachtet. Auch konnte die Oberflächenexpression durch die Stimulation mit einem inversen Agonisten wieder erhöht werden. In Übereinstimmung mit dem Zusammenhang zwischen Endozytose und axonalem Transport des CB1 Rezeptors wurde eine verstärkte axonale Polarisation des CB1F238L Rezeptors in transfizierten, primären Neuronen beobachtet. Obwohl die Endozytose und axonale Polarization des CB1F238L Rezeptors verstärkt ist, wurde eine verringerte basale Aktivität dieses Rezeptors gemessen. Eine Schlussfolgerung aus diesem Widerspruch ist, dass es neben der Rezeptoraktivität noch andere Mechanismen zur Regulation der axonalen Polarisation des Rezeptors gibt. Es ist bekannt, dass der CB1 Rezeptor mit dem Dopamin D2 Rezeptor heteromerisiert, wie in früheren Arbeiten mit Hilfe von Ko-Immunpräzipitationen und FRET gezeigt wurde. Die Aktivierung des CB1 Rezeptors reduziert die Affinität des D2 Rezeptors für Dopamin. In dieser Arbeit wurde die Ko-Immunpräzipitation des CB1 Rezeptors und des D2 Rezeptors reproduziert. Außerdem wurde gezeigt, dass bereits die Ko-Expression des CB1 Rezeptors die Affinität des D2 Rezeptors für Dopamin reduziert. In früheren Experimenten wurde ein verringertes Bindungspotential für einen D2 Rezeptor Radioliganden im Striatum der CB1F238L mutanten Ratte beobachtet, obwohl die D2 Rezeptor mRNA Menge unverändert war. Die durchgeführten Experimente ergaben ebenfalls widersprüchliche Ergebnisse hinsichtlich der D2 Rezeptor Proteinmenge im Striatum der CB1F238L mutanten Ratte. Da der CB1F238L Rezeptor veränderte Transporteigenschaften aufweist und da GPCRs in heteromeren ko-transportiert werden können, wurde eine Hypothese aufgestellt, die eine Veränderte subzelluläre Lokalisation des D2 Rezeptors mit einem veränderten Bindungspotential des D2 Rezeptor Radioliganden in Verbindung bringt. Für zukünftige Experimente zur Überprüfung dieser Hypothese wurden HEK293 Zellen hergestellt, die den D2 Rezeptor entweder allein, oder zusammen mit dem CB1wt Rezeptor oder dem CB1F238L Rezeptor exprimieren.159 S

The F238L Point Mutation in the Cannabinoid Type 1 Receptor Enhances Basal Endocytosis via Lipid Rafts

Defining functional domains and amino acid residues in G protein coupled receptors (GPCRs) represent an important way to improve rational drug design for this major class of drug targets. The cannabinoid type 1 (CB1) receptor is one of the most abundant GPCRs in the central nervous system and is involved in many physiological and pathophysiological processes. Interestingly, cannabinoid type 1 receptor with a phenylalanine 238 to leucine mutation (CB1F238L) has been already linked to a number of both in vitro and in vivo alterations. While CB1F238L causes significantly reduced presynaptic neurotransmitter release at the cellular level, behaviorally this mutation induces increased risk taking, social play behavior and reward sensitivity in rats. However, the molecular mechanisms underlying these changes are not fully understood. In this study, we tested whether the F238L mutation affects trafficking and axonal/presynaptic polarization of the CB1 receptor in vitro. Steady state or ligand modulated surface expression and lipid raft association was analyzed in human embryonic kidney 293 (HEK293) cells stably expressing either wild-type cannabinoid type 1 receptor (CB1wt) or CB1F238L receptor. Axonal/presynaptic polarization of the CB1F238L receptor was assessed in transfected primary hippocampal neurons. We show that in vitro the CB1F238L receptor displays increased association with lipid rafts, which coincides with increased lipid raft mediated constitutive endocytosis, leading to a reduction in steady state surface expression of the CB1F238L receptor. Furthermore, the CB1F238L receptor showed increased axonal polarization in primary hippocampal neurons. These data demonstrate that endocytosis of the CB1 receptor is an important mediator of axonal/presynaptic polarization and that phenylalanine 238 plays a key role in CB1 receptor trafficking and axonal polarization

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery