Novel Mechanisms in the Regulation of G Protein-coupled Receptor Trafficking to the Plasma Membrane

β₂-Adrenergic receptors (β₂-AR) are low abundance, integral membrane proteins that mediate the effects of catecholamines at the cell surface. Whereas the processes governing desensitization of activated β₂-ARs and their subsequent removal from the cell surface have been characterized in considerable detail, little is known about the mechanisms controlling trafficking of neo-synthesized receptors to the cell surface. Since the discovery of the signal peptide, the targeting of the integral membrane proteins to plasma membrane has been thought to be determined by structural features of the amino acid sequence alone. Here we report that localization of translationally silenced β₂-AR mRNA to the peripheral cytoplasmic regions is critical for receptor localization to the plasma membrane. β₂-AR mRNA is recognized by the nucleocytoplasmic shuttling RNA-binding protein HuR, which silences translational initiation while chaperoning the mRNA-protein complex to the cell periphery. When HuR expression is down-regulated, β₂-AR mRNA translation is initiated prematurely in perinuclear polyribosomes, leading to overproduction of receptors but defective trafficking to the plasma membrane. Our results underscore the importance of the spatiotemporal relationship between β₂-AR mRNA localization, translation, and trafficking to the plasma membrane, and establish a novel mechanism whereby G protein-coupled receptor (GPCR) responsiveness is regulated by RNA-based signals.     

LNA flow–FISH: A flow cytometry–fluorescence in situ hybridization method to detect messenger RNA using locked nucleic acid probes

We present a novel method using flow cytometry–fluorescence in situ hybridization (flow–FISH) to detect specific messenger RNA (mRNA) in suspended cells using locked nucleic acid (LNA)-modified oligonucleotide probes. β-Actin mRNA was targeted in whole A549 epithelial cells by hybridization with a biotinylated, LNA-modified probe. The LNA bound to β-actin was then stained using phycoerythrin-conjugated streptavidin and detected by flow cytometry. Shifts in fluorescence signal intensity between the β-actin LNA probe and a biotinylated, nonspecific control LNA were used to determine optimal conditions for this type of flow–FISH. Multiple conditions for permeabilization and hybridization were tested, and it was found that conditions using 3 μg/ml of proteinase K for permeabilization and 90 min hybridization at 60 °C with buffer containing 50% formamide allow cells containing the LNA-bound mRNA to be detected and differentiated from the control LNA with high confidence (< 14% overlap between curves). This combined method, called LNA flow–FISH, can be used for detection and quantification of other RNA species as well as for telomerase measurement and detection.     

The search for human pheromones: the lost decades and the necessity of returning to first principles

As humans are mammals, it is possible, perhaps even probable, that we have pheromones. However, there is no robust bioassay-led evidence for the widely published claims that four steroid molecules are human pheromones: androstenone, androstenol, androstadienone and estratetraenol. In the absence of sound reasons to test the molecules, positive results in studies need to be treated with scepticism as these are highly likely to be false positives. Common problems include small sample sizes, an overestimate of effect size (as no effect can be expected), positive publication bias and lack of replication. Instead, if we are to find human pheromones, we need to treat ourselves as if we were a newly discovered mammal, and use the rigorous methods already proven successful in pheromone research on other species. Establishing a pheromone relies on demonstration of an odour-mediated behavioural or physiological response, identification and synthesis of the bioactive molecule(s), followed by bioassay confirmation of activity. Likely sources include our sebaceous glands. Comparison of secretions from adult and pre-pubertal humans may highlight potential molecules involved in sexual behaviour. One of the most promising human pheromone leads is a nipple secretion from the areola glands produced by all lactating mothers, which stimulates suckling by any baby not just their own.     

Contribution of humoral immune responses to the antitumor effects mediated by anthracyclines

Immunogenic cell death induced by cytotoxic compounds contributes to the success of selected chemotherapies by eliciting a protective anticancer immune response, which is mediated by CD4⁺ and CD8⁺ T cells producing interferon-γ. In many instances, cancer progression is associated with high titers of tumor-specific antibodies, which become detectable in the serum, but whose functional relevance is elusive. Here, we explored the role of humoral immune responses in the anticancer efficacy of anthracyclines. Chemotherapy reduced the number of tumor-infiltrating B cells, and failed to promote humoral responses against immunodominant tumor antigens. Although anthracycline-based anticancer chemotherapies failed in T cell-deficient mice, they successfully reduced the growth of cancers developing in mice lacking B lymphocytes (due to the injection of a B-cell-depleting anti-CD20 antibody), immunoglobulins (Igs) or Ig receptors (Fc receptor) due to genetic manipulations. These results suggest that the humoral arm of antitumor immunity is dispensable for the immune-dependent therapeutic effect of anthracyclines against mouse sarcoma. In addition, we show here that the titers of IgA and IgG antibodies directed against an autoantigen appearing at the cell surface of tumor cells post chemotherapy (calreticulin, CRT) did not significantly increase in patients treated with anthracyclines, and that anti-CRT antibodies had no prognostic or predictive significance. Collectively, our data indicate that humoral anticancer immune responses differ from cellular responses in, thus far, that they do not contribute to the success of anthracycline-mediated anticancer therapies in human breast cancers and mouse sarcomas.     

Olfactory Neurons Obtained through Nasal Biopsy Combined with Laser-Capture Microdissection: A Potential Approach to Study Treatment Response in Mental Disorders

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium carbonate. Animal studies as well as human genetic studies indicate that lithium affects molecular targets that are involved in neuronal growth, survival and maturation, and notably molecules involved in Wnt signaling. Given the ethical challenge to obtaining brain biopsies for investigating dynamic molecular changes associated with lithium-response in the central nervous system (CNS), one may consider the use of neurons obtained from olfactory tissues to achieve this goal.The olfactory epithelium contains olfactory receptor neurons at different stages of development and glial-like supporting cells. This provides a unique opportunity to study dynamic changes in the CNS of patients with neuropsychiatric diseases, using olfactory tissue safely obtained from nasal biopsies. To overcome the drawback posed by substantial contamination of biopsied olfactory tissue with non-neuronal cells, a novel approach to obtain enriched neuronal cell populations was developed by combining nasal biopsies with laser-capture microdissection. In this study, a system for investigating treatment-associated dynamic molecular changes in neuronal tissue was developed and validated, using a small pilot sample of BD patients recruited for the study of the molecular mechanisms of lithium treatment response.     

Translocation of Activator of G-protein Signaling 3 to the Golgi Apparatus in Response to Receptor Activation and Its Effect on the trans-Golgi Network

Group II activators of G-protein signaling play diverse functional roles through their interaction with Gα_i, Gα_t, and Gα_o via a G-protein regulatory (GPR) motif that serves as a docking site for Gα-GDP. We recently reported the regulation of the AGS3-Gα_i signaling module by a cell surface, seven-transmembrane receptor. Upon receptor activation, AGS3 reversibly dissociates from the cell cortex, suggesting that it may function as a signal transducer with downstream signaling implications, and this question is addressed in the current report. In HEK-293 and COS-7 cells expressing the α_2A/D-AR and Gα_i3, receptor activation resulted in the translocation of endogenous AGS3 and AGS3-GFP from the cell cortex to a juxtanuclear region, where it co-localized with markers of the Golgi apparatus (GA). The agonist-induced translocation of AGS3 was reversed by the α₂-AR antagonist rauwolscine. The TPR domain of AGS3 was required for agonist-induced translocation of AGS3 from the cell cortex to the GA, and the translocation was blocked by pertussis toxin pretreatment or by the phospholipase Cβ inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122. Agonist-induced translocation of AGS3 to the GA altered the functional organization and protein sorting at the trans-Golgi network. The regulated movement of AGS3 between the cell cortex and the GA offers unexpected mechanisms for modulating protein secretion and/or endosome recycling events at the trans-Golgi network.