Free fatty acid receptor 2 (FFAR2) is activated by short-chain fatty acids and expressed widely, including in white adipocytes and various immune and enteroendocrine cells. Using both wild-type human ...FFAR2 and a designer receptor exclusively activated by designer drug (DREADD) variant we explored the activation and phosphorylation profile of the receptor, both in heterologous cell lines and in tissues from transgenic knock-in mouse lines expressing either human FFAR2 or the FFAR2-DREADD. FFAR2 phospho-site-specific antisera targeting either pSer
296
/pSer
297
or pThr
306
/pThr
310
provided sensitive biomarkers of both constitutive and agonist-mediated phosphorylation as well as an effective means to visualise agonist-activated receptors in situ. In white adipose tissue, phosphorylation of residues Ser
296
/Ser
297
was enhanced upon agonist activation whilst Thr
306
/Thr
310
did not become phosphorylated. By contrast, in immune cells from Peyer’s patches Thr
306
/Thr
310
become phosphorylated in a strictly agonist-dependent fashion whilst in enteroendocrine cells of the colon both Ser
296
/Ser
297
and Thr
306
/Thr
310
were poorly phosphorylated. The concept of phosphorylation bar-coding has centred to date on the potential for different agonists to promote distinct receptor phosphorylation patterns. Here, we demonstrate that this occurs for the same agonist-receptor pairing in different patho-physiologically relevant target tissues. This may underpin why a single G protein-coupled receptor can generate different functional outcomes in a tissue-specific manner.
•Several hypomorphic mutations are present in NPS receptor and ligand.•Archaic genomes contain only ancestral alleles for hypomorphic NPSR1 mutations.•Worldwide distribution of mutant NPSR1 alleles ...is consistent with out-of-Africa scenario.•A mutation reducing NPS ligand potency is common among Neandertals.•The NPS mutation may have entered the modern human gene pool by interbreeding.
The neuropeptide S (NPS) system plays an important role in fear and fear memory processing but has also been associated with allergic and inflammatory diseases. Genes for NPS and its receptor NPSR1 are found in all tetrapods. Compared to non-human primates, several non-synonymous single-nucleotide polymorphisms (SNPs) occur in both human genes that collectively result in functional attenuation, suggesting adaptive mechanisms in a human context. To investigate historic and geographic origins of these hypomorphic mutations and explore genetic signs of selection, we analyzed ancient genomes and worldwide genotype frequencies of four prototypic SNPs in the NPS system. Neandertal and Denisovan genomes contain exclusively ancestral alleles for NPSR1 while all derived alleles occur in ancient genomes of anatomically modern humans, indicating that they arose in modern Homo sapiens. Worldwide genotype frequencies for three hypomorphic NPSR1 SNPs show significant regional homogeneity but follow a gradient towards increasing derived allele frequencies that supports an out-of-Africa scenario. Increased density of high-frequency polymorphisms around the three NPSR1 loci suggests weak or possibly balancing selection. A hypomorphic mutation in the NPS precursor, however, was detected at high frequency in Eurasian Neandertal genomes and shows genetic signatures indicating that it was introgressed into the human gene pool, particularly in Southern Europe, by interbreeding with Neandertals. We discuss potential evolutionary scenarios including behavior and immune-based natural selection.
Background The clinical benefits of opioid drugs are counteracted by the development of tolerance and addiction. We provide in vivo evidence for the involvement of G protein–coupled receptor kinases ...(GRKs) in opioid dependence in addition to their roles in agonist-selective mu-opioid receptor (MOR) phosphorylation. Methods In vivo MOR phosphorylation was examined by immunoprecipitation and nanoflow liquid chromatography–tandem mass spectrometry analysis. Using the hot-plate and conditioned place preference test, we investigated opioid-related antinociception and reward effects in mice lacking GRK3 or GRK5. Results Etonitazene and fentanyl stimulated the in vivo phosphorylation of multiple carboxyl-terminal phosphate acceptor sites, including threonine 370, serine 375, and threonine 379, which was predominantly mediated by GRK3. By contrast, morphine promoted a selective phosphorylation of serine 375 that was predominantly mediated by GRK5. In contrast to GRK3 knockout mice, GRK5 knockout mice exhibited reduced antinociceptive responses after morphine administration and developed morphine tolerance similar to wild-type mice but fewer signs of physical dependence. Also, morphine was ineffective in inducing conditioned place preference in GRK5 knockout mice, whereas cocaine conditioned place preference was retained. However, the reward properties of morphine were evident in knock-in mice expressing a phosphorylation-deficient S375A mutation of the MOR. Conclusions These findings show for the first time that MOR phosphorylation is regulated by agonist-selective recruitment of distinct GRK isoforms that influence different opioid-related behaviors. Modulation of GRK5 function could serve as a new approach for preventing addiction to opioids, while maintaining the analgesic properties of opioid drugs at an effective level.
Termination of signaling of activated G protein-coupled receptors (GPCRs) is essential for maintenance of cellular homeostasis. It is well established that β-arrestin redistributes to phosphorylated ...GPCRs and thereby facilitates desensitization of classical G protein-dependent signaling. β-Arrestin in turn serves as a scaffold to initiate a second wave of signaling. Here, we report a molecular mechanism that regulates the termination of unconventional β-arrestin-dependent GPCR signaling. We identify protein phosphatase 1β (PP1β) as a phosphatase for the cluster of phosphorylated threonines (353TTETQRT359) within the sst2A somatostatin receptor carboxyl terminus that mediates β-arrestin binding using siRNA knock-down screening. We show that PP1β-mediated sst2A dephosphorylation is initiated directly after receptor activation at or near the plasma membrane. As a functional consequence of diminished PP1β activity, we find that somatostatin- and substance P-induced but not epidermal growth factor-induced ERK activation was aberrantly enhanced and prolonged. Thus, we demonstrate a novel mechanism for fine tuning unconventional β-arrestin-dependent GPCR signaling in that recruitment of PP1β to activated GPCRs facilitates GPCR dephosphorylation and, hence, leads to disruption of the β-arrestin-GPCR complex.
Pasireotide (SOM230) is currently under clinical evaluation as a successor compound to octreotide for the treatment of acromegaly, Cushing's disease, and carcinoid tumors. Whereas octreotide acts ...primarily via the sst(2A) somatostatin receptor, pasireotide was designed to exhibit octreotide-like sst(2A) activity combined with enhanced binding to other somatostatin receptor subtypes. In the present study, we used phophosite-specific antibodies to examine agonist-induced phosphorylation of the rat sst(2A) receptor. We show that somatostatin and octreotide stimulate the complete phosphorylation of a cluster of four threonine residues within the cytoplasmic (353)TTETQRT(359) motif in a variety of cultured cell lines in vitro as well as in intact animals in vivo. This phosphorylation was mediated by G protein-coupled receptor kinases (GRK) 2 and 3 and followed by rapid cointernalization of the receptor and ss-arrestin into the same endocytic vesicles. In contrast, pasireotide failed to promote substantial phosphorylation and internalization of the rat sst(2A) receptor. In the presence of octreotide or SS-14, SOM230 showed partial agonist behavior, inhibiting phosphorylation, and internalization of sst(2A). Upon overexpression of GRK2 or GRK3, pasireotide stimulated selective phosphorylation of Thr356 and Thr359 but not of Thr353 or Thr354 within the (353)TTETQRT(359) motif. Pasireotide-mediated phosphorylation led to the formation of relatively unstable beta-arrestin-sst(2A) complexes that dissociated at or near the plasma membrane. Thus, octreotide and pasireotide are equally active in inducing classical G protein-dependent signaling via the sst(2A) somatostatin receptor. Yet, we find that they promote strikingly different patterns of sst(2A) receptor phosphorylation and, hence, stimulate functionally distinct pools of beta-arrestin.
Some amphibians use chemical signals in addition to optical and acoustical signals to transmit information. Males of mantellid frogs from Madagascar and hyperoliid frogs from Africa emit complex, ...species‐ and sex‐specific bouquets of volatiles from their femoral or gular glands. We report here on the identification, synthesis, and determination of the absolute configuration of a macrocyclic lactone occurring in several species of both families, (S)‐3,7,11‐dodec‐6,10‐dien‐12‐olide (S‐14, frogolide). Macrolides are a preferred compound class of frog volatiles. Nevertheless, frogolide is the first macrocyclic lactone obviously derived from the terpene pathway, in contrast to known frog macrolides that are usually formed via the fatty acid biosynthetic pathway.
Frogolide is a new sesquiterpene macrolactone identified in gular and femoral scent glands of the African frog families Mantellidae and Hyperoliidae. Frogolide is the first frog macrolide of terpenoid origin. The identification, synthesis, and determination of the absolute configuration are presented.
Experimental work in the early 20th century showed that background albedo experienced by larvae of the fire salamander (Salamandra salamandra) induce a durable morphological modification of the ...postmetamorphic color pattern, which needed confirmation due to the controversies regarding Paul Kammerer's experiments. Such a carry‐over effect would be relevant as the black and yellow pattern of the alkaloid‐containing adult fire salamanders has been suggested to serve as an aposematic signal. Hence, we hypothesized that (a) adult coloration is conspicuous to potential predators under light conditions at night, given the nocturnal activity of this species, and (b) a condition affecting the salamander's coloration pattern would also affect its toxicity to maintain a quantitatively honest aposematic signal. To test the first hypothesis, we used spectrometry to model the vision of potential avian and snake predators and confirmed that fire salamander's black‐and‐yellow pattern is contrasting enough against the forest leaf litter to be considered conspicuous at night. To test the second hypothesis, we first confirmed the background carry‐over effect on black and yellow proportions in the dorsal skin of experimentally reared fire salamanders, using a rigorous experimental design. Then, we calculated the conspicuousness and determined the alkaloid profiles of these individuals. We did not find a correlation between conspicuousness and toxicity at the intrapopulation level. Moreover, there was no background carry‐over effect on the alkaloid profile. We discuss our results in a physiological, ecological, evolutionary, and historical context.
Fire salamanders are conspicuous to potential predators at night. Confirming historical experiments, the background albedo experienced by a larva has a carry‐over effect on its postmetamorphic black‐and‐yellow coloration but not on its toxicity.
Brassicadiene, a novel tricyclic diterpene hydrocarbon, was identified by a combination of mass spectrometry, microchemical tests, and analysis of NMR spectra. The compound constitutes >90% of the ...volatile organic compounds produced by cauliflower seedlings, Brassica oleracea var. botrytis. The invasive stink bug Bagrada hilaris is strongly attracted to brassicadiene, providing a mechanism for this herbivore, which specializes on cruciferous plants, to locate its hosts in a nutrient-rich and vulnerable stage.
Clinical narratives are typically produced under time pressure, which incites the use of abbreviations and acronyms. To expand such short forms in a correct way eases text comprehension and further ...semantic processing. We propose a completely unsupervised and data-driven algorithm for the resolution of non-lexicalised and potentially ambiguous abbreviations. Based on the lookup of word bigrams and unigrams extracted from a corpus of 30,000 pseudonymised cardiology reports in German, our method achieved an F1 score of 0.91, evaluated with a test set of 200 text excerpts. The results are statistically significantly better (p < 0.001) than a baseline approach and show that a simple and domain-independent strategy may be enough to resolve abbreviations when a large corpus of similar texts is available. Further work is needed to combine this strategy with sentence and abbreviation detection modules, to adapt it to acronym resolution and to evaluate it with different datasets.
Purpose
Whereas the different somatostatin receptor (SSTR) subtypes and the chemokine receptor CXCR4 are known to be expressed in a wide variety of human malignancies, comprehensive data are still ...lacking for MALT-type lymphomas.
Methods
Overall, 55 cases of MALT-type lymphoma of both gastric and extragastric origin were evaluated for the SSTR subtype and CXCR4 expression by means of immunohistochemistry using novel monoclonal rabbit antibodies. The stainings were rated by means of the immunoreactive score and correlated with clinical data.
Results
While the CXCR4 was detected in 92 % of the cases investigated, the SSTR subtypes were much less frequently present. The SSTR5 was expressed in about 50 % of the cases, followed by the SSTR3, the SSTR2A, the SSTR4 and the SSTR1, which were present in 35, 27, 18 or 2 %, respectively, of the tumors only. Gastric lymphomas displayed a significantly higher SSTR3, SSTR4 and SSTR5 expression than extragastric tumors. A correlation between CXCR4 and Ki-67 expression was seen in gastric lymphomas, whereas primarily in extragastric tumors SSTR5 negativity was associated with poor patient outcome.
Conclusions
The CXCR4 may serve as a promising target for diagnostics and therapy of MALT-type lymphomas, while the SSTRs appear not suitable in this respect.
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