Claudia Stäubert

A melanocortin 1 receptor allele suggests varying pigmentation among Neanderthals

The melanocortin 1 receptor (MC1R) regulates pigmentation in humans and other vertebrates. Variants of MC1R with reduced function are associated with pale skin color and red hair in humans of primarily European origin. We amplified and sequenced a fragment of the MC1R gene (mc1r) from two Neanderthal remains. Both specimens have a mutation that was not found in ∼3700 modern humans analyzed. Functional analyses show that this variant reduces MC1R activity to a level that alters hair and/or skin pigmentation in humans. The impaired activity of this variant suggests that Neanderthals varied in pigmentation levels, potentially on the scale observed in modern humans. Our data suggest that inactive MC1R variants evolved independently in both modern humans and Neanderthals.

Evolution of GPCR: change and continuity.

Once introduced into the very early eukaryotic blueprint, seven-transmembrane receptors soon became the central and versatile components of the evolutionary highly successful G protein-coupled transmembrane signaling mechanism. In contrast to all other components of this signal transduction pathway, G protein-coupled receptors (GPCR) evolved in various structural families, eventually comprising hundreds of members in vertebrate genomes. Their functional diversity is in contrast to the conserved transmembrane core and the invariant set of intracellular signaling mechanisms, and it may be the interplay of these properties that is the key to the evolutionary success of GPCR. The GPCR repertoires retrieved from extant vertebrate genomes are the recent endpoints of this long evolutionary process. But the shaping of the fine structure and the repertoire of GPCR is still ongoing, and signatures of recent selection acting on GPCR genes can be made visible by modern population genetic methods. The very dynamic evolution of GPCR can be analyzed from different perspectives: at the levels of sequence comparisons between species from different families, orders and classes, and at the level of populations within a species. Here, we summarize the main conclusions from studies at these different levels with a specific focus on the more recent evolutionary dynamics of GPCR.

Structural and Functional Evolution of the Trace Amine-Associated Receptors TAAR3, TAAR4 and TAAR5 in Primates

The family of trace amine-associated receptors (TAAR) comprises 9 mammalian TAAR subtypes, with intact gene and pseudogene numbers differing considerably even between closely related species. To date the best characterized subtype is TAAR1, which activates the Gs protein/adenylyl cyclase pathway upon stimulation by trace amines and psychoactive substances like MDMA or LSD. Recently, chemosensory function involving recognition of volatile amines was proposed for murine TAAR3, TAAR4 and TAAR5. Humans can smell volatile amines despite carrying open reading frame (ORF) disruptions in TAAR3 and TAAR4. Therefore, we set out to study the functional and structural evolution of these genes with a special focus on primates. Functional analyses showed that ligands activating the murine TAAR3, TAAR4 and TAAR5 do not activate intact primate and mammalian orthologs, although they evolve under purifying selection and hence must be functional. We also find little evidence for positive selection that could explain the functional differences between mouse and other mammals. Our findings rather suggest that the previously identified volatile amine TAAR3–5 agonists reflect the high agonist promiscuity of TAAR, and that the ligands driving purifying selection of these TAAR in mouse and other mammals still await discovery. More generally, our study points out how analyses in an evolutionary context can help to interpret functional data generated in single species.

Selective activation of G alpha i mediated signalling of S1P3 by FTY720-phosphate

The immune modulator FTY720 is phosphorylated in vivo to FTY720 phosphate (FTY-P), which activates four sphingosine 1-phosphate (S1P) receptors including S1P(3). Upon activation with S1P, S1P(3) couples to G(i)- and G(q)-protein-dependent signalling pathways. Here we show that FTY-P selectively activates the S1P(3)-mediated and G(i)-coupled inhibition of adenylyl cyclase. Contemporaneously, it antagonizes the S1P-induced activation of G(q) via S1P(3) in intracellular calcium flux measurements, GTP-binding experiments, and flow cytometric analyses of activation-induced receptor down-regulation. In contrast to S1P, pre-treatment with FTY-P did not desensitize S1P-induced calcium flux or chemotaxis via S1P(3). The lack of receptor desensitization prevented S1P(3)-mediated migration to FTY-P. Human umbilical vein endothelial cells express S1P(1) and S1P(3), and respond to S1P and FTY-P by ERK1/2 phosphorylation and by intracellular calcium release in a pertussis toxin-sensitive manner. But whereas a mixture of S1P and FTY-P was not affecting ERK1/2 phosphorylation, the intracellular calcium flux was hampered with increasing amounts of FTY-P, which points to a cross-talk between S1P(1) and S1P(3). FTY-P is therefore one of the rare ligands which bind to a receptor that couples multiple G-proteins but selectively activates only one signalling pathway.

Rewired Metabolism in Drug-resistant Leukemia Cells A METABOLIC SWITCH HALLMARKED BY REDUCED DEPENDENCE ON EXOGENOUS GLUTAMINE

Background: Drug resistance is a common problem in cancer chemotherapy. Results: Transcriptomic and metabolomic data show that resistant leukemia cells exhibit reduced glutamine dependence, enhanced glucose dependence, and altered fatty acid metabolism. Conclusion: The metabolism of resistant leukemia cells is fundamentally rewired. Significance: Understanding the metabolic cost of resistance can lead to novel therapeutic strategies. Cancer cells that escape induction therapy are a major cause of relapse. Understanding metabolic alterations associated with drug resistance opens up unexplored opportunities for the development of new therapeutic strategies. Here, we applied a broad spectrum of technologies including RNA sequencing, global untargeted metabolomics, and stable isotope labeling mass spectrometry to identify metabolic changes in P-glycoprotein overexpressing T-cell acute lymphoblastic leukemia (ALL) cells, which escaped a therapeutically relevant daunorubicin treatment. We show that compared with sensitive ALL cells, resistant leukemia cells possess a fundamentally rewired central metabolism characterized by reduced dependence on glutamine despite a lack of expression of glutamate-ammonia ligase (GLUL), a higher demand for glucose and an altered rate of fatty acid β-oxidation, accompanied by a decreased pantothenic acid uptake capacity. We experimentally validate our findings by selectively targeting components of this metabolic switch, using approved drugs and starvation approaches followed by cell viability analyses in both the ALL cells and in an acute myeloid leukemia (AML) sensitive/resistant cell line pair. We demonstrate how comparative metabolomics and RNA expression profiling of drug-sensitive and -resistant cells expose targetable metabolic changes and potential resistance markers. Our results show that drug resistance is associated with significant metabolic costs in cancer cells, which could be exploited using new therapeutic strategies.

The ligand specificity of the G-protein-coupled receptor GPR34.

Lyso-PS (lyso-phosphatidylserine) has been shown to activate the G(i/o)-protein-coupled receptor GPR34. Since in vitro and in vivo studies provided controversial results in assigning lyso-PS as the endogenous agonist for GPR34, we investigated the evolutionary conservation of agonist specificity in more detail. Except for some fish GPR34 subtypes, lyso-PS has no or very weak agonistic activity at most vertebrate GPR34 orthologues investigated. Using chimaeras we identified single positions in the second extracellular loop and the transmembrane helix 5 of carp subtype 2a that, if transferred to the human orthologue, enabled lyso-PS to activate the human GPR34. Significant improvement of agonist efficacy by changing only a few positions strongly argues against the hypothesis that nature optimized GPR34 as the receptor for lyso-PS. Phylogenetic analysis revealed several positions in some fish GPR34 orthologues which are under positive selection. These structural changes may indicate functional specification of these orthologues which can explain the species- and subtype-specific pharmacology of lyso-PS. Furthermore, we identified aminoethyl-carbamoyl ATP as an antagonist of carp GPR34, indicating ligand promiscuity with non-lipid compounds. The results of the present study suggest that lyso-PS has only a random agonistic activity at some GPR34 orthologues and the search for the endogenous agonist should consider additional chemical entities.

The Multitarget Ligand 3-Iodothyronamine Modulates β-Adrenergic Receptor 2 Signaling

Background: 3-Iodothyronamine (3-T₁AM), a signaling molecule with structural similarities to thyroid hormones, induces numerous physiological responses including reversible body temperature decline. One target of 3-T₁AM is the trace amine-associated receptor 1 (TAAR1), which is a member of the rhodopsin-like family of G protein-coupled receptors (GPCRs). Interestingly, the effects of 3-T₁AM remain detectable in TAAR1 knockout mice, suggesting further targets for 3-T₁AM such as adrenergic receptors. Therefore, we evaluated whether β-adrenergic receptor 1 (ADRB1) and 2 (ADRB2) signaling is affected by 3-T₁AM in HEK293 cells and in human conjunctival epithelial cells (IOBA-NHC), where these receptors are highly expressed endogenously. Methods: A label-free EPIC system for prescreening the 3-T₁AM-induced effects on ADRB1 and ADRB2 in transfected HEK293 cells was used. In addition, ADRB1 and ADRB2 activation was analyzed using a cyclic AMP assay and a MAPK reporter gene assay. Finally, fluorescence Ca²⁺ imaging was utilized to delineate 3-T₁AM-induced Ca²⁺ signaling. Results: 3-T₁AM (10^-5- 10^-10<smlcap>M</smlcap>) enhanced isoprenaline-induced ADRB2-mediated G_s signaling but not that of ADRB1-mediated signaling. MAPK signaling remained unaffected for both receptors. In IOBA-NHC cells, norepinephrine-induced Ca²⁺ influxes were blocked by the nonselective ADRB blocker timolol (10 µ<smlcap>M</smlcap>), indicating that ADRBs are most likely linked with Ca²⁺ channels. Notably, timolol was also found to block 3-T₁AM (10^-5<smlcap>M</smlcap>)-induced Ca²⁺ influx. Conclusions: The presented data support that 3-T₁AM directly modulates β-adrenergic receptor signaling. The relationship between 3-T₁AM and β-adrenergic signaling also reveals a potential therapeutic value for suppressing Ca²⁺ channel-mediated inflammation processes, occurring in eye diseases such as conjunctivitis.

Evolutionary Conservation of 3-Iodothyronamine as an Agonist at the Trace Amine-Associated Receptor 1

Objectives: The trace amine-associated receptor 1 (Taar1) is a Gs protein-coupled receptor activated by trace amines, such as β-phenylethylamine (β-PEA) and 3-iodothyronamine (T1AM). T1AM is an endogenous biogenic amine and thyroid hormone derivative that exerts several biological functions. However, the physiological relevance of T1AM acting via Taar1 is still under discussion. Therefore, we studied the structural and functional evolution of Taar1 in vertebrates to provide evidence for a conserved Taar1-mediated T1AM function. Study Design: We searched public sequence databases to retrieve Taar1 sequence information from vertebrates. We cloned and functionally characterized Taar1 from selected vertebrate species using cAMP assays to determine the evolutionary conservation of T1AM action at Taar1. Results: We found intact open reading frames of Taar1 in more than 100 vertebrate species, including mammals, sauropsids and amphibians. Evolutionary conservation analyses of Taar1 protein sequences revealed a high variation in amino acid residues proposed to be involved in agonist binding, especially in rodent Taar1 orthologs. Functional characterization showed that T1AM, β-PEA and p-tyramine (p-Tyr) act as agonists at all tested orthologs, but EC50 values of T1AM at rat Taar1 differed significantly when compared to all other tested vertebrate Taar1. Conclusions: The high structural conservation of Taar1 throughout vertebrate evolution highlights the physiological relevance of Taar1, but species-specific differences in T1AM potency at Taar1 orthologs suggest a specialization of rat Taar1 for T1AM recognition. In contrast, β-PEA and p-Tyr potencies were rather conserved throughout all tested Taar1 orthologs. We provide evidence that the observed differences in potency are related to differences in constraint during Taar1 evolution.

Examining the Dynamic Evolution of G Protein-Coupled Receptors

The valuable source of large-scale genomic information initiated attempts to identify the origin(s) of G protein-coupled receptors (GPCR), count and categorize those genes, and follow their evolutionary history. Being present in fungi, plants, and unicellular eukaryotes, GPCR must have evolved before the plant-fungi-animal split about 1.5 billion years ago. Phylogenetic analyses revealed several kinds of evolutionary patterns that occurred during GPCR evolution including one-to-one orthologous relationships, species-specific gene expansion, and episodic duplication of the entire GPCR repertoire in certain species lineages. These data document the highly dynamic process of birth and death of GPCR genes since hundreds of millions of years. Genetic drift and selective forces have shaped the individual structure of a given receptor gene but also of the species-specific receptor repertoire - a process that is still ongoing. These processes have left footprints in the genomic sequence that can be detected by bioinformatic methods and may help to interpret receptor function in the light of a given species in its environment. Reasonable intraspecies sequence variability in GPCR is either physiologically tolerated or promotes individual phenotypes and adaptation, but also susceptibilities for diseases. Therefore, the impact of GPCR variants in epistatic networks will be an important task of future GPCR research. The chapter summarizes evolutionary processes working on GPCR genes and sheds light on their consequences at the levels of receptor structure and function.

GPCR Signaling From Intracellular Membranes − A Novel Concept

The properties of the main psychoactive compound of the Cannabis plant, namely Δ-tetrahydrocannabinol (THC), have been recognized for a long time. Ever since the discovery of cannabinoid receptors (CB1R and CB2R) and the characterization of endogenous ligands acting at these receptors (endocannabinoids) they have been studied intensely and shown to be crucial regulators of brain and peripheral functions. CB1R is a G protein-coupled receptor (GPCR) with high and abundant cell surface expression in the central nervous system. Evidence of functional CB1R in brain mitochondria (mtCB1R) made it necessary to revisit the physiological impact of CB1R signaling in brain and to discuss possible downstream effects of mtCB1R activation. In their comprehensive overview DjeungouePetga et al. summarize the potential impact of mtCB1R on the regulation of brain physiology. CB1R regulates numerous brain tasks, including synaptic activity, perception and memory performance, and at least some of these functions might be mediated by mtCB1R. It is well established that cannabinoid administration impacts on mitochondrial structure. Djeungoue-Petga et al. hypothesize thatmtCB1Ractivationmight have a crucial role in this process, and showed that mtCB1R activation decreases mitochondrial mobility, an outcome that is probably linked to the reduced mitochondrial metabolism. Moreover, activation of mtCB1R decreases flux through the electron transport chain and thus mitochondrial oxygen consumption and energy production, which likely impacts on ATP generation, the production of the central neurotransmittersglutamateandGABAandsynaptic transmission. The classical concept of GPCR function and signaling includes the expression of the receptor exclusively on the plasma membrane and the recognition of extracellular agonists. This concept was recently challenged by accumulating evidence for signaling of GPCRs from intracellular membranes (reviewed in Ref. ) of endosomes, mitochondria (as discussed in the current issue of BioEssays for CB1R), the endoplasmatic reticulum (ER), the Golgi apparatus, and the nucleus. G-proteins and G protein-related signaling components have also been shown to be present in intracellular organelles including mitochondria (reviewed in Ref. ). One of the first examples