Elucidating regulators of astrocytic Ca2+ signaling via multi-threshold event detection (MTED).

Recent achievements in indicator optimization and imaging techniques promote the advancement of functional imaging to decipher complex signaling processes in living cells, such as Ca2+ activity patterns. Astrocytes are important regulators of the brain network and well known for their highly complex morphology and spontaneous Ca2+ activity. However, the astrocyte community is lacking standardized methods to analyze and interpret Ca2+ activity recordings, hindering global comparisons. Here, we present a biophysically-based analytical concept for deciphering the complex spatio-temporal changes of Ca2+ biosensor fluorescence for understanding the underlying signaling mechanisms. We developed a pixel-based multi-threshold event detection (MTED) analysis of multidimensional data, which accounts for signal strength as an additional signaling dimension and provides the experimenter with a comprehensive toolbox for a differentiated and in-depth characterization of fluorescence signals. MTED was validated by analyzing astrocytic Ca2+ activity across Ca2+ indicators, imaging setups, and model systems from primary cell culture to awake, head-fixed mice. We identified extended Ca2+ activity at 25°C compared to 37°C physiological body temperature and dissected how neuronal activity shapes long-lasting astrocytic Ca2+ activity. Our MTED strategy, as a parameter-free approach, is easily transferrable to other fluorescent indicators and biosensors and embraces the additional dimensionality of signaling activity strength. It will also advance the definition of standardized procedures and parameters to improve comparability of research data and reports.

Mechanisms underlying dual effects of serotonin during development of Helisoma trivolvis (Mollusca).

BACKGROUND: Serotonin (5-HT) is well known as widely distributed modulator of developmental processes in both vertebrates and invertebrates. It is also the earliest neurotransmitter to appear during neuronal development. In aquatic invertebrates, which have larvae in their life cycle, 5-HT is involved in regulation of stages transition including larval metamorphosis and settlement. However, molecular and cellular mechanisms underlying developmental transition in aquatic invertebrate species are yet poorly understood. Earlier we demonstrated that in larvae of freshwater molluscs and marine polychaetes, endogenous 5-HT released from the neurons of the apical sensory organ (ASO) in response to external stimuli retarded larval development at premetamorphic stages, and accelerated it at metamorphic stages. Here we used a freshwater snail Helisoma trivolvis to study molecular mechanisms underlying these dual developmental effects of 5-HT. RESULTS: Larval development of H. trivolvis includes transition from premetamorphic to metamorphic stages and shares the main features of metamorphosis with free-swimming aquatic larvae. Three types of 5-HT receptors (5-HT1-, 5-HT4- and 5-HT7-like) are functionally active at premetamorphic (trochophore, veliger) and metamorphic (veliconcha) stages, and expression patterns of these receptors and respective G proteins undergo coordinated changes during development. Stimulation of these receptors modulated cAMP-dependent regulation of cell divisions. Expression of 5-HT4- and 5-HT7-like receptors and their downstream Gs protein was down-regulated during the transition of pre- to metamorphic stage, while expression of 5-HT1 -like receptor and its downstream Gi protein was upregulated. In accordance with relative amount of these receptors, stimulation of 5-HTRs at premetamorphic stages induces developmental retardation, while their stimulation at metamorphic stages induces developmental acceleration. CONCLUSIONS: We present a novel molecular mechanism that underlies stage-specific changes in developmental tempo of H. trivolvis larvae in response to endogenous 5-HT produced by the neurons of the ASO. We suggest that consecutive changes in expression patterns of different receptors and their downstream partners in the course of larval development represent the molecular base of larval transition from premetamorphic (non-competent) to metamorphic (competent) state.

Heterodimerization of serotonin receptors 5-HT1A and 5-HT7 differentially regulates receptor signalling and trafficking.

Serotonin receptors 5-HT(1A) and 5-HT(7) are highly coexpressed in brain regions implicated in depression. However, their functional interaction has not been established. In the present study we show that 5-HT(1A) and 5-HT(7) receptors form heterodimers both in vitro and in vivo. Foerster resonance energy transfer-based assays revealed that, in addition to heterodimers, homodimers composed either of 5-HT(1A) or 5-HT(7) receptors together with monomers coexist in cells. The highest affinity for complex formation was obtained for the 5-HT(7)-5-HT(7) homodimers, followed by the 5-HT(7)-5-HT(1A) heterodimers and 5-HT(1A)-5-HT(1A) homodimers. Functionally, heterodimerization decreases 5-HT(1A)-receptor-mediated activation of G(i) protein without affecting 5-HT(7)-receptor-mediated signalling. Moreover, heterodimerization markedly decreases the ability of the 5-HT(1A) receptor to activate G-protein-gated inwardly rectifying potassium channels in a heterologous system. The inhibitory effect on such channels was also preserved in hippocampal neurons, demonstrating a physiological relevance of heteromerization in vivo. In addition, heterodimerization is crucially involved in initiation of the serotonin-mediated 5-HT(1A) receptor internalization and also enhances the ability of the 5-HT(1A) receptor to activate the mitogen-activated protein kinases. Finally, we found that production of 5-HT(7) receptors in the hippocampus continuously decreases during postnatal development, indicating that the relative concentration of 5-HT(1A)-5-HT(7) heterodimers and, consequently, their functional importance undergoes pronounced developmental changes.

Quantitative intensity-based FRET approaches--a comparative snapshot.

Förster resonance energy transfer (FRET) has become an important tool for analyzing different aspects of interactions among biological macromolecules in their native environments. FRET analysis has also been successfully applied to study the spatiotemporal regulation of various cellular processes using genetically encoded FRET-based biosensors. A variety of procedures have been described for measuring FRET efficiency or the relative abundance of donor-acceptor complexes, based on analysis of the donor fluorescence lifetime or the spectrally resolved fluorescence intensity. The latter methods are preferable if one wants to not only quantify the apparent FRET efficiencies but also calculate donor-acceptor stoichiometry and observe fast dynamic changes in the interactions among donor and acceptor molecules in live cells. This review focuses on a comparison of the available intensity-based approaches used to measure FRET. We discuss their strengths and weaknesses in terms of FRET quantification, and provide several examples of biological applications.

Homodimerization of the Src homology 3 domain of the calcium channel ß-subunit drives dynamin-dependent endocytosis.

Voltage-dependent calcium channels constitute the main entry pathway for calcium into excitable cells. They are heteromultimers formed by an α(1) pore-forming subunit (Ca(V)α(1)) and accessory subunits. To achieve a precise coordination of calcium signals, the expression and activity of these channels is tightly controlled. The accessory ß-subunit (Ca(V)ß), a membrane associated guanylate kinase containing one guanylate kinase (ß-GK) and one Src homology 3 (ß-SH3) domain, has antagonistic effects on calcium currents by regulating different aspects of channel function. Although ß-GK binds to a conserved site within the α(1)-pore-forming subunit and facilitates channel opening, ß-SH3 binds to dynamin and promotes endocytosis. Here, we investigated the molecular switch underlying the functional duality of this modular protein. We show that ß-SH3 homodimerizes through a single disulfide bond. Substitution of the only cysteine residue abolishes dimerization and impairs internalization of L-type Ca(V)1.2 channels expressed in Xenopus oocytes while preserving dynamin binding. Covalent linkage of the ß-SH3 dimerization-deficient mutant yields a concatamer that binds to dynamin and restores endocytosis. Moreover, using FRET analysis, we show in living cells that Ca(V)ß form oligomers and that this interaction is reduced by Ca(V)α(1). Association of Ca(V)ß with a polypeptide encoding the binding motif in Ca(V)α(1) inhibited endocytosis. Together, these findings reveal that ß-SH3 dimerization is crucial for endocytosis and suggest that channel activation and internalization are two mutually exclusive functions of Ca(V)ß. We propose that a change in the oligomeric state of Ca(V)ß is the functional switch between channel activator and channel internalizer.

Specific oligomerization of the 5-HT1A receptor in the plasma membrane.

In the present study we analyze the oligomerization of the 5-HT1A receptor within living cells at the sub-cellular level. Using a 2-excitation Förster Resonance Energy Transfer (FRET) method combined with spectral microscopy we are able to estimate the efficiency of energy transfer based on donor quenching as well as acceptor sensitization between CFP-and YFP-tagged 5-HT1A receptors at the plasma membrane. Through the analysis of the level of apparent FRET efficiency over the various relative amounts of donor and acceptor, as well as over a range of total surface expressions of the receptor, we verify the specific interaction of these receptors. Furthermore we study the role of acylation in this interaction through measurements of a palmitoylation-deficient 5-HT(1A) receptor mutant. Palmitoylation increases the tendency of a receptor to localize in lipid rich microdomains of the plasma membrane. This increases the effective surface density of the receptor and provides for a higher level of stochastic interaction.

5-HT7 receptor is coupled to G alpha subunits of heterotrimeric G12-protein to regulate gene transcription and neuronal morphology.

The neurotransmitter serotonin (5-HT) plays an important role in the regulation of multiple events in the CNS. We demonstrated recently a coupling between the 5-HT4 receptor and the heterotrimeric G13-protein resulting in RhoA-dependent neurite retraction and cell rounding (Ponimaskin et al., 2002). In the present study, we identified G12 as an additional G-protein that can be activated by another member of serotonin receptors, the 5-HT7 receptor. Expression of 5-HT7 receptor induced constitutive and agonist-dependent activation of a serum response element-mediated gene transcription through G12-mediated activation of small GTPases. In NIH3T3 cells, activation of the 5-HT7 receptor induced filopodia formation via a Cdc42-mediated pathway correlating with RhoA-dependent cell rounding. In mouse hippocampal neurons, activation of the endogenous 5-HT7 receptors significantly increased neurite length, whereas stimulation of 5-HT4 receptors led to a decrease in the length and number of neurites. These data demonstrate distinct roles for 5-HT7R/G12 and 5-HT4R/G13 signaling pathways in neurite outgrowth and retraction, suggesting that serotonin plays a prominent role in regulating the neuronal cytoarchitecture in addition to its classical role as neurotransmitter.

The GABA(B) receptor subunits R1 and R2 interact differentially with the activation transcription factor ATF4 in mouse brain during the postnatal development.

Gamma-aminobutyric acid type B receptors (GABA(B)R) belong to the family of G-protein-coupled receptors that mediate synaptic actions by modulation of different ion channels. Here, we demonstrate that the receptor subunits GABA(B)R1 and GABA(B)R2 interact directly with the soluble activating transcription factor 4 (ATF4) in different regions of the neonatal mouse brain. We found that about 5-12% of expressed ATF4 protein is involved in the complex formation with GABA(B) receptors. Confocal fluorescence microscopy showed that GABA(B)R and ATF4 are co-localized in several well-defined spots in neurons and in glial cells. Co-immunoprecipitation analysis also reveals that the interaction efficiency between GABA(B) receptors and ATF4 in the mouse brain markedly changed during postnatal development, and such changes in interaction were dependent on the GABA(B) receptor subtype.

Interplay between serotonin 5-HT1A and 5-HT7 receptors in depressive disorders.

Serotonin (5-hydroxytryptamine or 5-HT) is an important neurotransmitter regulating a wide range of physiological and pathological functions via activation of heterogeneously expressed 5-HT receptors. Besides the important role of 5-HT receptors in the pathogenesis of depressive disorders and in their clinical medications, underlying mechanisms are far from being completely understood. This review focuses on possible cross talk between two serotonin receptors, 5-HT1A and the 5-HT7 . Although these receptors are highly co-expressed in brain regions implicated in depression, and most agonists developed for the 5-HT1A or 5-HT7 receptors have cross-reactivity, their functional interaction has not been yet established. It has been recently shown that 5-HT1A and 5-HT7 receptors form homo- and heterodimers both in vitro and in vivo. From the functional point of view, heterodimerization has been shown to play an important role in regulation of receptor-mediated signaling and internalization, suggesting the implication of heterodimerization in the development and maintenance of depression. Interaction between these receptors is also of clinical interest, because both receptors represent an important pharmacological target for the treatment of depression and anxiety.

Serotonin 5-HT7 receptor is critically involved in acute and chronic inflammation of the gastrointestinal tract.

BACKGROUND: Intestinal inflammation is often associated with an increased level of serotonin (5-HT), an important gastrointestinal signaling molecule involved in gut homeostasis through stimulation of specific receptors. In this study, we investigated the role of 5-HT7 receptor (5-HT7R) in the induction and development of intestinal inflammation using a mouse model of acute and chronic colitis and human patients with Crohn's disease (CD). METHODS: Acute colitis was induced through administration of dextran sodium sulfate to wild-type, 5-HT7R-deficient mice and hematopoietic bone marrow chimera. Chronic colitis was induced in interleukin 10-deficient mice. The role of 5-HT7R in gut inflammation was assessed using agonist/antagonist treatment. We investigated expression and distribution of 5-HT7R, extent of gut inflammation with magnetic resonance imaging and histological analysis, survival rate, and disease activity index. Finally, biopsies from the large intestine of patients with CD were analyzed. RESULTS: Under basal conditions, 5-HT7R is expressed both in enteric neurons and CD11c cells of the large intestine. Expression of 5-HT7R significantly increased after induction of colitis in mice and in inflamed intestinal regions of patients with CD in CD11c/CD86 double-positive cells. Pharmacological blockade or genetic ablation of 5-HT7R resulted in increased severity of both acute and chronic dextran sodium sulfate-induced colitis, whereas receptor stimulation showed an anti-inflammatory effect. Analysis of bone marrow chimera indicated importance of 5-HT7R expressed by hematopoietic cells in intestinal inflammation. CONCLUSIONS: The 5-HT7R expressed on CD11c/CD86-positive myeloid cells modulates the severity of intestinal inflammation in an acute and chronic colitis and thus represents a potential therapeutic target for the treatment of inflammatory disorders such as CD.

G protein--mediated signaling: same receptor, multiple effectors.

The superfamily of G protein coupled receptors (GPCRs) comprises the largest group of cell surface receptors expressed by the human genome. Accordingly, these receptors are the target of a substantial portion of current pharmaceuticals. Over the past few decades there have been many substantial discoveries regarding GPCRs structure and function that have led to the current understanding of the complexity of the signal transduction which these receptors initiate. What was once generally believed to be a simple linear pathway, has become one with manifold bifurcations and multiple regulatory and feedback mechanisms. In the following we review the fundamental ground work upon which this field of research was established and the work that has more recently begun to uncover the complexity of GPCR signaling. The emerging signaling paradigm includes (i) the capacity of one receptor to couple to and initiate pathways through multiple G proteins, (ii) the capability of one G protein to activate many effectors, as well as (iii) the ability of a GPCR to transduce signals through G protein independent pathways. We also briefly touch upon some implications of GPCR oligomerization and discuss signaling cascades of two serotonin receptors, 5-HT(4) and 5-HT(7), whose pathways exemplify the richness and complexity of GPCR signaling mechanisms.

5-Hydroxytryptamine 4(a) receptor is coupled to the Galpha subunit of heterotrimeric G13 protein.

Serotonin (5-hydroxytryptamine (5-HT)) is an important neurotransmitter that regulates multiple events in the central nervous system. Many of the 5-HT functions are mediated via G protein-coupled receptors that are coupled to multiple heterotrimeric G proteins, including G(s), G(i), and G(q) subfamilies (Martin, G. R., Eglen, R. M., Hamblin, M. W., Hoyer, D., and Yocca, F. (1998) Trends Pharmacol. Sci. 19, 2-4). Here we show for the first time that the 5-hydroxytryptamine 4(a) receptor (5-HT(4(a))) is coupled not only to heterotrimeric G(s) but also to G(13) protein, as assessed both by biochemical and functional assays. Using reconstitution of 5-HT(4(a)) receptor with different G proteins in Spodoptera frugiperda (Sf.9) cells, we have proved that agonist stimulation of receptor-induced guanosine 5'-(3-O-thio)triphosphate binding to Galpha(13) protein. We then determined that expression of 5-HT(4(a)) receptor in mammalian cells induced constitutive- as well as agonist-promoted activation of a transcription factor, serum response element, through the activation of Galpha(13) and RhoA. Finally, we have determined that expression of 5-HT(4(a)) receptor in neuroblastoma x glioma NIE-115 cells cause RhoA-dependent neurite retraction and cell rounding under basal conditions and after agonist stimulation. These data suggest that by activating 5-HT(4(a)) receptor-G(13) pathway, serotonin plays a prominent role in regulating neuronal architecture in addition to its classical role in neurotransmission.

The 5-hydroxytryptamine(1A) receptor is stably palmitoylated, and acylation is critical for communication of receptor with Gi protein.

In the present study, we verified that the mouse 5-hydroxytryptamine(1A) (5-HT(1A)) receptor is modified by palmitic acid, which is covalently attached to the protein through a thioester-type bond. Palmitoylation efficiency was not modulated by receptor stimulation with agonists. Block of protein synthesis by cycloheximide resulted in a significant reduction of receptor acylation, suggesting that palmitoylation occurs early after synthesis of the 5-HT(1A) receptor. Furthermore, pulse-chase experiments demonstrated that fatty acids are stably attached to the receptor. Two conserved cysteine residues 417 and 420 located in the proximal C-terminal domain were identified as acylation sites by site-directed mutagenesis. To address the functional role of 5-HT(1A) receptor acylation, we have analyzed the ability of acylation-deficient mutants to interact with heterotrimeric G(i) protein and to modulate downstream effectors. Replacement of individual cysteine residues (417 or 420) resulted in a significantly reduced coupling of receptor with G(i) protein and impaired inhibition of adenylyl cyclase activity. When both palmitoylated cysteines were replaced, the communication of receptors with G alpha(i) subunits was completely abolished. Moreover, non-palmitoylated mutants were no longer able to inhibit forskolin-stimulated cAMP formation, indicating that palmitoylation of the 5-HT(1A) receptor is critical for the enabling of G(i) protein coupling/effector signaling. The receptor-dependent activation of extracellular signal-regulated kinase was also affected by acylation-deficient mutants, suggesting the importance of receptor palmitoylation for the signaling through the G beta gamma-mediated pathway, in addition to the G alpha(i)-mediated signaling.

Expression studies and functional characterization of renal human organic anion transporter 1 isoforms.

The human organic anion transporter 1 (hOAT1) facilitates the basolateral entry of organic anions such as endogenous metabolites, xenobiotics, and drugs into the proximal tubule cells. In the present study we investigated the general occurrence of hOAT1 isoforms in the kidneys and performed functional characterizations. Kidney specimens of 10 patients were analyzed by reverse transcription-polymerase chain reaction. We detected hOAT1-2 as the main transcript in almost all patients, and weak transcripts of hOAT1-1, hOAT1-3, and hOAT1-4 in many of them. An evaluation of the renal distribution showed all four mRNAs mostly restricted to the cortex. Western blot analysis of membrane fractions from two kidney specimens yielded two bands corresponding to the observed mRNA expression, suggesting hOAT1-3 and hOAT1-4 to be expressed on the protein level in vivo. This observation is further supported by immunofluorescence analyses of all four cloned hOAT1 isoforms transiently transfected in COS 7 cells. Functional characterizations did not show any transport activity of hOAT1-3 and hOAT1-4 for the tested substrates. Cotransfection studies of each of them with hOAT1-1 did not alter fluorescein uptake indicating no regulatory impact of these isoforms. Further functional comparisons of hOAT1-1 and hOAT1-2 in fluorescein uptake studies exhibited almost identical affinities for fluorescein with Michaelis constants of 11.6 +/- 3.7 microM (hOAT1-1) and 11.9 +/- 6.4 microM (hOAT1-2), and similar sensitivities to inhibition by p-aminohippurate [IC(50): 16 microM (hOAT1-1), 10 microM (hOAT1-2)], urate [IC(50): 440 microM (hOAT1-1), 385 microM (hOAT1-2)], and furosemide (IC(50): 14 microM (hOAT1-1), 20 microM (hOAT1-2)], implying functional equivalence.

5-HT4(a) receptors avert opioid-induced breathing depression without loss of analgesia.

Opiates are widely used analgesics in anesthesiology, but they have serious adverse effects such as depression of breathing. This is caused by direct inhibition of rhythm-generating respiratory neurons in the Pre-Boetzinger complex (PBC) of the brainstem. We report that serotonin 4(a) [5-HT4(a)] receptors are strongly expressed in respiratory PBC neurons and that their selective activation protects spontaneous respiratory activity. Treatment of rats with a 5-HT4 receptor-specific agonist overcame fentanyl-induced respiratory depression and reestablished stable respiratory rhythm without loss of fentanyl's analgesic effect. These findings imply the prospect of a fine-tuned recovery from opioid-induced respiratory depression, through adjustment of intracellular adenosine 3',5'-monophosphate levels through the convergent signaling pathways in neurons.

The 5-hydroxytryptamine(4a) receptor is palmitoylated at two different sites, and acylation is critically involved in regulation of receptor constitutive activity.

We have reported recently that the mouse 5-hydroxytryptamine(4a) (5-HT(4(a))) receptor undergoes dynamic palmitoylation (Ponimaskin, E. G., Schmidt, M. F., Heine, M., Bickmeyer, U., and Richter, D. W. (2001) Biochem. J. 353, 627-663). In the present study, conserved cysteine residues 328/329 in the carboxyl terminus of the 5-HT(4(a)) receptor were identified as potential acylation sites. In contrast to other palmitoylated G-protein-coupled receptors, the additional cysteine residue 386 positioned close to the COOH-terminal end of the receptor was also found to be palmitoylated. Using pulse and pulse-chase labeling techniques, we demonstrated that palmitoylation of individual cysteines is a reversible process and that agonist stimulation of the 5-HT(4(a)) receptor independently increases the rate of palmitate turnover for both acylation sites. Analysis of acylation-deficient mutants revealed that non-palmitoylated 5-HT(4(a)) receptors were indistinguishable from the wild type in their ability to interact with G(s), to stimulate the adenylyl cyclase activity and to activate cyclic nucleotide-sensitive cation channels after agonist stimulation. The most distinctive finding of the present study was the ability of palmitoylation to modulate the agonist-independent constitutive 5-HT(4(a)) receptor activity. We demonstrated that mutation of the proximal palmitoylation site (Cys(328) --> Ser/Cys(329) --> Ser) significantly increases the capacity of receptors to convert from the inactive (R) to the active (R*) form in the absence of agonist. In contrast, the rate of isomerization from R to R* for the Cys(386) --> Ser as well as for the triple, non-palmitoylated mutant (Cys(328) --> Ser/Cys(329) --> Ser/Cys(386) -->Ser) was similar to that obtained for the wild type.