Vagal nerve stimulation (VNS) has been approved for treatment-resistant depression. Many antidepressants increase expression of brain-derived neurotrophic factor (BDNF) in brain or activate, via phosphorylation, its receptor, TrkB. There have been no studies yet of whether VNS would also cause phosphorylation of TrkB.Western blot analysis was used to evaluate the phosphorylation status of TrkB in the hippocampus of rats administered VNS either acutely or chronically. Acute effects of VNS were compared with those caused by fluoxetine or desipramine (DMI) whereas its chronic effects were compared with those of sertraline or DMI.All treatments, given either acutely or chronically, significantly elevated phosphorylation of tyrosines 705 and 816 on TrkB in the hippocampus. However, only VNS increased the phosphorylation of tyrosine 515, with both acute and chronic administration causing this effect. Pretreatment with K252a, a nonspecific tyrosine kinase inhibitor, blocked the phosphorylation caused by acute VNS at all three tyrosines. Downstream effectors of Y515, namely Akt and ERK, were also phosphorylated after acute treatment with VNS, whereas DMI did not cause this effect.VNS rapidly activates TrkB phosphorylation and this effect persists over time. VNS-induced phosphorylation of tyrosine 515 is distinct from the effect of standard antidepressant drugs. Neurotrophins, particularly brain-derived neurotrophic factor (BDNF), are key molecules regulating neuronal survival, development, function and plasticity via the TrkB receptor is required for a behavioral response typically induced by antidepressants. In particular, they showed that administration of antidepressants to transgenic mice over-expressing the truncated TrkB-T1 isoform, which lacks the functional cytosolic signaling domain, does not produce less immobility in the forced swim test (FST).The neurotrophic hypothesis of depression and antidepressant action arose from evidence suggesting that BDNF levels are reduced in mood disorders Vagal nerve stimulation (VNS) has been approved by the Food and Drugs Administration (FDA) for treatment-resistant epilepsy (1997) and treatment-resistant depression (TRD) (2005). Over long time periods  it has been shown to be effective in reducing the severity of symptoms in patients with TRD ad libitum. Experimental protocols were approved by the IACUC in accordance with the guidelines of the Public Health Service, American Physiological Society, and the Society for Neuroscience.Experiments were carried out using adult male Sprague-Dawley rats, 250–350 g (Harlan). Rats were group housed and maintained in a temperature-controlled environment on a 14∶10 h light-dark cycle and had access to food and water Vagus nerve electrodes were implanted on the vagus nerve under aseptic conditions. The surgical procedure was similar to that described by Cunningham et al. This was done exactly as described previously Rats were administered K252a . K252a is a nonspecific tyrosine kinase inhibitor 2O, and 10 µl of protease inhibitor mixture (Sigma) per 100 mg tissue), incubated on ice for 30 min and then centrifuged . Protein levels of the collected supernates were measured using the Bradford assay . Proteins were separated in a SDS-PAGE gel and blotted onto a nitrocellulose membrane. Membranes were incubated at 4°C overnight with the following primary antibodies: anti-phoshoY515 , anti-phoshoY705 , anti-phoshoY816 , and anti-TrkB , anti-phoshoAkt , anti-phoshoAkt , anti-Akt , anti-p44/42 MAPK , anti-phosho-p42/44 MAPK . Equal loading was confirmed using anti-β-actin , which was routinely used for normalization. Membranes were washed with TBS/0.1% Tween (TBST) and incubated with horseradish peroxidase conjugated secondary antibody . Secondary antibodies were visualized using enhanced chemiluminescence kits  followed by an exposure to X-ray film for detection.Rats were sacrificed by rapid decapitation, brains quickly removed and hippocampi were dissected out on an ice-cooled dish. Samples were stored at −80°C until processed for Western blot analysis. Samples were homogenized in a lysis buffer  followed by Student's Newman-Keuls post-hoc analysis revealed that acute administration of either fluoxetine or DMI caused a significant increase in phosphorylation of tyrosines 705 and 816 in the hippocampus compared to that measured in vehicle-treated rats .In the first experiment, the effect of acute (2 hr) VNS on TrkB phosphorylation was compared with that produced by acute administration of a selective serotonin reuptake inhibitor (SSRI), fluoxetine , and a selective norepinephrine reuptake inhibitor (NRI), desipramine  . The druted rats . Acute ated rats . Total Tted rats , e.g., tGiven the importance of prolonged antidepressant and VNS treatments for optimal clinical response in depression, the next experiment examined the effect of longer administration, for 14 days, of VNS, sertraline  or DMI  on the phosphorylation of TrkB. The results were similar to what was seen acutely, namely the drugs and VNS significantly increased phosphorylation at Y705  and Y816 ; for VNS, F = 10.58, p<0.01] but only VNS increased phosphorylation of Y515  . WesternPost-hoc analysis revealed that administration of K252a alone did not alter TrkB phosphorylation in comparison to that measured in rats pretreated with vehicle. Total TrkB protein levels were not altered  and that the phosphorylation of tyrosine 515 is distinct from the effect of antidepressant drugs, there may be some differences in the mechanisms by which VNS and antidepressant drugs cause such effects. To determine whether VNS was causing tyrosine 515 to be phosphorylated through a mechanism not directly involving TrkB activation, i.e., not involving the autophosphorylation site, we tested the effect of an agent that prevents tyrosine kinase activity, K252a. It was administered intracerebroventricularly for 2 hours before the start of VNS, which was then given for 2 hours. K252a pretreatment blocked the effects of VNS on TrkB phosphorylation at all three tyrosines, namely Y705, Y816 and Y515 . Two-fac altered .Because of the VNS-induced phosphorylation of Y515, subsequent experiments compared the effect of VNS to that produced by DMI on phosphorylation of proteins downstream of the 515 site on TrkB, namely Akt and ERK2 Post hoc analysis indicated VNS caused significant increases in phosphorylation of both S475 and T308. In this case, though, K252a produced a complete, rather than partial, blockade of the effect of VNS  and ERK1/2 (at threonine 202 for ERK1 and Tyr204 for ERK2) in total hippocampus. VNS treatment consistently caused an increase in phosphorylation of both Akt and ERK2, whereas treatment with DMI failed to increase in Akt and ERK phosphorylation. Further studies using immunohistochemical detection instead of western blot analysis is needed to address whether or not DMI could potentially cause a more selective pattern of activation of specific subregions within hippocampus.If BDNF were mediating the effect of antidepressants, one might expect all three tyrosines to be phosphorylated. It is often assumed that the signaling molecule causing the increase in antidepressant-induced phosphorylation of TrkB is BDNF. However, a recent study by Rantamaki et al. However, as VNS increases phosphorylation of all three tyrosines and, in addition, induces the phosphorylation of Akt/ERK known to be coupled to Y515, it still remains possible that BDNF (and/or NT-4) is mediating its effect on TrkB. In addition, it is possible that VNS increases TrkB signaling by mechanisms in addition to its influencing BDNF or NT-4 release. Increased intracellular cAMP in vitro have shown that norepinephrine application in cultured hippocampal neurons is able to induce TrkB phosphorylation and downstream signaling (activation of ERK and PI3-K) via GPCR transactivation of TrkB in vivo approach. Cultured neurons lack all the physiological inputs that could be responsible for the mechanisms by which TrkB is differentially activated.It is also possible that the effects of VNS could be ligand-mediated, whereas the effects of DMI could potentially be mediated by transactivation of TrkB through G-protein coupled receptors (GPCRs). DMI is a tricyclic antidepressant that preferentially blocks norepinephrine transporters Other possible mechanisms underlying this difference between the effects of the antidepressants and VNS include membrane surface expression of TrkB isoforms. Alternative splicing of the TrkB pre-mRNA from its locus on DNA yields two isoforms Nocardiopsis, is frequently used as a nonspecific protein kinase inhibitor for blocking Trk activity. It was first described as an inhibitor of protein kinase C K252a, an alkaloid-like compound isolated from In conclusion, VNS phosphorylates tyrosine 515 on TrkB and also the activation of effectors coupled to this Y515 site (Akt/ERK), and this effect is distinct from those phosphorylated by standard antidepressant drugs. What role, if any, this plays in the behavioral effect of VNS remains to be clarified by future experiments.


Here, we determined the distributions of inhibitory postsynaptic sites on the dendritic arbors of individual neurons, and compared their developmental patterns with that of excitatory postsynaptic sites. We focused on retinal ganglion cells (RGCs), the output neurons of the retina, which receive excitatory input from bipolar cells and inhibitory input from amacrine cells. To visualize and map inhibitory postsynaptic sites, we generated transgenic mice in which RGCs express fluorescently tagged Neuroligin 2 (YFP-NL2) under the control of the Thy1 promoter. By labeling RGC dendrites biolistically in YFP-NL2-expressing retinas, we were able to map the spatial distribution and thus densities of inhibitory postsynaptic sites on the dendritic arbors of individual large-field RGCs across ages.Neuronal output is shaped by a balance of excitation and inhibition. How this balance is attained in the central nervous system during development is not well understood, and is complicated by the fact that, A receptor and glycine receptors. YFP-NL2 puncta were apposed to the vesicular inhibitory transmitter transporter VGAT but not to CtBP2, a marker of presynaptic ribbons found at bipolar cell terminals. Similar patterns of co-localization with synaptic markers were observed for endogenous NL2. We also verified that expression of YFP-NL2 in the transgenic line did not significantly alter spontaneous inhibitory synaptic transmission onto RGCs. Using these mice, we found that, on average, the density of inhibitory synapses on individual arbors increased gradually until eye opening . A small centro-peripheral gradient in density found in mature arbors was apparent at the earliest age we examined . Unexpectedly, the adult ratio of inhibitory/excitatory postsynaptic sites was rapidly attained, shortly after glutamatergic synaptogenesis commenced .We demonstrate that YFP-NL2 is present at inhibitory synapses in the inner plexiform layer by its co-localization with gephyrin, the γ2 subunit of the GABAOur observations suggest that bipolar and amacrine cell synaptogenesis onto RGCs appear coordinated to rapidly attain a balanced ratio of excitatory and inhibitory synapse densities prior to the onset of visual experience. The normal functioning of the nervous system requires balanced excitatory and inhibitory neurotransmission. If excitation or inhibition is perturbed, neurons undergo alterations in their intrinsic excitability and synaptic transmission in order to restore a balance, and prevent their circuits from undergoing epileptiform activity ,2. Whilein vivo development have not been charted. Because mature inhibitory synapse distributions vary across cell types [Many studies have tracked the distribution of glutamatergic synapses on a given neuron by imaging spines -9 or by ll types  and evenll types , it is ill types .Inhibition in the inner retina is provided by amacrine interneurons that primarily use either GABA or glycine as their neurotransmitter ,19. SeriThe ratio of amacrine to bipolar cell synapses is not known for developing RGCs. EM of the mouse retina suggested that conventional (presumed amacrine) synapses are present shortly after birth, and ribbon (bipolar) synapses appear only after postnatal day (P)10 . ConventWe previously expressed PSD95 fluorescently tagged with yellow fluorescent protein (PSD95-YFP) in RGCs to label their glutamatergic postsynaptic densities . Here, wA receptor [A receptor , or an Fristchy . SimilarFristchy , we fount Figure .A and glycine receptors . Our results indicate that there is a positive correlation of endogenous NL2 signal and immunolabeled gephyrin, GABAs Figure . We confe et al.  demonstre et al.  indicatiThy1 promoter drives expression of NL2 fused to YFP. Retinas from Tg(Thy1-YFP-NL2) mice showed punctate expression of YFP-NL2 in a large population of cells in the ganglion cell layer. Bright YFP puncta were present along dendrites of the RGCs, as well as on their somata  receptors [NL2 is one of the earliest components of the postsynaptic specialization of inhibitory synapses, and helps recruit gephyrin ,45 and Gse sites ,40. In tnous NL2 . Like eneceptors . In the eceptors  and is peceptors . We obseThy1-YFP-NL2 mice showed no significant change in the median amplitude and frequency of spontaneous inhibitory synaptic currents. Moreover, transgenic YFP-NL2 animals showed grossly normal behavioral traits, life spans and offspring production. This is in contrast to what was described for transgenic lines with high levels of NL2 over-expression (50% or more above endogenous levels), which displayed a variety of phenotypes, including altered synaptic function, early postnatal death, and behavioral changes such as limb clasping, repetitive behavior and anxiety [One concern that we had was that in our transgenic line, expression of YFP-NL2 may alter synapse number because NL2 over-expression increases the number of both inhibitory and excitatory synaptic connections . However anxiety . Thus, wTo date, the densities and distribution patterns of amacrine synapses onto the dendrites of RGCs in the mature retina have been obtained largely by serial EM. As such, only a handful of cells can be fully reconstructed. More recently, combining immunostaining for transmitter receptors with single cell labeling has enabled comparisons of the distributions of excitatory and inhibitory synapses on adult primate RGCs ,28. HereA receptor suggest that inhibitory synapses constitute at least one-third of all synapses onto these RGCs [Serial EM and partial three-dimensional reconstructions of RGC dendritic trees in the cat, marmoset and rabbit retina have also revealed a considerable variation in the ratio of inhibitory and excitatory synapses onto individual RGCs -24,50. Aese RGCs ,50. In oese RGCs . Thus, tin vivo, GABAergic and glycinergic synaptic connections are formed before glutamatergic connections. For example, in the hippocampus, the apical dendrite of CA1 pyramidal cells is first contacted by GABAergic inteneurons prior to extension of these dendrites into the stratum lacunosum-moleculare, where they later receive input from glutamatergic afferents of the perforant pathway [A receptors, glycine receptor subunits or gephyrin on RGCs, for example in rat and rabbit retina [It is evident from morphological and physiological studies that,  pathway ,5. Likew pathway  (but see pathway ). Unlike pathway -55. Thus pathway . Rapid at retina ,56.in vitro [Our observations here show that the time course in the increase in YFP-NL2 puncta density with age parallels that of PSD95-YFP on the dendrites of large-field RGCs , resultiin vitro ,58. It sin vitro .What mechanisms regulate the ratio of inhibitory to excitatory synapses on dendrites? It is likely that apart from glutamatergic transmission ,61, GABAThy1-YFP-NL2 transgenic mouse line we generated, we found that the density of inhibitory amacrine synapses on the arbors of large-field ON RGCs increased gradually, until around eye opening (P15). By comparing the spatial densities of YFP-NL2 with PSD95-YFP across the dendritic arbors of these RGCs, we discovered that their adult ratio of inhibitory/excitatory postsynaptic sites was rapidly attained, shortly after bipolar cells form synapses in the IPL (P7). Our observations suggest that bipolar and amacrine cell synaptogenesis onto RGCs are coordinated, and a balanced ratio of excitatory and inhibitory synapse densities is established prior to the onset of visual experience.Based on YFP-NL2 expression in a et al. [Thy1 promoter [Thy1 promoter fragment into shuttle vector LNL, which contained the needed restriction sites for transgene release. YFP-NL2 was digested with HindIII and Afl II and blunted into XhoI cut and blunted thy1-LNL. The Thy1-YFP-NL2 transgene was released from the vector backbone sequence by restriction digestion with AscI and PmeI and injected into B6/CBA F1 hybrid pronuclei to generate founder mice.YFP-NL2 expressed here was modified from Graf et al.  and conspromoter  was usedThy1-YFP-NL2  mice were deeply anesthetized with 5% isofluorane and decapitated. Eyes were removed and placed in ice cold mouse artificial cerebrospinal fluid , 20 mM HEPES, pH = 7.4). After removing the lens and vitreous, the eye cup was fixed in 4% paraformaldehyde for 15 to 30 minutes. After fixation, the eye cups were rinsed in 0.1 M PBS. The retina was removed from the eye cup, embedded in 4% low-melting point agarose and cut into 60 μm thick sections using a vibratome. Sections were mounted and used for imaging or processed for immunostaining as follows: blocked in 10% NGS in PBS for 1 hour followed by overnight incubation in 5% NGS, 0.5% Triton-X100, with the corresponding primary antibodies. The primary antibodies used were: rabbit anti-NL2 antibody  [C57BL/6 (P21) and N Brose) ,40, guinThy1-YFP-NL2 mice were deeply anesthetized with 5% isofluorane and decapitated. Eyes were removed and placed in ice cold mouse mACSF. Retinas were removed from the eye cup and mounted RGC side up on black nitrocellulose filter paper . Gold particles were coated with 20 μg CMV:tdtomato DNA (gift of R Tsien) and delivered using a Bio-Rad Helios gene-gun as previously described [escribed . RetinasImages were obtained using a 1.35 NA 60× oil objective (Olympus). Images were acquired at 0.069 × 0.069 × 0.3 μm for double labeling immunohistochemistry Figures , and 0.1In order to assess whether NL2 signal significantly overlaps with other synapse markers, a custom Matlab program was used to calculate the two-dimensional cross-correlation coefficients of the signals from both channels (Josh Morgan and Daniel Kerschensteiner). This approach does not require identification of puncta, yet allows us to determine whether the fluorescent signals in the two channels are spatially correlated, or are randomly associated (random association determined by rotating one image 180° relative to the other image).Within an optical plane, the signal intensity of a pixel in the green channel was compared with the signal intensity of the corresponding pixel in the red channel . To obtain the two-dimensional correlation plot, the intensity of a green pixel was compared with the intensity of red pixels (or vice versa) to the right, left, top and bottom, displaced from the reference pixel up to 2.8 μm. A pixel intensity threshold of 20 to 35, defining the background, was used while analyzing images obtained from wild-type mouse retina. Thresholding was not used when analyzing images obtained from the YFP-NL2 transgenic mouse line due to the lower immunohistochemistry background level in the transgenic retina.Representative examples shown in the figures are cross-correlation coefficients calculated based on 20 to 45 image planes (z-step size = 0.3 μm) of an imaged field of view. The equation used to calculate the correlation coefficient is:where R is the correlation coefficient of the red and green channel and C is the covariance of the corresponding channels.For two identical images, the correlogram peak is 1. The value of the positive peak for two identical images is only weakly influenced by the absolute intensities of the pixels within an image but strongly corresponds to how spatially 'similar' the two images are. For overlapping green and red puncta, the peak of the two-dimensional correlation plot falls off symmetrically on all sides. The correlograms with positive peaks have a full-width at half maximum of less than 1 μm, suggesting that there exist structures in the two images within less than 1 μm overlap in space.2, 1 mM MgCl2, 1.25 mM NaH2PO4, 11 mM glucose and 26 mM NaHCO3 (equilibrated with 95% O2 and 5% CO2). Whole-cell recordings were performed with electrodes (4 to 8 MΩ) filled with 120 mM Cs-gluconate, 1 mM CaCl2, 1 mM MgCl2, 10 mM Na-HEPES, 11 mM EGTA, and 10 mM TEA-Cl (pH 7.2 adjusted with CsOH). For some experiments 2 mM QX314 was also included in the patch pipette. A liquid junction potential of 15 mV was corrected before the cell was attached, and series resistance was not compensated. Data were acquired using an Axopatch 200 B amplifier (Molecular Devices), low-pass filtered at 2 kHz and digitized at 5 kHz. sIPSCs were recorded at -0 mV, the reversal potential of cation currents in our recording conditions. Area and amplitude thresholds  were optimized to detect > 90% of the events identified by eye for the entirety of recordings analyzed. For overlapping events, the baseline for amplitude measurement of each event was estimated from exponential decay extrapolation of the previous event.Retinal flat mounts were prepared as described above for cell transfection, but mounted on white filter paper  for better visualization. Recordings were performed at room temperature and retinas were maintained in bicarbonate-buffered mACSF containing 125 mM NaCl, 2.5 mM KCl, 2 mM CaClCNS: central nervous system; CtBP2: carboxy-terminal binding protein 2; EM: electron microscopy; GABA: γ-aminobutyric acid; IPL: inner plexiform layer; mACSF: mouse artificial cerebrospinal fluid; NL2: Neuroligin 2; P: postnatal day; PBS: phosphate-buffered saline; RGC: retinal ganglion cell; sIPSC: spontaneous inhibitory postsynaptic current; VGAT: vesicular inhibitory transmitter transporter; YFP: yellow fluorescent protein.The authors declare that they have no competing interests.FS and AB carried out the experiments. FS, AB and DK performed the analysis. RL, YK, and AMC generated the transgenic mice. FS and ROLW designed and coordinated the work. All authors read and approved the final manuscript.


Gap junctions are comprised of connexins that form cell-to-cell channels which couple neighboring cells to accommodate the exchange of information. The need for communication does, however, change over time and therefore must be tightly controlled. Although the regulation of connexin protein expression by transcription and translation is of great importance, the trafficking, channel activity and degradation are also under tight control. The function of connexins can be regulated by several post translational modifications, which affect numerous parameters; including number of channels, open probability, single channel conductance or selectivity. The most extensively investigated post translational modifications are phosphorylations, which have been documented in all mammalian connexins. Besides phosphorylations, some connexins are known to be ubiquitinated, SUMOylated, nitrosylated, hydroxylated, acetylated, methylated, and γ-carboxyglutamated. The aim of the present review is to summarize our current knowledge of post translational regulation of the connexin family of proteins. Figure 1). Six connexins oligomerize to form a connexon, which, once inserted in the plasma membrane, may dock with a connexon from a neighboring cell and form an intercellular gap junctional channel. When many intercellular channels aggregate, they form a gap junction plaque, which present a typical penta-laminar structure when viewed in transmission electron microscope images . In some cases, free connexons may open directly to the extracellular medium in which case they are often referred to as hemichannels. This review will primarily deal with intercellular gap junctional channels.Connexins are a family of membrane proteins forming gap junctional channels. Each connexin is a four membrane spanning protein with two extracellular loops, an intracellular loop and intracellular N- and C-termini . The most widely investigated PTMs in connexins are phosphorylations and ubiquitinations, but in recent years studies have shown how other PTM such as ubiquitination, SUMOylation, nitrosylation, hydroxylation, acetylation, methylation, and γ-carboxyglutamation also play important roles in the regulation of intercellular communication.Intercellular coupling is determined by both the number of channels incorporated in gap junctions, their activity and selectivity. The number of channels is regulated by transcription, translation, oligomerization, trafficking to and from the membrane, and degradation. Pre-translational events (transcription and translation) does of course not involve PTM  is not only the most widely expressed in mammalian cells, it is also the most intensively studied connexin. Cx43 is translated as a 40 kDa protein, which becomes phosphorylated to a 41 kDa form soon after synthesis . The earal Golgi . A more al Golgi , hemodynal Golgi  and diabal Golgi , may affFigure 2 illustrates all the specific phosphorylation sites identified in Cx43, as well as the specific kinases that facilitate their phosphorylation. In addition to the sites and kinases shown in Figure 2, bioinformatics has identified additional phosphorylation consensus sites in Cx43 and heat map analysis reveal that the majority of the phosphorylation sites may be recognized by multiple kinases , are targets for in vitro phosphorylation by Ca2+/calmoduline-dependent kinase II (CaMKII). CaMKII is involved in a variety of cellular processes, such as Ca2+ homeostasis, transcription and apoptosis  in cardiomyocytes . ZO-1 isBased on the complex nature of Src kinase regulation of Cx43 gap junction channels, it is not surprising that Src induced alterations in gap junction coupling is found in several pathological conditions. Since gap junction communication is essential for controlled cell growth and cell differentiation, Src kinase induced Cx43 phopshorylation seems to play an essential role in tumorigenesis  was associated with a decreased macroscopic and single channel conductance in SKHep1 cells  induced stimulation of protein kinase C  in cultured cardiomyocytes induces Ser262 phosphorylation along with increased cardiomyocyte proliferation . The samin vitro  responsible for phosphorylation of Ser296, Ser297, and Ser306 is still a matter of debate; While That Cx43 phosphorylation is involved in the regulation of trafficking, assembly and dis-assembly, as well as the localization of Cx43 gap junction channels was first suggested by Musil and Goodenough . Since tIn isolated Langendorff perfused rat hearts, Ser330 becomes phosphorylated within 7 min of global no-flow ischemia, whereas it returns to the dephosphorylated state between 30 and 45 min of ischemia . In addiChronic hemodynamic overload also causes dephosphorylation and delocalization of atrial Cx43 in both rats and humans, which may be related to the development of atrial fibrillation . FurtherSer364 is phosphorylated by protein kinase A PKA; , while SBesides being a potential target for both PKA and PKC, Ser365 is proposed to be a “gatekeeper,” which controls the ability of other serine residues in Cx43 to become phosphorylated. More specifically, it was reported that phosphorylation of Ser365 causes a conformational change in the c-terminal region of Cx43, which prevents PKC induced phosphorylation of Ser368 . These dO-tetradecanoyl-phorbol 13-acetate (TPA), is also known to increase cardiac macroscopic conductance along with reduced single-channel conductance  . Akt indin vitro. However, it is still unknown whether CaMKII phosphorylates Cx43 in vivo and the potential physiological role of CaMKII induced Cx43 phosphorylation remains to be established.Finally, the previously discussed mass spectrometry study by Most experimental studies have focused on the protein kinases, which are responsible for Cx43 phosphorylation. Nevertheless, regulation of Cx43 phosphorylation is not only dependent on the kinases, but also on the equilibrium between protein phosphatase and kinase activity. Even so, experimental data regarding the phosphatases that dephosphorylate Cx43 remain limited.Under normal physiological conditions, both protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) co-localize with Cx43 in rabbit hearts , indicatin vivo. Based on the available data, it seems that PP1 is the main player during acute dephosphorylation of Cx43, as seen during cardiac ischemia, whereas PP2A may become the dominant player during long-term pathological alterations such as non-ischemic heart failure. The different results may, however, also be species dependent and further studies are needed before the final conclusion can be drawn.Both PP1 and PP2A are also expressed in mini pig hearts, but in contrast to what was found in rabbit hearts, only PP2A co-localizes with Cx43 in the mini pig . FurtherAs outlined above, phosphorylation of Cx43 has been a subject for intense investigation for more than three decades. These investigations have clarified that the regulation of Cx43 gap junction channels by phosphorylation is an extremely complex matter, which includes at least 21 different phosphorylation sites and a growing list of more than 10 different kinases and phosphatases. In addition, it is now clear that the different phosphorylation sites may interact and depend upon each other and site specific changes in phosphorylation may exert different effects on electric and metabolic coupling. Furthermore, it seems that the effects of altered Cx43 phosphorylation may be both species and cell type dependent. All of these considerations should be taken into account when planning future studies on Cx43 phosphorylation, as well as during the interpretation of such studies.For many years, it was generally thought that Cx26 was not a phospho-protein, because of its fairly short C-terminal tail, consisting of only 11 amino acids. A mass spectrometry study, however, showed that Cx26 is phosphorylated in the intracellular N-terminal tail at either position Asp2, Thr5, or Ser8 when expressed in HeLa cells . ImportaAs for Cx26, Cx32 also contains phosphorylation sites in the N-terminal domain, more specifically Thr4, Tyr7, Thr8, or Ser11, which was identified as phosphorylated in Cx32 from both HeLa cells and mouse liver . In addiFurther experimental studies are needed in order to reveal to physiological role of Cx32 phosphorylation in biological systems.32 into Cx40 in transfected human cells and that 8-Br-cAMP induced PKA activation causes an electrophoretic mobility shift of Cx40. Furthermore, Cx40 is also regulated by post-translational phosphorylation , but theRecently, it was also shown that reduced serine phosphorylation of Cx40 correlates with reduced electrical coupling between microvascular endothelial cells (EC) during sepsis . This prSince Cx40 is the predominant connexin found in the atria, it can be hypothesized that dephosphorylation of Cx40 and decreased electrical coupling in the atria may play a role in the patogenisis of atrial fibrillation. This, however, remains an interesting question for future research.32P-orthophosphate. Even when all the above mentioned carboxy-terminal serine residues of Cx45 were replaced, it did not interfere with intracellular trafficking or assembly of gap junction channels at the plasma membrane. Furthermore, HeLa cells transfected with the Cx45 mutant showed the same extend of dye transfer as cells transfected with wild type Cx45  to a target protein begins with activation of ubiquitin by an E1 ubiquitin activating enzyme that forms a bond between the carboxyl group of the C-terminal glycine residue (Gly 76) of ubiquitin and an E1 cysteine. This step is followed by transfer of ubiquitin from E1 to a cysteine residue of the ubiquitin-conjugating enzyme E2. Finally, the E2-ubiquitin conjugated enzyme associates with an E3 ubiquitin-protein ligase  and/or phosphoserine and phosphothreonin containing motifs and thereby mediate target recognition .An ubiquitin molecule contains a total of seven lysine residues. Following addition of a single ubiquitin to a protein (monoubiquitination), further ubiquitin molecules can be conjugated to lysine residues in the bound ubiquitin, resulting in a polyubiquitin chain. The first identified type of polyubiquitin chains were linked via lysine 48 (Lys48 forming an isopeptide bond to Gly76), however, a wide variety of linkages involving all possible lysine residues  have been demonstrated, reviewed by . FurtherThe various types of ubiquitin modifications have been linked to distinct physiological functions in cells. Multi-monoubiquitination is associated with DNA repair and receptor endocytosis, and lysosomal degradation or recycling to the surface  whereas Ubiquitination may be involved in several important steps in the life cycle of connexins  was the first E3 ubiquitin ligase to be shown to interact with connexin . Nedd4 iNedd4 belongs to the HECT family of E3 ubiquitin ligases  and contMany growth factors, such as EGF and tumor promotors, including TPA inhibits intercellular communication by inducing Cx43 ubiquination followed by endocytosis and degradation . In rat Besides stimulating the MAP kinase pathway, EGF activates phospholipase C, resulting in hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol DAG; . ReducedThe tumor promoting phobol ester TPA activates the MAP kinase pathway, as well as PKC . This inRecently, the role of ubiquitination in regulation of Cx43 gap junction turnover was questioned in a study by Small ubiquitin-like modifier (SUMO) proteins are a small family of proteins that are structurally and functionally related to ubiquitin. In humans, the SUMO protein family consists of 3 members (SUMO1-3). SUMO 2 and 3 show a high degree of similarity . The strAnalog to ubiquitination, SUMOylation is controlled by an enzymatic cascade resulting in covalently attachment of SUMO to lysine residues in the target protein  plays an important role in coordinating the activity of EC and vascular smooth muscle cells VSMC; . The MEJS-nitrosylation play an important role in regulating hemichannel permeability in astrocytes (Xenopus oocytes caused an increased voltage sensitivity and current amplitude (Nitrosylation may also play a role in the regulation of hemichannel function. Ischemia and/or hypoxia are associated with an increased production of NO, and at the same time an increase in Cx43 and Cx46 hemichannel permeability has been observed , 2009. Ttrocytes . The sammplitude . On the Although methylation and acetylation are mainly known for their epigenetic regulation of gene transcription by DNA methylation and histone acetylation, there is emerging evidence that connexins may be directly modified. By mass spectrometry, Cx26 was shown to be both methylated and acetylated . Five meBesides methylation, Cx26 is also acetylated on six sites including Lys15 and Lys102 for which disease causing mutations/deletions are known (K15T, del102K; The data from Cx26 and Cx43 clearly shows that methylation and acetylation will likely emerge as important PTMs in the regulation of intercellular communication mediated by these and other connexins.2+ binding site. This indicates that γ-carboxylation of Cx26 may be involved in the regulation of Cx26 Ca2+ sensitivity and, in this context, it is noteworthy that mutations at Glu42 and Glu47 cause Cx26 dependent deaf mutations (2+ sensitive connexins, such as Cx43 and Cx50, are also subject to γ-carboxylation.The conversion of glutamic acid to γ-carboxyglutamic acid is a vitamin K dependent PTM, which was originally described for blood clotting proteins . The masutations . This imThe literature clearly shows the importance of PTMs in modifying gap junctional coupling and dependent on the connexin and the PTM type, may either increase or decrease intercellular coupling. This is achieved by regulating connexin function at all levels, including oligomerization, trafficking, channel activity and connexin degradation. The importance of these phenomena is underpinned by the fact that several disease causing mutations affect amino acids known to be PTM sites, as described above.The large and increasing number of PTMs that regulate connexins comprises a major challenge in unraveling their function and importance. For instance, Cx43 contains at least 24 PTM sites and the number of combinations thereof is overwhelming and their investigation beyond the reach of current methods for studying connexins. Most studies address only one or a few modifications at a time using site directed phosphospecific antibodies or mutagenesis. Such approaches carry the risk of overlooking confounding influence of other modifications and may explain some of the contradicting results in the literature. The field of proteomics is rapidly evolving and may eventually disclose quantitative important PTMs and combinations thereof occurring in physiology and pathophysiology. At present, the published mass spectrometry studies of connexins are limited to determinations of whether a certain modification is detectable or not. The threshold for such detection is undefined and not necessarily linked to the threshold for achieving a significant physiological effect although this is often implied. In some cases, such as for Ser306 in Cx43, one may find a change in phosphorylation that passes the threshold for detection after some intervention, in this case ischemia , and be The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Therefore, regulation of tyrosine hydroxylase (TH) activity may be particularly critical for somatodendritic DA signaling. Phosphorylation of TH at ser31 or ser40 can increase activity, but their impact on L-DOPA biosynthesis in vivo is unknown. Thus, determining their relationship with L-DOPA tissue content could reveal a mechanism by which DA signaling is normally maintained. In Brown-Norway Fischer 344 F1 hybrid rats, we quantified TH phosphorylation versus L-DOPA accumulation. After inhibition of aromatic acid decarboxylase, L-DOPA tissue content per recovered TH protein was greatest in NAc, matched by differences in ser31, but not ser40, phosphorylation. The L-DOPA per catecholamine and DA turnover ratios were significantly greater in SN and VTA, suggesting greater reliance on de novo DA biosynthesis therein. These compartmental differences reflected an overall autonomy of DA regulation, as seen by decreased DA content in SN and VTA, but not in striatum or NAc, following short-term DA biosynthesis inhibition from local infusion of the TH inhibitor α-methyl-p-tyrosine, as well as in the long-term process of aging. Such data suggest ser31 phosphorylation plays a significant role in regulating TH activity in vivo, particularly in somatodendritic regions, which may have a greater reliance on de novo DA biosynthesis. Thus, to the extent that somatodendritic DA release affects behavior, TH regulation in the midbrain may be critical for DA bioavailability to influence behavior.Measures of dopamine-regulating proteins in somatodendritic regions are often used only as static indicators of neuron viability, overlooking the possible impact of somatodendritic dopamine (DA) signaling on behavior and the potential autonomy of DA regulation between somatodendritic and terminal field compartments. DA reuptake capacity is less in somatodendritic regions, possibly placing a greater burden on  Two factors, however, challenge the concept that ser40 phosphorylation plays a singular role in regulating TH. First, while depolarizing stimuli increase striatal ser19, ser31, and ser40 phosphorylation in vivoin situTyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis in vivo. In support of this concept, ser31 phosphorylation co-varies with DA tissue content in vivode novo DA biosynthesis may be comparatively more critical to maintain normal DA bioavailability, ser31 phosphorylation could have considerable impact on DA regulation and, consequently, upon DA-influenced behaviors. In fact, together both ser31 phosphorylation status and TH protein content in the substantia nigra (SN) have significant correlation to locomotor activity in vivo is still an open question, because either phosphorylation site can affect L-DOPA biosynthesis and because DA tissue content is influenced by both DAT and TH function Direct involvement of increased ser19 phosphorylation in L-DOPA biosynthesis is not likely in vivo pharmacological approach, and by assessing the impact of aging on TH and DA tissue content. We provide evidence that in somatodendritic compartments, DA tissue content may have greater dependence on de novo biosynthesis and that ser31 TH phosphorylation likely plays a major role in the regulation thereof. The results also indicate that the regulation of DA biosynthesis and metabolism is distinct and autonomous between the neuronal compartments, an observation that has critical implications for the modeling of DA-influenced behaviors.The basal differences in TH phosphorylation and DA reuptake between somatodendritic and terminal field regions imply that regulation of DA bioavailability may be autonomous between these compartments. We investigated this potential autonomy in three approaches: by examining TH phosphorylation versus L-DOPA and DA tissue content in CNS tissues, by determining how local TH inhibition affected DA tissue content using an 1 hybrid rats (BNF) of 6, 18, and 24 months old were obtained from NIA and given food and water ad libitum for at least seven days prior to tissue collection. Male BNF rats of 12 months old were purchased from Harlan and were used in the L-DOPA vs. TH phosphorylation component of the study. Additional BNF rats (<6 months old) were used along with Sprague-Dawley rats in the striatal AMPT infusion experiments. These strains were selected because the BNF rat is a proven model for aging and the Sprague-Dawley is a commonly used strain. All procedures were approved by the LSUHSC Institutional Animal Care and Use Committee under approval number P-09-055 and carried out in accordance with the NIH “Principles of laboratory animal care” (NIH publication no. 85-23).Male Sprague-Dawley rats, ages 8–12 months, were purchased from Harlan and used in the AMPT-infusion component of the study. Male Brown-Norway Fischer 344 FBNF rats of 12 months old were given a 50 mg/kg i.p. injection of the aromatic amino acid decarboxylase (AADC) inhibitor NSD-1015 , cat # 54880) dissolved in 0.9% saline. One hour later, striatum, nucleus accumbens, SN, and VTA were dissected from ice-cold chilled brain, as previously described p-tyrosine (AMPT) methyl ester hydrochloride , cat # M3281) was dissolved in Krebs buffer to a concentration of 0.92 mM and infused into the midbrain of anesthetized rats at coordinates relative to Bregma ) in a volume of 3 µl to deliver 1.38 nmoles of AMPT. Vehicle was infused in the contralateral SN to serve as an inherently matched control, meaning that baseline DA levels in each test subject (following vehicle-infusion) were used to quantify AMPT effects. At 90 min after infusion, the striatum, nucleus accumbens, SN, and VTA were dissected from ice-cold chilled brain, as previously described Alpha-methyl-4-EDTA buffer. Aliquots from these protein-precipitated supernatants were analyzed for DA, DOPAC, L-DOPA, and/or NE tissue content by HPLC Dissected brain tissues were kept frozen at −70°C until sonicated in ice-cold 0.1 M HClO125-protein A (high-specific activity). The blots are exposed to Kodak film to reveal immunoreactive areas, which are excised and counted for gamma radioactivity. In the case of quantification of the TH protein, the value is expressed as cpm per ug total protein loaded Protein quantities were determined by BCA method. Protein concentration in each aliquot was determined and multiplied by the total volume of SDS to determine the entire protein quantity (as mg) recovered in each sample. Total protein content is used to normalize DA tissue content and TH protein from each sample. For TH protein and TH phosphorylation determination, samples are prepared in reducing (dithiothreitol used as reducing agent) sample buffer containing SDS and are subjected to SDS gel electrophoresis on 10% gels and transferred to nitrocellulose. Protein loads are verified by Ponceau S stain and blots are put into polyvinylpyrrolidone-based Tris buffer for at least 2 hr prior to antibody exposure. For each blot immunolabeling experiment, after treatment with primary and secondary antibody , the detection method uses Ist Century Biochemicals). Antibodies specific for phosphorylated ser19 and ser40 were purchased from Phosphosolutions . The ser31 primary antibody was previously validated for phosphorylation-state specificity Tyrosine hydroxylase phosphorylation is quantified by western blot using affinity-purified primary antibodies developed to the specific phosphorylation site. We have our own affinity-purified ser31 phosphorylation primary  with regard to endogenous L-DOPA tissue content, ratio of L-DOPA per catecholamine, DA tissue content, ratio of DOPAC per DA, TH protein, and TH phosphorylation used a Repeated measures ANOVA followed by Bonferroni's Multiple Comparison Test. Pearson correlational analysis was run on comparisons of L-DOPA per TH vs. TH phosphorylation stoichiometries and ser19 TH phosphorylation stoichiometry vs. ser31 and ser40 TH phosphorylation stoichiometries. To determine if DA/TH in each of the four regions was significantly different from the phosphorylation stoichiometries, comparing str vs. SN, str vs. NAc, str vs. VTA, SN vs. NAc, SN vs. VTA, and NAc vs. VTA, we used a Repeated Measures ANOVA followed by Dunnett's Multiple Comparison Test. Determination of statistical significance in the AMPT-related work used a paired Student's t-test, comparing pmols DA per mg protein in the AMPT-infused hemisphere versus pmols DA per mg protein in the matched contralateral vehicle-infused hemisphere. For assessment of aging-related effects on DA and TH measures, we assumed normal distribution and used Repeated Measures ANOVA, ranking the values obtained in each age group from highest to lowest, followed by Bonferroni's Multiple Comparison Test to determine if significant differences existed between the age groups.in vivo. However, L-DOPA is converted so efficiently into DA that L-DOPA is not detectable in CNS tissue unless aromatic amino acid decarboxylase (AADC) is inhibited. We verified linearity of L-DOPA detection in dopaminergic tissue using the HPLC analysis described in test rats that were given the AADC inhibitor NSD-1015. To compare L-DOPA to TH phosphorylation profiles, we then administered NSD-1015 (50 mg/kg i.p.) to 12-month old BNF rats one hour prior to dissection of dopaminergic brain regions. Total L-DOPA recovery (per protein) was greatest in terminal field compartments, being highest in the NAc (298±12 pmols L-DOPA per mg protein) followed by the striatum (269±4 pmols L-DOPA per mg protein). Somatodendritic compartments had comparatively less, being least in SN (40±3 pmols L-DOPA per mg protein) and then the VTA (172±9 pmols L-DOPA per mg protein)  . The recprotein) . Notablyprotein) .To further examine differences in basal TH activity and how site-specific phosphorylation affected L-DOPA content, we normalized L-DOPA to TH content , providiWe emphasize that these observations were made during inhibition of L-DOPA conversion to DA by the AADC inhibitor NSD-1015. Thus, the inhibition of DA formation in the neuropil may have influenced the TH phosphorylation observed in each region. For example, ser40 phosphorylation in the SN was over 0.10, which exceeds previous observations de novo DA biosynthesis in order to maintain normal DA bioavailability. Conversely, the striatum, which had the lowest L-DOPA per catecholamine ratio (0.17±0.003), may have the least dependence on de novo DA biosynthesis.Although L-DOPA recovery from the somatodendritic regions was much less than the terminal fields , in compde novo DA biosynthesis in cell body compartments was further supported by a greater degree of DA turnover in the somatodendritic regions  (1.4 nmols) into the midbrain produced a consistent and significant decrease in nigral DA tissue content (90 minutes later) in comparison to content in the contralateral SN, averaging ∼30% . This result further supports our findings that there is an overall autonomy in DA regulation between the somatodendritic and terminal field compartments.in vivo as evidenced by three major observations: 1) compartmental differences in TH phosphorylation at ser31 are seen under both basal conditions and conditions of AADC inhibition, 2) short-term local inhibition of TH reduces DA only in a targeted compartment without effect on DA in the cognate compartment of the same pathway, and 3) the long-term natural process of aging affects DA and TH phosphorylation in somatodendritic compartments but not in terminal field compartments.Previous work highlighting differences in DA reuptake suggests a dichotomy in how DA is regulated between cell body and terminal field regions of the same neuronal pathway. Our work further supports that such compartmental dichotomies exist The differences in L-DOPA  and DA  between The impact of TH activity on DA tissue content in each compartment of the dopaminergic pathways is also autonomously maintained within both short- and long-term temporal dynamics as seen in the AMPT and aging components of this study. Short-term pharmacological inhibition of TH by AMPT in the somatodendritic regions decreases DA tissue content therein without effect in terminal field regions and vice versa. One might speculate that decreased nigral DA resulting from AMPT infusion could lead to reduced DA release and subsequently less stimulation of autoreceptors, thus, enhancing nigrostriatal activity. Indeed, antipsychotics, which block the action of DA on the autoreceptor, can increase TH activity and neuronal activity Long-term reduction of TH phosphorylation and DA in the somatodendritic regions, as seen in aging, is also autonomous, as aging does not similarly impact terminal field regions. The compartmental autonomy of TH regulation demonstrated here has definite implications for the interpretation of behavioral outcomes influenced by DA release within either compartment of the nigrostriatal or mesoaccumbens pathways. Our data clearly indicate that any change in TH protein, TH phosphorylation, or DA in one compartment does not necessarily reflect similar changes in the other compartment in either DA pathway.in vivo. Previous reports support these results. Even before the discovery of ser31 as a TH phosphorylation site in vivo study also supports the contention that ser31 regulates basal TH activity in vivoOur results indicate that ser31 phosphorylation influences TH activity (from the L-DOPA work) and DA tissue content in vivo. While our data do not summarily dismiss a role for ser40 influencing basal TH activity or DA tissue content, they do not indicate that ser40 affects DA biosynthesis under basal conditions. One reason may be that basal ser40 phosphorylation stoichiometry in brain may be less than that necessary for TH activation and thus, substantial increases above basal levels may be required to increase L-DOPA biosynthesis. In PC12 cells, three-fold increases in ser40 phosphorylation increase L-DOPA biosynthesis, but two-fold increases are without effect in vivo results since basal ser40 phosphorylation levels in PC12 cells or chromaffin cells are ∼0.03 in vivo (∼0.02–0.06) in vivo. Thus, pharmacological agents such as antipsychotics in vivo. A thorough investigation could determine how much change in phosphorylation at ser31 versus ser40 is required to affect L-DOPA biosynthesis in vivo.However, the premise that ser40 phosphorylation is the dominant mechanism regulating TH activity still garners wide acceptance, and many studies measure only ser40 phosphorylation as an index of TH activity 2+-dependent increases in TH phosphorylation in situA potentially novel relationship between ser19 and ser31 phosphorylation in the somatodendritic regions  may poinOur results have illustrated the significant impact of TH protein and TH activity on DA bioavailability and illuminated how TH activity could be comparatively more critical for maintaining DA in the somatodendritic regions. Following AADC inhibition by NSD-1015, the L-DOPA per catecholamine ratio was greater in the somatodendritic compartments, indicating much less DA reserve therein . There wThis distinct difference in how TH may regulate DA between striatum and SN may be of significant consequence in models of Parkinson's disease If TH activity in the somatodendritic region is a critical regulatory component of DA bioavailability therein, it stands to reason that alterations in TH activity would significantly influence DA signaling in SN and VTA and, hence, behaviors affected by DA signaling in these regions. Somatodendritic release of DA is well established In summary, our findings support the idea that TH activity, as regulated by ser31 phosphorylation, contributes to autonomous regulation of DA between the somatodendritic and terminal field compartments of the nigrostriatal and mesoaccumbens pathways. In particular, TH activity may be more important to the maintenance of DA bioavailability in somatodendritic regions as previously demonstrated by less DA reuptake and DAT protein expression and, as revealed here, by a greater L-DOPA per catecholamine ratio and higher DA turnover. This dichotomy of TH regulation is further supported by the compartment-specific loss of DA following local inhibition of TH activity and the specific loss of DA and TH phosphorylation only in somatodendritic regions during the natural process of aging. These results shed a new light on the importance of the significance of the neuroanatomical loci of TH measures and how the autonomy of DA and TH regulation between somatodendritic and terminal field regions may contribute to behavioral outcomes.


Phosphorylation of G protein coupled receptors (GPCRs) by G protein coupled receptor kinases (GRKs) and the subsequent recruitment of β-arrestins are important for their desensitization. Using shRNA-mediated gene silencing strategy, we have recently shown that GRK2, GRK3 and β-arrestin-2 promote C3a receptor (C3aR) desensitization in human mast cells. We also demonstrated that β-arrestin-2 provides an inhibitory signal for NF-κB activation. C3aR possesses ten potential phosphorylation sites within its carboxyl terminus but their role on desensitization, β-arrestin recruitment and NF-κB activation has not been determined.2+ mobilization, degranulation and NF-κB activation when compared to the wild-type receptor. Interestingly, co-expression of MT7 with a constitutively active mutant of β-arrestin (R169E) inhibited C3a-induced degranulation by 28±2.4% and blocked NF-κB activation by 80±2.4%.We utilized a site directed mutagenesis approach in transfected HEK293 cells to determine the role of receptor phosphorylation on β-arrestin-2 recruitment and RBL-2H3 cells for functional studies. We found that although Ala substitution of Ser475/479, Thr480/481 residues resulted in 58±3.8% decrease in agonist-induced C3aR phosphorylation there was no change in β-arrestin-2 binding or receptor desensitization. By contrast, Ala substitution of Thr463, Ser465, Thr466 and Ser470 led to 40±1.3% decrease in agonist-induced receptor phosphorylation but this was associated with 74±2.4% decreases in β-arrestin-2 binding, significantly reduced desensitization and enhanced NF-κB activation. Combined mutation of these Ser/Thr residues along with Ser459 (mutant MT7), resulted in complete loss of receptor phosphorylation and β-arrestin-2 binding. RBL-2H3 cells expressing MT7 responded to C3a for greater CaThis study demonstrates that although C3a causes phosphorylation of its receptor at multiple sites, Ser459, Thr463, Ser465, Thr466 and Ser470 participate in C3aR desensitization, β-arrestin-2 recruitment and inhibition of NF-κB activity. Furthermore, β-arrestin-2 inhibits C3a-induced NF-κB activation via receptor desensitization-dependent and independent pathways. Cross-linking of high affinity IgE receptors (FcεRI) on mast cells is known to play an important role in allergic and hypersensitive diseases It is well established that following activation by agonists, most GPCRs are phosphorylated by a family of protein kinases, collectively known as G protein coupled receptor kinases (GRKs) Following agonist-induced GPCR phosphorylation, β-arrestins uncouple the receptor from G protein, leading to receptor desensitization and facilitate their clathrin-mediated internalization The goal of the present study was to extend our previous findings with shRNA-mediated silencing of GRKs and β-arrestins in human mast cells 32P and C3a-induced receptor phosphorylation was determined. As shown in Human C3aR possesses ten potential phosphorylation sites, of which eight are present in two distinct clusters as depicted in Our next goal was to determine the role of Ser449 and Ser459, which are present outside the two main clusters of potential phosphorylation sites , on agon2+ mobilization/degranulation and therefore cannot be used to study C3aR regulation in mast cells. We have previously performed intracellular Ca2+ mobilization and degranulation assays in a transfected mast cell line, RBL-2H3 cells, in order to determine the role of receptor phosphorylation on desensitization 2+ mobilization and greater degranulation when compared to cells expressing wild-type receptors. To test the role of site-specific C3aR phosphorylation on desensitization, we generated stable transfectants in RBL-2H3 cells expressing HA-tagged WT-C3aR, mutants MT1, MT2 and MT7 at equivalent levels  caused ∼10% degranulation . This reAla MT8, . RBL-2H3lization  and degrlization . These fIn addition to desensitization, agonist-induced phosphorylation of many GPCRs, results in the recruitment of β-arrestin and receptor internalization Although we used HEK293 cells for receptor phosphorylation and β-arrestin-2 recruitment studies, C3aR does not undergo internalization in this system C3a causes degranulation and chemokine production in a highly differentiated mast cell line, LAD2 cells Given that C3aR phosphorylation and β-arrestin recruitment promote receptor desensitization it can be speculated that enhanced NF-κB activation/chemokine generation reflects lack of receptor desensitization. To test this possibility, we took advantage of a constitutively active mutant of β-arrestin (β-arrestin-R169E) that has been shown to associate with phosphorylation-deficient mutants of various GPCRs in vivo by further promoting mast cell activation via C3aR The anaphylatoxin C3a is generated following cross-linking of IgE-receptors on mast cells and contributes allergic responses Settmacher et al., 2+ mobilization and degranulation. Unlike wild-type C3aR or MT1 and MT2, mutant MT7 did not bind β-arrestin-2 and was completely resistant to agonist-induced receptor internalization. These findings demonstrate that although C3a causes phosphorylation of its receptor at multiple sites Ser459 and Thr463 within the residues Ser459, Thr463, Ser465, Thr466 and Ser470 play particularly important role in C3aR desensitization, β-arrestin-2 recruitment and internalization.In the present study, we showed that Thr463, Ser465, Thr466 and Ser470 contribute to 40±1.3% of agonist-induced receptor phosphorylation and play a significant role on β-arrestin-2 binding, desensitization and internalization. Our phosphorylation studies with mutants MT3–MT6 indicate that Thr463 may participate in these responses. Interestingly, replacement of Ser459 to Ala alone had no effect on receptor desensitization but when combined with the eight phosphorylation sites in mutants MT1 and MT2 (Mutant M7) resulted in complete loss of phosphorylation, which was associated with substantial receptor desensitization, as demonstrated by greatly enhanced Caβ-arrestins have been shown to promote or inhibit NF-κB activity depending on the cell type and receptors utilized Our studies with a constitutively active mutant of β-arrestin (R169E) indicated that the ability of β-arrestin-2 to inhibit NF-κB activation involves both receptor desensitization-dependent and independent pathways. Thus, expression β-arrestin (R169E) in MT7-RBL-2H3 cells resulted in the inhibition of C3a-induced mast cell degranulation by 28±2.4%. However, under the same condition, C3a-induced NF-κB reporter activity was blocked by 80±2.4%. The mechanism by which β-arrestin inhibits the C3aR desensitization-independent transcription factor activity is unknown. Gao et al., In summary, we have identified the phosphorylation signature within the carboxyl terminus of C3aR that mediates receptor desensitization and internalization. It is generally accepted that GPCR phosphorylation and β-arrestin recruitment to the plasma membrane promote receptor desensitization by uncoupling them from G proteins and that receptor internalization promotes downstream ERK signaling All tissue culture reagents were purchased from Invitrogen . Amaxa cell transfection kits and reagents were purchased from Lonza . Anti- HA (HA-7) agarose beads were purchased from Sigma-Aldrich . Anti-human C3aR antibody was obtained from Santa Cruz Biotechnology . PE-labeled donkey anti-mouse IgG was purchased from eBioscience . Indo-1 AM was from Molecular Probes . SuperSignal® West Femto Maximum Sensitivity Substrate and HRP-labeled goat anti-rabbit IgG were from Thermo Scientific . Purified native C3a was purchased from Complement Technologies . p-nitrophenyl-N-acetyl-β-D-glucosamine (PNAG) and anti-Flag monoclonal antibody were from Sigma-Aldrich . CCL2 ELISA kit was from Peprotech .Rat basophilic leukemia, (RBL-2H3) and human embryonic kidney (HEK-293) cells  were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, glutamine (2 mM), penicillin (100 IU/mL) and streptomycin (100 µg/mL) 6) were transfected with receptor plasmids (1 µg) using the Amaxa Nucleofector device (Amaxa kit T) according to the manufacturer's protocol. Following nucleofection, cells were cultured in the presence of G418 (1 mg/ml) and cells expressing equivalent receptors were sorted by flow cytometry using anti-C3aR specific antibody and used in subsequent studies.Generation of C3aR (C3aR) and C-terminal C3aR mutants in pcDNA3.1 vector were described previously 32P] orthophosphate for 90 min and stimulated with 100 nM C3a at 37°C for 5 min. The cells were washed with ice-cold PBS and lysed in immunoprecipitation buffer . The pre-cleared cell lysates were incubated with 15 µl of anti-HA agarose beads for 2 h. Samples were washed three times with lysis buffer, resolved by 10% SDS-PAGE, transferred onto nitrocellulose membrane and 32P-incorporated protein bands were imaged by autoradiography. Levels of phosphorylation were normalized with their respective total levels as determined by Western blotting on the same membrane using monoclonal anti-C3aR antibody.Transient transfections were performed on 65–70% confluent HEK-293 monolayers in 60-mm dishes with 1 µg of plasmid DNA using Lipofectamine reagent (Invitrogen). Receptor phosphorylation experiments were performed via modification of procedures described previously HEK 293 cells transiently expressing HA-C3aR or its mutants and Flag-β-arrestin-2 were stimulated with C3a (100 nM) at 37°C for 5 min and washed with PBS. The cells were then incubated with disuccinimidyl suberate (0.1 mM) at room temperature for 30 min. The reaction was terminated by addition of ice-cold Tris-HCl (pH 7.4), to a final concentration of 10 mM and incubated for an additional 15 min. The samples were analyzed by immunoprecipitation and Western blotting. The lysates were centrifuged at 12,000 g for 15 min, and the supernatants were incubated with anti-HA agarose (Sigma) overnight at 4°C. After washing with immunoprecipitation buffer, the C3aR/β-arrestin-2 complexes adsorbed onto anti-HA agarose were eluted in sample buffer , and the presence of Flag-β-arrestin-2 was detected by Western blotting analysis using anti-Flag monoclonal antibody.2+ mobilization was determined as described previously 6) were washed twice with HEPES buffer  containing 1 mg/ml BSA and incubated with 1 µM of Indo-1 for 30 min in dark. Cells were then washed and re-suspended in 1.5 ml of the same buffer and time course of Ca2+ mobilization (0–5 min) was determined using Hitachi F-2500 Fluoro spectrophotometer  with an excitation wavelength of 355 nm and an emission wavelength of 410 nm. For degranulation assay, RBL-2H3 cells (5×104) were seeded into 96-well plates and incubated overnight. The following day, cells were washed twice with HEPES buffer saline and re-suspended in a total volume of 50 µl buffer containing 1 mg/ml BSA and exposed to different concentrations of C3a . For total β-hexosaminidase release, control cells were lysed in 50 µl of 0.1% Triton X-100. Aliquots (20 µl) of supernatants or cell lysates were incubated with 20 µl of 1 mM p-nitrophenyl-N-acetyl-β-D-glucosamine for 1.5 h at 37°C. The reaction was stopped by adding 250 µl of 0.1 M Na2CO3/0.1 M NaHCO3 buffer and absorbance was measured at 405 nm Ca5) stably expressing WT-C3aR or mutants were stimulated with or without C3a (100 nM) at 37°C. Cells were washed twice and re-suspended in 50 µl of ice-cold FACS buffer (PBS containing 2% FBS). Anti-C3aR antibody or isotype control was added and the cells were incubated on ice for 1 h. Cells were washed twice and re-suspended in ice-cold FACS buffer. PE-labeled donkey anti-mouse was added and incubated on ice for 1 h. Cells were washed twice with ice-cold FACS buffer and fixed in 250 µl of 2% formaldehyde. Receptor internalization was quantified as the loss of cell-surface receptors, as analyzed by BD LSR II flow cytometer (BD Biosciences).RBL-2H3 cells (2.5×106) stably expressing WT-C3aR or mutant C3aRs were co-transfected with NF-κB luciferase reporter gene construct  (in a 10∶1 ratio) using Amaxa Nucleofector device and kit T as per the manufacturer's protocol. After 24 h of incubation in complete medium, cells were serum starved (16 h), and then stimulated with 100 nM C3a for 6 h. Cells were washed in ice-cold PBS and finally lysed in passive lysis buffer . NF-κB luciferase activity was measured using Turner biosystem 20/20 Luminometer . The firefly luciferase activity was normalized to Renilla luciferase activity and is expressed as relative luciferase units (RLU). Chemokine release assay was performed as previously described 5 cells) were serum starved for 4 h and stimulated with 100 nM C3a for 6 h. Supernatants were collected and stored frozen at −80°C until analysis. CCL2 chemokine levels were quantified by sandwich ELISA according to the manufacturer's protocol.RBL-2H3 cells  with Dunnett's multiple comparison post-hoc test, and ANOVA with Bonferroni's post-hoc test.The results are expressed as ± S.E.M for values obtained from at least three independent experiments. GraphPad Prism software  was used to analyze the data and statistical significance was determined by student's two-tailed