Corrigendum: A reversible cell penetrating peptide-cargo linkage allows dissection of cell penetrating peptide-and cargo- dependent effects on internalization and identifies new functionalities of putative endolytic peptides.

[This corrects the article DOI: 10.3389/fphar.2022.1070464.].

BK Channels Function in Nematocyst Discharge from Vibration-Sensitive Cnidocyte Supporting Cell Complexes of the Sea Anemone Diadumene lineata.

AbstractIntegrated chemo- and mechanosensory pathways, along with activated calcium influxes, regulate nematocyst discharge from sea anemone tentacles. Discharge from vibration-sensitive Type A cnidocyte supporting cell complexes use calcium-conducting transient receptor potential V4-like channels. Because calcium influxes often couple with calcium-activated, large-conductance potassium (BK) channels, we hypothesized that BK channels function in nematocyst discharge. To verify this hypothesis, we first tested five selective BK channel blockers on nematocyst-mediated prey killing in Diadumene lineata (aka Haliplanella luciae). All tested BK channel blockers inhibited prey killing at concentrations comparable to their inhibition of vertebrate BK channels. In addition, the BK channel blocker paxilline selectively inhibited prey killing mediated by vibration-sensitive Type A cnidocyte supporting cell complexes. We queried a mammalian BKα amino acid sequence to the Exaiptasia diaphena database, from which we identified a putative anemone, pore-forming BKα subunit sequence. Using the E. diaphena BKα sequence as a template, we assembled a BKα transcript from our assembled D. lineata transcriptome. In addition, the hydra homolog of D. lineata BKα localizes to nematocytes on the hydra single-cell RNA sequencing map. Our findings suggest that D. lineata expresses BK channels that play a role in vibration-sensitive nematocyst discharge from Type A cnidocyte supporting cell complexes. We believe this is the first functional demonstration of BK channels in nonbilaterians. Because stimulated chemoreceptors frequency tune Type A cnidocyte supporting cell complexes to frequencies matching swimming movements of prey via a protein kinase A signaling pathway and protein kinase A generally activates BK channels, we suggest that D. lineata BK channels may participate in protein kinase A-mediated frequency tuning.

A reversible cell penetrating peptide-cargo linkage allows dissection of cell penetrating peptide- and cargo-dependent effects on internalization and identifies new functionalities of putative endolytic peptides.

Cell penetrating peptides (CPPs) are a promising technology for therapeutic delivery of macromolecular cargos. CPPs have generally used covalent linkages to cargo, ensuring a common fate as one molecule. Conversely, our CPP-adaptor, TAT-CaM, noncovalently binds calmodulin binding sequence (CBS)-containing cargos in calcium rich media then dissociates in the calcium-poor endosomal environment following internalization, enhancing endosomal escape relative to standard CPPs. In this study, we report cell entry of positively charged protein cargos that were not increased by TAT-CaM while cargos based on the negatively charged maltose binding protein (MBP) displayed little intrinsic internalization but were internalized by TAT-CaM. In addition, association of positively charged proteins with negatively charged nucleic acids reduced internalization. This evidence points to the dominant role cargo charge plays in apparent CPP effectiveness. There has been little systematic investigation as to how interaction between CPPs and cargos impacts internalization efficiency. Our adaptors provide a tool that allows combinatorial assays to detect emergent properties. Toward this end we added 4 endolytic peptide (EP) sequences between cargo CBS and MBP moieties to create 4 new cargos and between TAT and CaM to create 4 new adaptors. The new cargos were assayed for internalization alone and with a panel of CPP-adaptors to identify combinations that displayed increased internalization efficiency or other properties. Among the most important results, addition of the EP LAH4 improved adaptor performance and provided some CPP capability to cargos. MBP-LAH4-CBS was internalized more effectively by most adaptors, suggesting this sequence has general stimulatory ability. Two other EPs, Aurein 1.2 and HA2, also provided some CPP capability to their MBP cargos but were unexpectedly antagonistic to internalization by most adaptors due to retention of adaptor/cargo complexes on the cell surface. We thus identified LAH4 as stimulator of internalization in both adaptors and cargos and uncovered new functionality for Aurein 1.2 and HA2, which may be related to their identification as EPs. Future experiments will test new endolytic capabilities made possible with combinatorial approaches.

Efficient Synthesis for Altering Side Chain Length on Cannabinoid Molecules and Their Effects in Chemotherapy and Chemotherapeutic Induced Neuropathic Pain.

(1) Background: Recently, a number of side chain length variants for tetrahydrocannabinol and cannabidiol have been identified in cannabis; however, the precursor to these molecules would be based upon cannabigerol (CBG). Because CBG, and its side chain variants, are rapidly converted to other cannabinoids in the plant, there are typically only small amounts in plant extracts, thus prohibiting investigations related to CBG and CBG variant therapeutic effects. (2) Methods: To overcome this, we developed an efficient synthesis of corresponding resorcinol fragments using the Wittig reaction which, under acid catalyzed coupling with geraniol, produced the desired side chain variants of CBG. These compounds were then tested in an animal model of chemotherapeutic-induced neuropathic pain and to reduce colorectal cancer cell viability. (3) Results: We found that all side-chain variants were similarly capable of reducing neuropathic pain in mice at a dose of 10 mg/kg. However, the molecules with shorter side chains (i.e., CBGV and CBGB) were better at reducing colorectal cancer cell viability. (4) Conclusions: The novel synthesis method developed here will be of utility for studying other side chain derivatives of minor cannabinoids such as cannabichromene, cannabinol, and cannabielsoin.

Cannabigerol (CBG) attenuates mechanical hypersensitivity elicited by chemotherapy-induced peripheral neuropathy.

BACKGROUND: Cannabigerol (CBG) is a non-psychoactive phytocannabinoid produced by the plant Cannabis sativa with affinity to various receptors involved in nociception. As a result, CBG is marketed as an over-the-counter treatment for many forms of pain. However, there is very little research-based evidence for the efficacy of CBG as an anti-nociceptive agent. METHODS: To begin to fill this knowledge gap, we assessed the anti-nociceptive effects of CBG in C57BL/6 mice using three different models of pain; cisplatin-induced peripheral neuropathy, the formalin test, and the tail-flick assay. RESULTS: Using the von Frey test, we found that CBG-attenuated mechanical hypersensitivity evoked by cisplatin-induced peripheral neuropathy in both male and female mice. Additionally, we observed that this CBG-induced reduction in mechanical hypersensitivity was attenuated by the α2 -adrenergic receptor antagonist atipamezole (3 mg/kg, i.p.) and the CB1 R antagonist, AM4113 (3 mg/kg, i.p.), and blocked by the CB2 R antagonist/inverse agonist, SR144528 (10 mg/kg, i.p.). We found that the TRPV1 antagonist, SB705498 (20 mg/kg, i.p.) was unable to prevent CBG actions. Furthermore, we show that CBG:CBD oil (10 mg/kg, i.p.) was more effective than pure CBG (10 mg/kg) at reducing mechanical hypersensitivity in neuropathic mice. Lastly, we show that pure CBG and CBG:CBD oil were ineffective at reducing nociception in other models of pain, including the formalin and tail flick assays. CONCLUSIONS: Our findings support the role of CBG in alleviating mechanical hypersensitivity evoked by cisplatin-induced peripheral neuropathy, but highlight that these effects may be limited to specific types of pain. SIGNIFICANCE: There are few effective treatments for neuropathic pain and neuropathic pain is projected to increase with the aging population. We demonstrate that CBG (cannabigerol) and CBG:CBD oil attenuate neuropathy-induced mechanical hypersensitivity mice. Second, we identify receptor targets that mediate CBG-induced reduction in mechanical hypersensitivity in neuropathic mice. Third, we demonstrate that an acute injection of CBG is anti-nociceptive specifically for neuropathic pain rather than other forms of pain, including persistent pain and thermal pain.

Evaluating the Antinociceptive Efficacy of Cannabidiol Alone or in Combination with Morphine Using the Formalin Test in Male and Female Mice.

Introduction: Phytocannabinoids have emerged as a potential alternative treatment option for individuals experiencing persistent pain. However, evidence-based research regarding their clinical utility in both males and females remains incomplete. In addition, it is unknown whether combining readily available cannabinoids with opioids has a synergistic or subadditive effect on pain modulation. To begin to fill this knowledge gap, we investigated the antinociceptive effects of the phytocannabinoid, CBD, either alone or in combination with opioids in male and female C57BL/6J mice. Results: Using the formalin test, our results show that CBD (10 mg/kg, i.p.) treatment evoked antinociception in phase I, but not in phase II, of the formalin test in male mice. However, in female mice, CBD showed no significant antinociceptive effect. In addition, a direct sex comparison showed that CBD evoked a significant increase in nociceptive behaviors in female versus male mice during phase I of the formalin test. Furthermore, we show that CBD (10 mg/kg, i.p.) in combination with low-dose morphine (1 mg/kg, i.p.) was ineffective at eliciting a synergistic antinociceptive response in both male and female mice. Lastly, consistent with previous literature, we showed that females treated with a relatively higher dose of morphine (10 mg/kg, i.p.) displayed a significant increase in the variability of nociceptive behaviors compared to morphine-treated male mice. Conclusion: Overall, our results suggest that CBD treatment may have beneficial antinociceptive effects during the acute phase of persistent pain, but these effects are more beneficial to males than females. We provide further pre-clinical support that treatments geared toward reducing nociceptive behaviors differentially affect males and females.

A real-time assay for cell-penetrating peptide-mediated delivery of molecular cargos.

Cell-penetrating peptides (CPPs) are capable of transporting molecules to which they are tethered across cellular membranes. Unsurprisingly, CPPs have attracted attention for their potential drug delivery applications, but several technical hurdles remain to be overcome. Chief among them is the so-called 'endosomal escape problem,' i.e. the propensity of CPP-cargo molecules to be endocytosed but remain entrapped in endosomes rather than reaching the cytosol. Previously, a CPP fused to calmodulin that bound calmodulin binding site-containing cargos was shown to efficiently deliver cargos to the cytoplasm, effectively overcoming the endosomal escape problem. The CPP-adaptor, "TAT-CaM," evinces delivery at nM concentrations and more rapidly than we had previously been able to measure. To better understand the kinetics and mechanism of CPP-adaptor-mediated cargo delivery, a real-time cell penetrating assay was developed in which a flow chamber containing cultured cells was installed on the stage of a confocal microscope to allow for observation ab initio. Also examined in this study was an improved CPP-adaptor that utilizes naked mole rat (Heterocephalus glaber) calmodulin in place of human and results in superior internalization, likely due to its lesser net negative charge. Adaptor-cargo complexes were delivered into the flow chamber and fluorescence intensity in the midpoint of baby hamster kidney cells was measured as a function of time. Delivery of 400 nM cargo was observed within seven minutes and fluorescence continued to increase linearly as a function of time. Cargo-only control experiments showed that the minimal uptake which occurred independently of the CPP-adaptor resulted in punctate localization consistent with endosomal entrapment. A distance analysis was performed for cell-penetration experiments in which CPP-adaptor-delivered cargo showing wider dispersions throughout cells as compared to an analogous covalently-bound CPP-cargo. Small molecule endocytosis inhibitors did not have significant effects upon delivery. The real-time assay is an improvement upon static endpoint assays and should be informative in a broad array of applications.

Long-Term High-Altitude Hypoxia and Alpha Adrenoceptor-Dependent Pulmonary Arterial Contractions in Fetal and Adult Sheep.

Autonomic innervation of the pulmonary vasculature triggers vasomotor contractility predominately through activation of alpha-adrenergic receptors (α-ARs) in the fetal circulation. Long-term hypoxia (LTH) modulates pulmonary vasoconstriction potentially through upregulation of α1-AR in the vasculature. Our study aimed to elucidate the role of α-AR in phenylephrine (PE)-induced pulmonary vascular contractility, comparing the effects of LTH in the fetal and adult periods on α-AR subtypes and PE-mediated Ca2+ responses and contractions. To address this, we performed wire myography, Ca2+ imaging, and mRNA analysis of pulmonary arteries from ewes and fetuses exposed to LTH or normoxia. Postnatal maturation depressed PE-mediated contractile responses. α2-AR activation contracted fetal vessels; however, this was suppressed by LTH. α1A- and α1B-AR subtypes contributed to arterial contractions in all groups. The α1D-AR was also important to contractility in fetal normoxic vessels and LTH mitigated its function. Postnatal maturity increased the number of myocytes with PE-triggered Ca2+ responses while LTH decreased the percentage of fetal myocytes reacting to PE. The difference between myocyte Ca2+ responsiveness and vessel contractility suggests that fetal arteries are sensitized to changes in Ca2+. The results illustrate that α-adrenergic signaling and vascular function change during development and that LTH modifies adrenergic signaling. These changes may represent components in the etiology of pulmonary vascular disease and foretell the therapeutic potential of adrenergic receptor antagonists in the treatment of pulmonary hypertension.

Temporal Dissection of Rate Limiting Transcriptional Events Using Pol II ChIP and RNA Analysis of Adrenergic Stress Gene Activation.

In mammals, increasing evidence supports mechanisms of co-transcriptional gene regulation and the generality of genetic control subsequent to RNA polymerase II (Pol II) recruitment. In this report, we use Pol II Chromatin Immunoprecipitation to investigate relationships between the mechanistic events controlling immediate early gene (IEG) activation following stimulation of the α1a-Adrenergic Receptor expressed in rat-1 fibroblasts. We validate our Pol II ChIP assay by comparison to major transcriptional events assessable by microarray and PCR analysis of precursor and mature mRNA. Temporal analysis of Pol II density suggests that reduced proximal pausing often enhances gene expression and was essential for Nr4a3 expression. Nevertheless, for Nr4a3 and several other genes, proximal pausing delayed the time required for initiation of productive elongation, consistent with a role in ensuring transcriptional fidelity. Arrival of Pol II at the 3' cleavage site usually correlated with increased polyadenylated mRNA; however, for Nfil3 and probably Gprc5a expression was delayed and accompanied by apparent pre-mRNA degradation. Intragenic pausing not associated with polyadenylation was also found to regulate and delay Gprc5a expression. Temporal analysis of Nr4a3, Dusp5 and Nfil3 shows that transcription of native IEG genes can proceed at velocities of 3.5 to 4 kilobases/min immediately after activation. Of note, all of the genes studied here also used increased Pol II recruitment as an important regulator of expression. Nevertheless, the generality of co-transcriptional regulation during IEG activation suggests temporal and integrated analysis will often be necessary to distinguish causative from potential rate limiting mechanisms.

Stimulation of α1a adrenergic receptors induces cellular proliferation or antiproliferative hypertrophy dependent solely on agonist concentration.

Stimulation of α1aAdrenergic Receptors (ARs) is known to have anti-proliferative and hypertrophic effects; however, some studies also suggests this receptor can increase cell proliferation. Surprisingly, we find the α1aAR expressed in rat-1 fibroblasts can produce either phenotype, depending exclusively on agonist concentration. Stimulation of the α1aAR by high dose phenylephrine (>10(-7) M) induces an antiproliferative, hypertrophic response accompanied by robust and extended p38 activation. Inhibition of p38 with SB203580 prevented the antiproliferative response, while inhibition of Erk or Jnk had no effect. In stark contrast, stimulation of the α1aAR with low dose phenylephrine (∼10(-8) M) induced an Erk-dependent increase in cellular proliferation. Agonist-induced Erk phosphorylation was preceded by rapid FGFR and EGFR transactivation; however, only EGFR inhibition blocked Erk activation and proliferation. The general matrix metalloprotease inhibitor, GM6001, blocked agonist induced Erk activation within seconds, strongly suggesting EGFR activation involved extracellular triple membrane pass signaling. Erk activation required little Ca(2+) release and was blocked by PLCß or PKC inhibition but not by intracellular Ca(2+) chelation, suggesting Ca(2+) independent activation of novel PKC isoforms. In contrast, Ca(2+) release was essential for PI3K/Akt activation, which was acutely maximal at non-proliferative doses of agonist. Remarkably, our data suggests EGFR transactivation leading to Erk induced proliferation has the lowest activation threshold of any α1aAR response. The ability of α1aARs to induce proliferation are discussed in light of evidence suggesting antagonistic growth responses reflect native α1aAR function.

Kinetic characterization of Salmonella FliK-FlhB interactions demonstrates complexity of the Type III secretion substrate-specificity switch.

The bacterial flagellum is a complex macromolecular machine consisting of more than 20 000 proteins, most of which must be exported from the cell via a dedicated Type III secretion apparatus. At a defined point in flagellar morphogenesis, hook completion is sensed and the apparatus switches substrate specificity type from rod and hook proteins to filament ones. How the switch works is a subject of intense interest. FliK and FlhB play central roles. In the present study, two optical biosensing methods were used to characterize FliK-FlhB interactions using wild-type and two variant FlhBs from mutants with severe flagellar structural defects. Binding was found to be complex with fast and slow association and dissociation components. Surprisingly, wild-type and variant FlhBs had similar kinetic profiles and apparent affinities, which ranged between 1 and 10.5 microM, suggesting that the specificity switch is more complex than presently understood. Other binding experiments provided evidence for a conformational change after binding. Liquid chromatography-mass spectrometry (LC-MS) and NMR experiments were performed to identify a cyclic intermediate product whose existence supports the mechanism of autocatalytic cleavage at FlhB residue N269. The present results show that while autocatalytic cleavage is necessary for proper substrate specificity switching, it does not result in an altered interaction with FliK, strongly suggesting the involvement of other proteins in the mechanism.

Lipid rafts constrain basal alpha(1A)-adrenergic receptor signaling by maintaining receptor in an inactive conformation.

We have reported that the alpha(1A)-adrenergic receptor (alpha(1A)AR) in rat-1 fibroblasts is a lipid raft protein. Here we examined whether disrupting lipid rafts by methyl-beta-cyclodextrin (MCD) sequestration of cholesterol affects alpha(1A)AR signaling. Unexpectedly, MCD increased alpha(1A)AR-dependent basal inositol phosphate formation and p38 mitogen-activated protein kinase activation in a cholesterol-dependent manner. It also initiated internalization of surface alpha(1A)AR, which was partially blocked by receptor inhibition. Binding assays revealed MCD-mediated increases in receptor agonist affinity as well as reciprocal decreases in inverse agonist affinity, a behavior that is usually interpreted as a shift toward the active receptor conformation. In untreated cells a fraction of the receptor was found to be present in preassociated receptor/G protein complexes, which rapidly dissociate upon receptor stimulation. Consistent with MCD-induced signaling, raft disruption resulted in an increase in receptor/G protein complexes. These results strongly suggest that lipid rafts constrain basal alpha(1A)AR activity; however, preassembled receptor/G protein complexes could still provide a mechanism for accelerating alpha(1A)AR signaling following stimulation.

Study of pH-triggered heteroaggregation and gel formation within mixed dispersions.

This work involves an investigation of pH-triggered heteroaggregation and gelation within mixed dispersions of polystyrene (PS) and pigment particles. The PS particles were stabilised by a carboxylated alkyl ethoxylate surfactant which is pH-responsive. The pigment used was beta-copper phthalocyanine. The pigment particles contained a co-surfactant system consisting of the carboxylated alkyl ethoxylate and a non-ionic surfactant. The latter was a beta-naphthol ethoxylate. The PS and pigment particles were characterised using SEM, TEM, photon correlation spectroscopy and electrophoretic mobility measurements. The PS dispersions exhibited pH-triggered aggregation when the pH was decreased to below a critical value (pH(crit)), which was 1.9. Concentrated PS dispersions formed stable particle gels at pH values less than or equal to pH(crit). Dilute pigment dispersions were found to have a pH(crit) of 3.45. However, concentrated pigment dispersions did not form gels when the pH was decreased to below pH(crit). A phase diagram for the mixed dispersions was constructed which showed a gel phase existed at pH values between 2.0 and 3.0, which corresponds to a pH region higher than pH(crit) for the PS particles. This implicates PS-pigment inter-particle bonds in the gel structure. The heteroaggregate gels were investigated using dynamic rheological measurements and it was apparent that the highest elastic modulus values were obtained in the pH range of approximately 2 to 3. SEM images provided evidence of heteroaggregates with diameters of a few micrometers. These primary heteroaggregates are suggested to be the network forming unit for the gels formed in mixed dispersions. The data from the study are used to propose a conceptual model for the structure of the heteroaggregate gels.

The alpha1a-adrenergic receptor occupies membrane rafts with its G protein effectors but internalizes via clathrin-coated pits.

The alpha(1a)-adrenergic receptor (alpha(1a)AR) occupies intracellular and plasma membranes in both native and heterologous expression systems. Based on multiple independent lines of evidence, we demonstrate the alpha(1a)AR at the cell surface occupies membrane rafts but exits from rafts following stimulation. In non-detergent raft preparations, basal alpha(1a)AR is present in low density membrane rafts and colocalizes with its G protein effectors on density gradients. Raft disruption by cholesterol depletion with methyl-beta-cyclodextrin eliminates these light rafts. To confirm the presence of the alpha(1a)AR in plasma membrane rafts, fluorescence resonance energy transfer measurements were used to demonstrate colocalization of surface receptor and the raft marker, cholera toxin B. This colocalization was largely lost following alpha(1a)AR stimulation with phenylephrine. Similarly, receptor stimulation causes exit of the alpha(1a)AR from light rafts within 3-10 min in contrast to the G proteins, which largely remain in light rafts. Importantly, this delayed exit of the alpha(1a)AR suggests acute receptor signaling and desensitization occur entirely within rafts. Interestingly, both confocal analysis and measurement of surface alpha(1a)AR levels indicate modest receptor internalization during the 10 min following stimulation, suggesting most of the receptor has entered non-raft plasma membrane. Nevertheless, activation does increase the rate of receptor internalization as does disruption of rafts with methyl-beta-cyclodextrin, suggesting raft exit enables internalization. Confocal analysis of surface-labeled hemagglutinin-alpha(1a)AR reveals that basal and stimulated receptor occupies clathrin pits in fixed cells consistent with previous indirect evidence. The evidence presented here strongly suggests the alpha(1a)AR is a lipid raft protein under basal conditions and implies agonist-mediated signaling occurs from rafts.

Epigenetic regulation of human alpha1d-adrenergic receptor gene expression: a role for DNA methylation in Sp1-dependent regulation.

A growing body of evidence implicates alpha1-adrenergic receptors (alpha1ARs) as potent regulators of growth pathways. The three alpha1AR subtypes (alpha1aAR, alpha1bAR, alpha1dAR) display highly restricted tissue expression that undergoes subtype switching with many pathological stimuli, the mechanistic basis of which remains unknown. To gain insight into transcriptional pathways governing cell-specific regulation of the human alpha1dAR subtype, we cloned and characterized the alpha1dAR promoter region in two human cellular models that display disparate levels of endogenous alpha1dAR expression (SK-N-MC and DU145). Results reveal that alpha1dAR basal expression is regulated by Sp1-dependent binding of two promoter-proximal GC boxes, the mutation of which attenuates alpha1dAR promoter activity 10-fold. Mechanistically, chromatin immunoprecipitation data demonstrate that Sp1 binding correlates with expression of the endogenous gene in vivo, correlating highly with alpha1dAR promoter methylation-dependent silencing of both episomally expressed reporter constructs and the endogenous gene. Further, analysis of methylation status of proximal GC boxes using sodium bisulfite sequencing reveals differential methylation of proximal GC boxes in the two cell lines examined. Together, the data support a mechanism of methylation-dependent disruption of Sp1 binding in a cell-specific manner resulting in repression of basal alpha1dAR expression.

Evidence that phosphorylation of the RNA polymerase II carboxyl-terminal repeats is similar in yeast and humans.

Using an improved chromatin immunoprecipitation assay designed to increase immunoprecipitation efficiency, we investigated changes in RNA polymerase II (Pol II) density and carboxyl-terminal domain (CTD) phosphorylation during transcription of the cyclophilin A (PPIA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and several androgen-responsive genes in LNCaP cells. As generally observed in higher eukaryotes, promoter proximal pausing of Pol II appeared to occur on the PPIA and GAPDH genes, but apparently not on the androgen-responsive genes PSA and NKX3-1. Unlike some mammalian studies, we found that the CTD of Pol II in promoter regions contains little phosphorylation at Ser-2 of the heptad repeat, suggesting that Ser-2 phosphorylation is not involved in polymerase exit from the promoter region. In contrast, Pol II near the promoter displayed high levels of Ser-5 phosphorylation, which decreased as polymerase transcribed beyond the promoter region of the PPIA and GAPDH genes. However, total Pol II levels appear to decrease as much or more, suggesting that Ser-5 phosphorylation is maintained. In support of this conclusion, a phosphoserine 5-specific antibody quantitatively immunoprecipitates native hyperphosphorylated Pol II, suggesting that all polymerase with phosphoserine 2 also contains phosphoserine 5. Given reports indicating that phosphoserine 5 is present during elongation in yeast, our data suggest that gross changes in CTD phosphorylation patterns during transcription may be more conserved in yeast and humans than recognized previously.

Novel human alpha1a-adrenoceptor single nucleotide polymorphisms alter receptor pharmacology and biological function.

We identified nine naturally-occurring human single nucleotide polymorphisms (SNPs) in the alpha(1a)-adrenoceptor (alpha(1a)AR) coding region, seven of which result in amino acid change. Utilizing rat-1 fibroblasts stably expressing wild type alpha(1a)AR or each SNP at both high and low levels, we investigated the effect of these SNPs on receptor function. Compared with wild type, two SNPs (R166K, V311I) cause a decrease in binding affinity for agonists norepinephrine, epinephrine, and phenylephrine, and also shift the dose-response curve for norepinephrine stimulation of inositol phosphate (IP) production to the right (reduced potency) without altering maximal IP activity. In addition, SNP V311I and I200S display altered antagonist binding. Interestingly, a receptor with SNP G247R (located in the third intracellular loop) displays increased maximal receptor IP activity and stimulates cell growth. The increased receptor signaling for alpha(1a)AR G247R is not mediated by altered ligand binding or a deficiency in agonist-mediated desensitization, but appears to be related to enhanced receptor-G protein coupling. In conclusion, four naturally-occurring human alpha(1a)AR SNPs induce altered receptor pharmacology and/or biological activity. This finding has potentially important implications in many areas of medicine and can be used to guide alpha(1a)AR SNP choice for future clinical studies.

Cellular trafficking of human alpha1a-adrenergic receptors is continuous and primarily agonist-independent.

Alpha1a-adrenergic receptors (alpha1aARs) are present intracellularly and at the cell surface in cultured and natural cell models, where they are subject to agonist-mediated desensitization and internalization. To explore alpha1aAR trafficking, a hemagglutinin (HA)-tagged alpha1aAR/enhanced green fluorescent protein (EGFP) fusion protein was expressed in rat-1 fibroblasts and tracked by EGFP fluorescence and antibody labeling of surface receptors. Confocal analysis of antibody-labeled surface receptors revealed unexpected constitutive internalization in the absence of agonist stimulation. In partial agreement, the inverse agonist prazosin also caused a modest 20 +/- 2% increase in surface receptor levels, suggesting a partial block of constitutive internalization caused by decreased basal activation. However, prazosin was unable to prevent internalization of antibody-tagged surface receptors observed by confocal microscopy or cause obvious redistribution of intracellular receptor to the surface, suggesting that the alpha1aAR is internalizing even in a basal-inactive state. In contrast to the alpha1aAR, surface labeling of an HA-tagged alpha1b-EGFP fusion protein did not result in any apparent constitutive internalization. Constitutive internalization of the alpha1aAR seems to occur alongside reversible agonist-induced internalization, and both seem to involve clathrin-mediated endocytosis but not degradation in lysozymes. Surface receptor density must be maintained by recycling, because the protein synthesis inhibitor cycloheximide has no effect on total or surface receptor density in agonist-treated or untreated cells for 6 h. Constitutive agonist-independent trafficking of alpha1aARs may provide a novel mechanism by which an internal pool of alpha1aARs are maintained and recycled to allow continuous agonist-induced signaling.

Acute agonist-mediated desensitization of the human alpha 1a-adrenergic receptor is primarily independent of carboxyl terminus regulation: implications for regulation of alpha 1aAR splice variants.

Despite important roles in myocardial hypertrophy and benign prostatic hyperplasia, little is known about acute effects of agonist stimulation on alpha(1a)-adrenergic receptor (alpha(1a)AR) signaling and function. Regulatory mechanisms are likely complex since 12 distinct human alpha(1a)AR carboxyl-terminal splice variants have been isolated. After determining the predominance of the alpha(1a-1)AR isoform in human heart and prostate, we stably expressed an epitope-tagged alpha(1a-1)AR cDNA in rat-1 fibroblasts and subsequently examined regulation of signaling, phosphorylation, and internalization of the receptor. Human alpha(1a)AR-mediated inositol phosphate signaling is acutely desensitized in response to both agonist and phorbol 12-myristate 13-acetate (PMA) exposure. Concurrent with desensitization, alpha(1a)ARs in (32)P(i)-labeled cells are rapidly phosphorylated in response to both NE and PMA stimulation. Despite the ability of PKC to desensitize alpha(1a)ARs when directly activated with PMA, inhibitors of PKC have no effect on agonist-mediated desensitization. In contrast, involvement of GRK kinases is suggested by the ability of GRK2 to desensitize alpha(1a)ARs. Internalization of cell surface alpha(1a)ARs also occurs in response to agonist stimulation (but not PKC activation), but is initiated more slowly than receptor desensitization. Significantly, deletion of the alpha(1a)AR carboxyl terminus has no effect on receptor internalization or either agonist-induced or GRK-mediated receptor desensitization. Because mechanisms underlying acute agonist-mediated regulation of human alpha(1a)ARs are primarily independent of the carboxyl terminus, they may be common to all functional alpha(1a)AR isoforms.