John R. Raymond

Multiplicity of mechanisms of serotonin receptor signal transduction

The serotonin (5-hydroxytryptamine, 5-HT) receptors have been divided into 7 subfamilies by convention, 6 of which include 13 different genes for G-protein-coupled receptors. Those subfamilies have been characterized by overlapping pharmacological properties, amino acid sequences, gene organization, and second messenger coupling pathways. Post-genomic modifications, such as alternative mRNA splicing or mRNA editing, creates at least 20 more G-protein-coupled 5-HT receptors, such that there are at least 30 distinct 5-HT receptors that signal through G-proteins. This review will focus on what is known about the signaling linkages of the G-protein-linked 5-HT receptors, and will highlight some fascinating new insights into 5-HT receptor signaling.

The recombinant 5-HT1A receptor: G protein coupling and signalling pathways.

The 5‐hydroxytryptamine 5‐HT1A receptor was one of the first G protein coupled receptors whose cDNA and gene were isolated by molecular cloning methods. Transfection of the cDNA of this receptor into cells previously bearing no 5‐HT receptors has resulted in the acquisition of large amounts of information regarding potential signal transduction pathways linked to the receptor, correlations of receptor structure to its various functions, and pharmacological properties of the receptor. Transfection studies with the 5‐HT1A receptor have generated critical new information that might otherwise have been elusive. This information notably includes the discovery of unsuspected novel signalling linkages, the elucidation of the mechanisms of receptor desensitization, the refinement of models of the receptor pharmacophore, and the development of silent receptor antagonists, among others. The current review summarizes the most important studies of the recombinant 5‐HT1A receptor in the decade since the identificiation of its cDNA.

Urine Biomarkers Predict the Cause of Glomerular Disease

Diagnosis of the type of glomerular disease that causes the nephrotic syndrome is necessary for appropriate treatment and typically requires a renal biopsy. The goal of this study was to identify candidate protein biomarkers to diagnose glomerular diseases. Proteomic methods and informatic analysis were used to identify patterns of urine proteins that are characteristic of the diseases. Urine proteins were separated by two-dimensional electrophoresis in 32 patients with FSGS, lupus nephritis, membranous nephropathy, or diabetic nephropathy. Protein abundances from 16 patients were used to train an artificial neural network to create a prediction algorithm. The remaining 16 patients were used as an external validation set to test the accuracy of the prediction algorithm. In the validation set, the model predicted the presence of the diseases with sensitivities between 75 and 86% and specificities from 92 to 67%. The probability of obtaining these results in the novel set by chance is 5 x 10(-8). Twenty-one gel spots were most important for the differentiation of the diseases. The spots were cut from the gel, and 20 were identified by mass spectrometry as charge forms of 11 plasma proteins: Orosomucoid, transferrin, alpha-1 microglobulin, zinc alpha-2 glycoprotein, alpha-1 antitrypsin, complement factor B, haptoglobin, transthyretin, plasma retinol binding protein, albumin, and hemopexin. These data show that diseases that cause nephrotic syndrome change glomerular protein permeability in characteristic patterns. The fingerprint of urine protein charge forms identifies the glomerular disease. The identified proteins are candidate biomarkers that can be tested in assays that are more amenable to clinical testing.

Serotonin 5-HT1A receptor-mediated Erk activation requires calcium/calmodulin-dependent receptor endocytosis.

Many receptors that couple to heterotrimeric guanine nucleotide-binding (G) proteins mediate rapid activation of the mitogen-activated protein kinases, Erk1 and Erk2. The Gi-coupled serotonin (5-hydroxytryptamine (5-HT)) 5-HT1A receptor, heterologously expressed in Chinese hamster ovary or human embryonic kidney 293 cells, mediated rapid activation of Erk1/2 via a mechanism dependent upon both Ras activation and clathrin-mediated endocytosis. This activation was attenuated by chelation of intracellular Ca2+ and Ca2+/calmodulin (CAM) inhibitors or the CAM sequestrant protein calspermin. The CAM-dependent step in the Erk1/2 activation cascade is downstream of Ras activation, because inhibitors of CAM antagonize Erk1/2 activation induced by constitutively activated mutants of Ras and c-Src but not by constitutively activated mutants of Raf and MEK (mitogen and extracellular signal-regulated kinase). Inhibitors of the classical CAM effectors myosin light chain kinase, CAM-dependent protein kinases II and IV, PP2B, and CAM-sensitive phosphodiesterase had no effect upon 5-HT1A receptor-mediated Erk1/2 activation. Because clathrin-mediated endocytosis was required for 5-HT1A receptor-mediated Erk1/2 activation, we postulated a role for CAM in receptor endocytosis. Inhibition of receptor endocytosis by use of sequestration-defective mutants of β-arrestin1 and dynamin attenuated 5-HT1Areceptor-stimulated Erk1/2 activation. Inhibition of CAM prevented agonist-dependent endocytosis of epitope-tagged 5-HT1A receptors. We conclude that CAM-dependent activation of Erk1/2 through the 5-HT1A receptor reflects its role in endocytosis of the receptor, which is a required step in the activation of MEK and subsequently Erk1/2.

Inhibition of Phosphatidylinositol 3-Kinase Induces Nitric-oxide Synthase in Lipopolysaccharide- or Cytokine-stimulated C6 Glial Cells

Nitric oxide (NO) produced by inducible nitric-oxide synthase (iNOS) in different cells including brain cells in response to proinflammatory cytokines plays an important role in the pathophysiology of demyelinating and neurodegenerative diseases. The present study underlines the importance of phosphatidylinositol 3-kinase (PI 3-kinase) in the expression of iNOS in C6 glial cells and rat primary astrocytes. Bacterial lipopolysaccharide (LPS) or interleukin-1β (IL-1β) was unable to induce the expression of iNOS and the production of NO in rat C6 glial cells. Similarly, wortmannin and LY294002, compounds that inhibit PI 3-kinase, were also unable to induce the expression of iNOS and the production of NO. However, a combination of wortmannin or LY294002 with LPS or IL-1β induced the expression of iNOS and the production of NO in C6 glial cells. Consistent with the induction of iNOS, wortmannin also induced iNOS promoter-derived chloramphenicol acetyltransferase activity in LPS- or IL-1β-treated C6 glial cells. The expression of iNOS by LPS in C6 glial cells expressing a dominant-negative mutant of p85α, the regulatory subunit of PI 3-kinase, further supports the conclusion that inhibition of PI 3-kinase provides a necessary signal for the induction of iNOS. Next we examined the effect of wortmannin on the activation of mitogen-activated protein (MAP) kinase and nuclear factor NF-κB in LPS- or IL-1β-stimulated C6 glial cells. In contrast to the inability of LPS and IL-1β alone to induce the expression of iNOS, both LPS and IL-1β individually stimulated MAP kinase activity and induced DNA binding and transcriptional activity of NF-κB. Wortmannin alone was unable to activate MAP kinase and NF-κB. Moreover, wortmannin had no effect on LPS- or IL-1β-mediated activation of MAP kinase and NF-κB, suggesting that wortmannin induced the expression of iNOS in LPS- or IL-1β-stimulated C6 glial cells without modulating the activation of MAP kinase and NF-κB. Similar to C6 glial cells, wortmannin also stimulated LPS-mediated expression of iNOS and production of NO in astrocytes without affecting the LPS-mediated activation of NF-κB. Taken together, the results from specific chemical inhibitors and dominant-negative mutant expression studies demonstrate that apart from the activation of NF-κB, inhibition of PI 3-kinase is also necessary for the expression of iNOS and production of NO.

AGONIST ANTAGONIST INTERACTIONS WITH CLONED HUMAN 5-HT1A RECEPTORS - VARIATIONS IN INTRINSIC ACTIVITY STUDIED IN TRANSFECTED HELA-CELLS

SummaryThe characteristics of 5-HT1A-recognition sites and receptor-mediated release of intracellular calcium were established in two transfected HeLa cell lines (HA 6 and HA 7) expressing different levels of human 5-HT1A receptors (about 3000 and 500 fmol/mg protein, Fargin et al. 1989; 1991; Raymond et al. 1989).The pharmacological profiles of the binding (determined with [3H]8-OH-DPAT) and the calcium response (measured using Fura-2) were clearly of the 5-HT1A type. Compounds such as 5-HT, 5-CT and 8-OH-DPAT acted as full agonists on the calcium response in both HeLa cell lines. In addition, methiothepin, pindolol, NAN 190 and SDZ 216-525 (Seiler et al. 1991) acted as silent and potent antagonists.Marked differences were observed in the responses mediated in the two cell lines. EC50 values of agonists (particularly 5-HT, 5-CT, flesinoxan and 8-OH-DPAT) were higher in HA 7 cells (up to 80-fold) than in other 5-HT1A receptor models (e.g. inhibition of adenylate cyclase in calf hippocampus). Further, a variety of compounds (ipsapirone, buspirone, spiroxatrine, MDL 73005) acted as agonists in HA 6 cells, whereas they behaved as silent antagonists in HA 7 cells (which express fewer receptors). By contrast, KB values for antagonists were comparable in HA 6 and HA 7 cells.The present data show that EC50 values and intrinsic activity for a given drug are subject to large variations depending on the number of receptors expressed in the target tissue. The results obtained in HA 6 cells are comparable with respect to both potency and efficacy to those observed, in calf or mouse hippocampus (inhibition of forskolin stimulated adenylate cyclase), whereas the results obtained in HA 7 cells are similar to those reported in mouse cortex (which was suggested to represent an atypical subtype of the 5-HT1A receptor).Since the agonist activity of a given compound at the same receptor can vary markedly, the present data show that intrinsic activity is not only ligand-dependent but also varies with the receptor-effector system studied. In addition, there seems to be no simple way to make predictions about intrinsic activity, since that feature is model-dependent.

5-HT2A Receptor Induces ERK Phosphorylation and Proliferation through ADAM-17 Tumor Necrosis Factor-α-converting Enzyme (TACE) Activation and Heparin-bound Epidermal Growth Factor-like Growth Factor (HB-EGF) Shedding in Mesangial Cells

In this study, we present multiple lines of evidence to support a critical role for heparin-bound EGF (epidermal growth factor)-like growth factor (HB-EGF) and tumor necrosis factor-α-converting enzyme (TACE) (ADAM17) in the transactivation of EGF receptor (EGFR), ERK phosphorylation, and cellular proliferation induced by the 5-HT2A receptor in renal mesangial cells. 5-hydroxy-tryptamine (5-HT) resulted in rapid activation of TACE, HB-EGF shedding, EGFR activation, ERK phosphorylation, and longer term increases in DNA content in mesangial cells. ERK phosphorylation was attenuated by 1) neutralizing EGFR antibodies and the EGFR kinase inhibitor, AG1478, 2) neutralizing HB-EGF, but not amphiregulin, antibodies, heparin, or CM197, and 3) pharmacological inhibitors of matrix-degrading metalloproteinases or TACE small interfering RNA. Exogenously administered HB-EGF stimulated ERK phosphorylation. Additionally, TACE was co-immunoprecipitated with HB-EGF. Small interfering RNA against TACE also blocked 5-HT-induced increases in ERK phosphorylation, HB-EGF shedding, and DNA content. In aggregate, this work supports a pathway map that can be depicted as follows: 5-HT → 5-HT2A receptor → TACE → HB-EGF shedding → EGFR → ERK → increased DNA content. To our knowledge, this is the first time that TACE has been implicated in 5-HT-induced EGFR transactivation or in proliferation induced by a G protein-coupled receptor in native cells in culture.

Adrenergic receptors. Models for regulation of signal transduction processes.

Adrenergic receptors are prototypic models for the study of the relations between structure and function of G protein-coupled receptors. Each receptor is encoded by a distinct gene. These receptors are integral membrane proteins with several striking structural features. They consist of a single subunit containing seven stretches of 20-28 hydrophobic amino acids that represent potential membrane-spanning alpha-helixes. Many of these receptors share considerable amino acid sequence homology, particularly in the transmembrane domains. All of these macromolecules share other similarities that include one or more potential sites of extracellular N-linked glycosylation near the amino terminus and several potential sites of regulatory phosphorylation that are located intracellularly. By using a variety of techniques, it has been demonstrated that various regions of the receptor molecules are critical for different receptor functions. The seven transmembrane regions of the receptors appear to form a ligand-binding pocket. Cysteine residues in the extracellular domains may stabilize the ligand-binding pocket by participating in disulfide bonds. The cytoplasmic domains contain regions capable of interacting with G proteins and various kinases and are therefore important in such processes as signal transduction, receptor-G protein coupling, receptor sequestration, and down-regulation. Finally, regions of these macromolecules may undergo posttranslational modifications important in the regulation of receptor function. Our understanding of these complex relations is constantly evolving and much work remains to be done. Greater understanding of the basic mechanisms involved in G protein-coupled, receptor-mediated signal transduction may provide leads into the nature of certain pathophysiological states.

Expression of a Dominant‐Negative Mutant of p21ras Inhibits Induction of Nitric Oxide Synthase and Activation of Nuclear Factor‐κB in Primary Astrocytes

Abstract: The present study underlines the importance of p21ras in regulating the inducible nitric oxide synthase (iNOS) in primary astrocytes. Bacterial lipopolysaccharides induced the GTP loading of p21ras, and the expression of a dominant‐negative mutant of p21ras (Δp21ras) inhibited lipopolysaccharide‐induced GTP loading in rat primary astrocytes. To delineate the role of p21ras in the induction of iNOS, we examined the effect of Δp21ras on the expression of iNOS and the production of nitric oxide. It is interesting that expression of Δp21ras markedly inhibited the production of nitric oxide and the expression of iNOS in lipopolysaccharide‐ and proinflammatory cytokine (tumor necrosis factor‐α, interleukin‐1β; interferon‐γ)‐stimulated rat and human primary astrocytes. Inhibition of iNOS promoter‐derived chloramphenicol acetyltransferase activity by Δp21ras suggests that p21ras is involved in the transcription of iNOS. As activation of nuclear factor‐κB (NF‐κB) is necessary for the transcription of iNOS, we examined the effect of Δp21ras on the activation of NF‐κB. Expression of Δp21ras inhibited the DNA binding as well as the transcriptional activity of NF‐κB in activated astrocytes, suggesting that Δp21ras inhibits the expression of iNOS by inhibiting the activation of NF‐κB. These studies also suggest that inhibitors of p21ras may be used as therapeutics in nitric oxide‐ and cytokine‐mediated neuroinflammatory diseases.

Changing the Future of Health Professions: Embedding Interprofessional Education Within an Academic Health Center

Institutions are increasingly considering interprofessional education (IPE) as a means to improve health care and reduce medical errors in the United States. Effective implementation of IPE within health professions education requires a strategic institutional approach to ensure longevity and sustainability. In 2007, the Medical University of South Carolina (MUSC) established Creating Collaborative Care (C3), an IPE initiative that takes a multifaceted approach to weaving interprofessional collaborative experiences throughout MUSCs culture to prepare students to participate in interprofessional, collaborative health care and research settings. In this article, the authors describe C3s guiding conceptual foundation and student learning goals. They present its implementation framework to illustrate how C3 is embedded within the institutional culture. It is housed in the provosts office, and an overarching implementation committee functions as a central coordinating group. Faculty members develop and implement C3 activities across professions by contributing to four collaborating domains—curricular, extracurricular, faculty development, and health care simulation—each of which captures an IPE component. The authors provide examples of IPE activities developed by each domain to illustrate the breadth of IPE at MUSC. The authors believe that MUSCs efforts, including the conceptual foundation and implementation framework, can be generalized to other institutions intent on developing IPE within their organizational cultures.