Biochemical Society Transactions (200 I ) Volume 29, Part 3
79 Regulation of endogenous secretin receptor responsiveness by P K C R, Ghadessy and E. Kelly Department of Pharmacology, School of Medical Sciences, University of Bristol, Bristol BS8 The G,-coupled secretin receptor endogenously expressed in NG108-15 mouse neuroblastoma x rat glioma cells exhibits agonist-induced desensitization; the role of PKC in this process was investigated. Wild type NG108-15 cells were seeded into 24 well plates. The PKC inhibitor GF109203X, the PKC activator phorbol 12-myristate 13-acetate (PMA) o r the purinoceptor agonist U T P were added to the wells 15-30 min prior to agonist addition. Pretreatment of cells with PMA decreased both secretinand forskolin-stimulated cAMP accumulation to 66 2 9 % and 44 2 2 % (n=4-8; mean ? s.e.mean), that of the control agonist response in the absence of PMA, respectively. Pretreatment with U T P also attenuated secretin and forskolin responsiveness to 66 2 3% and 81 2 5% that of the control agonist response, respectively (n=13-16). PMA- and UTP-induced attenuation of secretin and forskolin responsiveness was reversed by co-addition of GF109203X. Interestingly, cAMP formation via adenosine A, or IP prostanoid receptor activation with N E C A o r iloprost respectively, was unaffected by PMA or U T P pretreatment. The results indicate that PKC can regulate heterologous desensitization of secretin receptor responsiveness. The selective effects of PKC activation o n secretin and forskolin responsiveness suggest compartmentalization of G, signalling components in these cells.
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A role for receptor internalisation in M3 muscarinic 81 receptor association with ARF and activation of phospholipase D D.N. Robertson, M.S. Johnson, E.M. Lutz, P.J.Holland and R. Mitchell MRC Membrane and Adapter Proteins Co-op, Membrane Biology -_ Group, Dept of Biomedicai Sciences, University of Edinburgh. EH8 9XD Many G protein-coupled receptors (GPCRs) including the M, receptor, can associate directly with ARF and thereby facilitate their activation of phospholipase D (PLD). In 1321N1 cells, PLD (but not PLC) activation by the native M, receptor was attenuated by monodansylcadaverine and hypertonic sucrose (inhibitors of G P C R internalisation through clathrin coated pits) whereas that mediated by the TXA, receptor (which appears to be ARF-independent) was unaffected. In C O S 7 cells transiently transfected with the M, receptor (signal-FLAG tagged at the Nterminus), cross linking of the surface M, receptor to insoluble Protein G (by means of FLAG antibody) inhibited [3H]oxotremorine internalisation and PLD, but not PLC, activation. C O S 7 cells were also cotransfected with the signalFLAG-M, receptor and ARFl (with a C-terminus H A tag) and solubilised using CHAPS/DOC-containing buffer. FLAGdirected immunoprecipitates displayed clear ARF1-HA-immunoreactivity which was greatly reduced in cells incubated with monodansylcadaverine. M, receptor docking of ARFl and P L D activation may depend on receptor trafficking.
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82 In vivo imaging of ryanodine receptor-mediated release of
80 Localization of presynaptic merabotropic glutamate receptors at the Calyces of Held
B. Pilkineton, M. Cuttle, J. Chad, V. O'Connor Cell Sciences, School of Biological Sciences, University of Sou thampton Metabotropic glutamate receptors (mGluRs) are synaptically localized G-protein coupled receptors that regulate neurotransmission. In particular Group I1 (2/3) and Group I11 (4,7 and 8) mGluRs are implicated in the regulation of release from many presynaptic structures in brain. We have investigated the molecular basis of identified mGluR dependent modulation of glutamate transmission at the trapezoid body, a giant synapse found in the mammalian brainstem (Barnes-Davies and Forsythe, 1995 J. Physiol. 488.2 387). We identified mGluR 4 and mGluR 2/3 immunoreactivity in the homogenates of brainstem membranes. Using immunocytochemistry and confocal microscopy we show that both mGluR 4 and mGluR 2/3 immunoreactivities are localized at the Calyx of Held, the presynaptic terminal of this giant synapse. This indicates that both mGluR4 and mGluR 2/3 contribute an autoreceptor function at the giant synapse of the trapezoid body. These observations will support investigation of the molecular mechanisms of mGluR and G-protein coupled receptor function in the context of a physiologically tractable synapse.
Caz+ from dense core secretory vesicles K.Mitchel1, A.Varadi, P.Pinton**, T.Pozzan**, R.Rizzuto*, G.A.Rutter Department of Biochemistry, University of Bristol, U.K.,;*Departmentof Experimental and Diagnostic Medicine, Ferrara, Italy, ""Department of Biomedical Sciences, Padova, Italy. The role of dense core secretory vesicles as a mobilizable Ca2+ store is controversial. I n order to measure the free Ca2+concentration in the secretory vesicle matrix ([Ca2+Isv),, we have generated c D N A encoding recombinant aequorin, fused at the Cterminus of vesicle-associated membrane protein (VAMP). In M I N 6 p-cells, [Ca2+],, was 51 2 7.5 p.M (mean 2 S.E.M., n=3), - 5-fold lower than in the endoplasmic reticulum (ER; [CaZ+IEp 249 ? 12.9 pM, n=3). Secretory vesicle Ca2+uptake was insensitive to inhibitors of the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) pump activity, but sensitive to the P-type Ca2+pump inhibitor, orthovanadate. In contrast to the ER, the vesicular Ca2+store was unaffected by inositol (1,4,5) trisphosphate, however, both caffeine and the ryanodine receptor agonist, 4-chloroeythylphenol, induced Ca2+ release from the secretory vesicles and ER. Thus, secretory vesicles sequester CaZ+ via an ATP-dependent Ca2+ pump, distinct from SERCAs. Dense core vesicles may then act as an intracellular Ca2+store which could contribute to Ca2+-induced Ca2+release during exocytosis.
0 2001 Biochemical Society
