Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:4.6.1.1 (adenylate cyclase)
 19,190 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A new strategy has been successfully applied to reconstitute the brain specific serotonin 5-HT1A receptor-G protein-adenylate cyclase complex. A mild method of tissue preparation gave a stable, membrane-bound form of the receptor (SBP) which retained its natural lipid content. Treatment of SBP with serotonin (1 microM) and 3-[(3-cholamidopropyl) dimethyl ammonio]-1-propanesulphonate (CHAPS) (2%) solubilized the ligand-receptor-G protein-ligand complex along with the associated phospholipids and cholesterol. Dialysis of this extract (SBDS) against buffer containing 25% ethylene glycol produced a stable, reconstituted and active preparation (SBDSE) of vesicles which upon centrifugal separation followed by gentle resuspension retained 95-100% [3H] 8-OH-DPAT binding activity as well as 60% [3H] GppNHp binding and adenylate cyclase activities of SBDSE. The reconstituted receptor preparation compared well with the membrane-bound form in displaying a similar value for KD (2.1 nM) and a single affinity state for [3H] 8-OH-DPAT binding (Bmax = 118 fmol/mg). However, in sharp contrast to the membrane-bound receptor which was negatively coupled to adenylate cyclase, agonist treatment of the solubilized and reconstituted receptor resulted in an increase in adenylate cyclase. This change in receptor-adenylate cyclase coupling following reshuffling of membrane lipids during solubilization and reconstitution suggested that membrane lipids could have a profound effect on receptor-effector coupling. To study the effect of membrane lipid composition on receptor-mediated signal transduction in a stabler and more natural system, neural cells derived from different parts of the brain (hippocampus, HN2; CNS, NCB-20; dorsal root ganglion, F-11) and a non-neural cell line (CHO), all with differing membrane lipid compositions, were selected. Since no known cell line contains the serotonin 5-HT1A receptor (5-HT1A-R), stable transfection of the selected cell lines with a DNA construct encoding the human 5-HT1A-R was carried out and this resulted in a late increase of [3H] 8-OH-DPAT binding in the stationary phase only in the cell lines of neural origin. In the non-neural cell line (CHO), which also displayed marked difference in membrane lipids, the receptor was positively coupled to the phospholipase C-IP3-[Ca2+]i cascade. Even though GPLC was present in the NCB-20 and F-11 cells as evidenced by a bradykinin receptor-mediated increase in inositol phosphates in these cells 8-OH-DPAT treatment resulted in no change in phospholipase C in any of the cell lines of neural origin.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Role of lipids in receptor mediated signal transduction. 800 19

Stable expression of neuronal receptors in cell lines of neural origin is important for studies of neurotransmitter mediated signal transduction. We have achieved this for the first time in three cell lines which are derived from various tissues of neural origin (hippocampus, HN2; chinese hamster brain explant, NCB-20; rat dorsal root ganglion, F-11). Following electroporation assisted transfer of a construct containing the hippocampal serotonin 5-HT1A receptor (5-HT1AR) DNA, one neural cell line, NG-108-15 (murine neuroblastoma x C6 glioma), failed to express the transfected activity, while three others as well as the non-neural CHO (chinese hamster ovary) cells expressed high levels of the receptor. Upon normalization to coexpressed human beta-hexosaminidase B activity, it was found that the human 5-HT1AR, which is normally concentrated in the hippocampus and at a lesser density in the brain, was expressed at the highest level (15.7 x 10(4) receptors/cell) in the HN2 followed by the NCB-20 (8.3 x 10(4) receptors/cell), F-11 (4.4 x 10(4) receptors/cell) and lastly the non-neuronal CHO (4.2 x 10(4) receptors/cell) cells. Ten-twelve days after passage, a striking increase in expression of the receptor was observed only in the cell lines of neural origin. By contrast, there was no appreciable increase in expression of the transfected 5-HT1AR in the non-neural CHO cells over time. This late increase in expression was eliminated in cells which had been maintained in low glucose (1 g/L) for the first two days after passage, thus establishing a vital role of glucose in expression of the transfected 5-HT1AR in cell lines of neural origin. In all cases the 5-HT1AR was negatively coupled to adenylate cyclase, as evidenced by an agonist mediated decrease in prostaglandin E1 stimulated cyclic AMP levels.
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PMID:Heterologous expression of the serotonin 5-HT1A receptor in neural and non-neural cell lines. 847 11

Signal transduction cascades initiated by the neuronal kappa opioid receptor were studied following transfection of a neuronal (hippocampal) line, HN2, and the non-neural CHOs. Retinoic-acid mediated differentiation resulted in intense staining of the HN2 cells with a neurofilament protein antibody SMI 33 but not with an antibody to GFAP, thus establishing neuronal characteristics of the HN2 cell line. The kappa opioid receptor was stably expressed in the two cell lines by electroporation mediated transfer of a Cytomegalovirus-promoter driven construct, pCMV-kappa, harboring the kappa-opioid receptor cDNA. Positive clones (HN2 kappa 24 and CHO kappa 18) from both lines showed high expression of the kappa opioid receptor, as identified by [3H] U-69,593 binding to membranes prepared from HN2 kappa 24 and CHO kappa 18. Scatchard analysis revealed the presence of high affinity kappa opioid receptors in both engineered cell lines (KD=1.3 nM for HN2 kappa 24 and 2.1 nM for CHO kappa 18). Functional coupling to adenylate cyclase was displayed by 1 microM U-69,593 mediated inhibition (55-63%) of prostaglandin E1-stimulated intracellular cAMP levels. A major difference between the two clones was observed in functional coupling of the expressed kappa opioid receptor to phospholipases C (PL-C) and D (PL-D). U-69,593 (1 microM) treatment stimulated PL-C, but not PL-D, in HN2 kappa 24 cells, whereas PL-D, but not PL-C, was stimulated following such treatment of CHO kappa 18 cells. Our results using the model neuronal system, HN2 kappa 24, demonstrate cell-type specific, positive coupling of the kappa opioid receptor to the major Ca2+ mobilizing system, the PL-C cascade, which regulates neuronal firing.
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PMID:Stable expression and heterologous coupling of the kappa opioid receptor in cell lines of neural and nonneural origin. 861 81