Photoaffinity labelling of dopamine D<sub>2</sub> receptors by [<sup>3</sup>H]azidomethylspiperone

Niznik, Hyman B.; Grigoriadis, Dimitri E.; Seeman, Philip

doi:10.1016/0014-5793(86)81086-9

Cited by 28 publications

(12 citation statements)

References 17 publications

(6 reference statements)

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“…This is greater than the 39-kD predicted molecular weight of unmodified H218, but is not surprising given that most GPRs display apparent molecular weights from SDS-PAGE analysis that are significantly greater than their predicted molecular weights. Several examples have been reported in which this difference is comparable to or greater than that observed for H218 Niznik et al, 1986;Jarvie et al, 1988Jarvie et al, , 1989Huang et al, 1992;Shigemoto et al, 1993;Vigna et al, 1994;Pan et al, 1995]. Much of the additional mass of mature GPRs likely results from the N-glycosylation of consensus sites found in the N-terminal domains of most GPRs including H218 [Stiles et al, 1984;Jarvie et al, 1988Jarvie et al, , 1989Barrington et al, 1990;MacLennan et al, 1994;Garzon et al, 1995].…”

Section: Generation Of Pc12 Cell Lines Containing Reduced Levels Of Nmentioning

confidence: 95%

Antisense Studies in PC12 Cells Suggest a Role for H218, a Sphingosine 1-Phosphate Receptor, in Growth-Factor-Induced Cell-Cell Interaction and Neurite Outgrowth

et al. 2000

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Our previous studies of H218, a sphingosine 1-phosphate (S1P) receptor and a member of the G-protein-coupled receptor superfamily, suggest that it may participate in mammalian nervous system development. Thus, brain levels of H218 mRNA are higher during early neurogenesis than postnatally. In addition, embryonic H218 immunoreactivity is preferentially localized in young neuronal cell bodies during their early stages of differentiation and in axons during their extension. This report describes the morphological effects of reducing native H218 levels in PC12 cells. Western blot analyses demonstrated that PC12 cells stably transfected with an expression vector carrying an antisense-oriented H218 cDNA contain less H218 protein than vector-transfected control cells. When differentiated with growth factors, the antisense-H218 cells display more neurite production and form less cell-cell contacts than the control cells. Therefore, these data, along with our previous H218 expression studies and a recent, independent study of H218 overexpression, support the possibility that H218 contributes to developmental processes regulating neuronal interaction and axon growth. The data are also consistent with reports that H218 is a S1P receptor, that S1P is present in serum, like that used in our PC12 cell cultures, and that it causes PC12 cell neurite retraction. Finally, and in agreement with a S1P receptor role for H218, we find that the antisense-H218 cells display less S1P-induced neurite retraction than control cells.

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Section: Generation Of Pc12 Cell Lines Containing Reduced Levels Of Nmentioning

confidence: 95%

Antisense Studies in PC12 Cells Suggest a Role for H218, a Sphingosine 1-Phosphate Receptor, in Growth-Factor-Induced Cell-Cell Interaction and Neurite Outgrowth

et al. 2000

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“…['HIAzido methylspiperone ([. 'H]AMS) is one such photoaffinity label whose binding to striatal membranes (where it labels an M , 9 2 000 protein) has been extensively characterized and found to be Dz dopaminergic in nature (Seeman & Niznik, 1986). We have found that ['IAMS photolabels a protein of M , 94000 in both crude membranes and soluble prepara-tions.…”

Section: Sds/i'age Of An Eluute From Sequential Ligand Ufinify and mentioning

confidence: 88%

“…The reported molecular masses of the labelled receptor range from 85000 Da (Redouane et al, 1985) to 120000 Da (Amlaiky & Caron, 1986). However, the majority of data would suggest a molecular mass of 02-04000 Da (Amlaiky & Caron, 1985, 1986Seeman & Niznik, 1986;Niznik et ul., 1986). ['HIAzido methylspiperone ([.…”

Section: Sds/i'age Of An Eluute From Sequential Ligand Ufinify and mentioning

confidence: 99%

Characterization of calcium channel subunit

Burgess

Norman

1988

Biochemical Society Transactions

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“…The reported molecular masses of the labelled receptor range from 85000 Da (Redouane et al, 1985) to 120000 Da (Amlaiky & Caron, 1986). However, the majority of data would suggest a molecular mass of 02-04000 Da (Amlaiky & Caron, 1985Seeman & Niznik, 1986;Niznik et ul., 1986). ['HIAzido methylspiperone ([.…”

Section: Photolabelling Studiesmentioning

confidence: 99%

Partial purification of the D2 dopamine receptor from bovine caudate nucleus

Williamson

Worrall

Chazot

et al. 1988

Biochemical Society Transactions

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The D2 dopamine receptor mediates many of the physiological effects of the neurotransmitter dopamine, being involved in control of motor function and certain aspects of behaviour in the brain (Iversen, 1976) and of prolactin and a-melanocyte stimulating hormone release in the pituitary (Caron et a/., 1978;Cote et a[., 1984). Several secondmessenger systems have been proposed for D, dopamine receptor signal transduction. The most well documented of these involves coupling to adenylate cyclase via a pertussis toxin sensitive inhibitory guanine nucleotide regulatory protein (Sibley et al., 1982; Enjalbert & Bockaert, 1983: Cote et al., 1984. However, the receptor may also function through inhibition of polyphosphoinositide metabolism (Simmonds & Strange, 1985;Enjalbert et al., 1986) and stimulation of potassium channels (Israel et al., 1985; Memo et a/., 1987). A much greater understanding of the receptor signalling system would be gained if the receptor could be isolated in pure form and reconstituted together with purified effector molecules in defined artificial membrane environments. In addition, microsequencing of the pure receptor protein would permit cloning of the receptor gene.In view of these considerations we have attempted to purify iI2 dopamine receptors from bovine caudate nuclei. Affinity matricesThe D2 receptor is present at extremely low concentrations in the caudate nucleus (approximately 400 fmol/mg of membrane protein: Leonard et a/., 1987), hence ligand affinity chromatography is likely to be an essential step in purification. We have synthesized a series of affinity matrices in which the carboxymethoxyloxime derivative of the D? dopaminergic antagonists haloperidol or spiperone (type I coupling) or the hemisuccinate derivative of haloperidol (type I1 coupling) were immobilized on Sepharose via spacer arms of differing lengths or composition.Type I1 coupling was the more efficient technique, immobilizing, for example, 709 k 1 14 nmol haloperidol/ml w-aminohexyl Sepharose (mean t-S.E.M. of five observations) as compared to 229 k 29 nmol/ml for type I coupling. It is not possible, using conventional ligand binding techniques, to determine the receptor affinity of the Sepharose-coupled ligands. However, we were able to gain some measure of the effect of coupling on affinity using ligands coupled to bovine serum albumin (BSA) in competition binding studies. Both coupling methods reduced the affinity of the ligands for receptor, though the effect of type I1 coupling was less marked than type 1 ( K , values for the inhibition of [3H]spiperone binding to solubilized receptor were 33 nM for haloperidol, 1900 nM for the BSA-haloperidol type 1 conjugate Abbreviations used: BSA, bovine serum albumin; WGL, wheat germ lectin; SDS/PAGE, SDS/polyacrylamide-gel electrophoresis; AMS, azidomethylspiperone. and 1010 nM for the BSA-haloperidol type I1 conjugate). The type I spiperone conjugate had the highest affinity for receptor ( K , 270 nM) though this is still considerably below its affinity in the uncoupled...

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Photoaffinity labelling of dopamine D₂ receptors by [³H]azidomethylspiperone

Cited by 28 publications

References 17 publications

Antisense Studies in PC12 Cells Suggest a Role for H218, a Sphingosine 1-Phosphate Receptor, in Growth-Factor-Induced Cell-Cell Interaction and Neurite Outgrowth

Antisense Studies in PC12 Cells Suggest a Role for H218, a Sphingosine 1-Phosphate Receptor, in Growth-Factor-Induced Cell-Cell Interaction and Neurite Outgrowth

Characterization of calcium channel subunit

Partial purification of the D2 dopamine receptor from bovine caudate nucleus

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Photoaffinity labelling of dopamine D2 receptors by [3H]azidomethylspiperone

Cited by 28 publications

References 17 publications

Antisense Studies in PC12 Cells Suggest a Role for H218, a Sphingosine 1-Phosphate Receptor, in Growth-Factor-Induced Cell-Cell Interaction and Neurite Outgrowth

Antisense Studies in PC12 Cells Suggest a Role for H218, a Sphingosine 1-Phosphate Receptor, in Growth-Factor-Induced Cell-Cell Interaction and Neurite Outgrowth

Characterization of calcium channel subunit

Partial purification of the D2 dopamine receptor from bovine caudate nucleus

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Photoaffinity labelling of dopamine D₂ receptors by [³H]azidomethylspiperone