Linked Life Data

8952462

Source:http://linkedlifedata.com/resource/pubmed/id/8952462

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
48
pubmed:dateCreated
1997-1-9
pubmed:abstractText
In order to evaluate the baculovirus expression system as a means for high-yield production of homogeneous D2S receptor, we have expressed various D2S receptor constructs in two Spodoptera frugiperda cell lines, a Trichoplusia ni and a Mammestra brassicae cell line. To improve expression yield, the environment of the polyhedrin gene translational initiation site was retained by fusing the first 12 codons of the polyhedrin gene to the 5'-end of the D2S receptor coding sequence. The pharmacological profile of the expressed D2S receptor was similar to that reported for neuronal D2 receptors. Sf9 and Tn cells were best suited for overexpression, yielding about 2 x 10(6) and 4 x 10(6) receptors/cell, respectively, corresponding to 6 pmol/mg of cell protein in Sf9 cells and 10 pmol/mg of cell protein in Tn cells. We have developed a D2 receptor-specific anti-peptide antibody to study glycosylation, palmitoylation, and localization of the heterologously produced receptor. Immunoprecipitation of digitonin/cholate-solubilized receptor from control and tunicamycin-treated Sf9, Tn, and Mb cells revealed an apparent molecular mass of 47-48 kDa for the glycosylated receptor and of 39-40 kDa for the unglycosylated receptor. Although pulse-chase studies showed that glycosylation occurred rapidly and efficiently, the glycosylated receptor only constituted a small fraction of the overall produced receptor protein, which was mainly located intracellularly. The glycosylation of the receptor was of the high-mannose-type in contrast to the complex-type glycosylation found in native tissue. The glycosylated D2S receptor was palmitoylated. Glycosylation, however, was not a prerequisite for palmitoylation which was insensitive to tunicamycin, brefeldin A, and monensin. NH2-terminal addition of the signal sequence of prepromelittin to the D2S receptor increased expression levels 2-3-fold and significantly enhanced membrane insertion and processing, resulting in increased targeting of the synthesized receptor to the plasma membrane.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Dec
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
3
pubmed:volume
35
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
15149-61
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
1996
pubmed:articleTitle
Glycosylation, palmitoylation, and localization of the human D2S receptor in baculovirus-infected insect cells.
pubmed:affiliation
Max-Planck-Institut für Biophysik, Frankfurt, Germany.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't