Purification and mass spectrometric analysis of the kappa opioid receptor.

Purification and mass spectrometric analysis of the kappa opioid receptor. Research Abstract Details 

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Purification and mass spectrometric analysis of the kappa opioid receptor. Abstract Text:

A clonal human embryonic kidney (HEK) 293 cell line was established that stably expressed the rat kappa-opioid receptor (rKOR) with a FLAG epitope at the amino terminus. The K(d) for [(3)H]diprenorphine was 1.1+/-0.2 nM, and the B(max) was 2.6+/-0.4 pmol/mg. Dynorphin A (1-13), U69,593 and naloxone competitively inhibited [(3)H]diprenorphine binding with K(i) values of 2.0, 18 and 18 nM, respectively, in good agreement with previously reported affinities for the unmodified receptor. U69,593 stimulated [(35)S]GTPgammaS binding in a concentration-dependent manner and caused phosphorylation of mitogen-activated protein (MAP) kinase, indicating that the activated epitope-tagged receptor triggered appropriate signaling pathways. Immunoblot analysis demonstrated that two immunoreactive receptor species with apparent molecular masses of 42 and 52 kDa were expressed. Previous studies indicated that the 42 kDa protein was localized intracellularly and was a precursor of the 52 kDa receptor, which was present at the cell surface. rKOR was extracted from transfected HEK 293 cell membranes with n-dodecyl-beta-d-maltopyranoside. Sequential use of wheat germ agglutinin chromatography, Sephacryl S300 gel filtration chromatography, anti-FLAG immunoaffinity chromatography and SDS/PAGE permitted purification of the 52 kDa receptor. MALDI-TOF mass spectrometry was used to identify peptides derived from rKOR following sequential in-gel digestion with trypsin and cyanogen bromide. Eighteen rKOR peptides were detected, corresponding to 27.1% coverage of the receptor. Precursor-selective MS/MS confirmed the identity of most of these peptides. In addition, we have identified heat shock protein 70 (HSP70) as a rKOR-interacting protein.

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Purification and mass spectrometric analysis of the kappa opioid receptor. Journal Published:

PUBLICATION TYPE: Journal Article

Journal: Brain research

VOLUME: 1230

Page Numbers: 13-26

Journal Abbreviation: Brain Res.

ISSN: 0006-8993

DAY: 12

MONTH: 07

YEAR: 2008

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LANGUAGE: eng

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Grant and Affiliation Information for Purification and mass spectrometric analysis of the kappa opioid receptor.

AFFILIATION: Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey-Graduate School of Biomedical Science, Newark, NJ, USA.

Country: Netherlands

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MEDLINETA: Brain Res

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