Purification and mass spectrometric analysis of the κ opioid receptor

Abstract

A clonal human embryonic kidney (HEK) 293 cell line was established that stably expressed the rat κ-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 pmoles/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]GTPγS 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-β-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.

Proteomic profiling of renal allograft rejection in serum using magnetic bead–based sample fractionation and MALDI-TOF MS

“…As a convenient, fast proteomic technique, MB-WCX purification and MALDI-TOF analysis, in combination with bioinformatics software, facilitates identification of novel biomarkers. As a powerful tool for surveying complex patterns of biologically informative molecules, MALDI-TOF MS protein profiling has been applied in proteomics biomarker research [37][38][39] and become a promising tool in nephrology research [40][41][42][43]. In our study, we divided serum samples into four groups.…”

Naltrindole Inhibits Human Multiple Myeloma Cell Proliferation In Vitro and in a Murine Xenograft Model In Vivo

“…Our laboratory has purified and identified -, ␦-, and -opioid receptors by using mass spectrometry (Christoffers et al, 2003(Christoffers et al, , 2005Wannemacher et al, 2008); however, our efforts to purify and identify the naltrindole binding site in multiple myeloma cells have been compromised by the loss of binding activity in subcellular fractions. Investigations into developing binding assays in subcellular fractions continue, along with efforts to identify the binding site by expression cloning or microRNA knockdown.…”

Опиоидные Κ-Рецепторы Как Молекулярная Мишень Для Создания Нового Поколения Обезболивающих Лекарственных Средств

“…Áåëîê k-ðåöåïòîðà ïðèíàäëåaeèò ê À êëàññó ñóïåð-ñåìåéñòâà ìåòàáîòðîïíûõ GPCR [4,5], ñîñòîèò èç 380 àìèíîêèñëîòíûõ îñòàòêîâ, 7 ãèäðîôîáíûõ Ü-ñïèðà-ëåé, èíòåãðèðîâàííûõ â êëåòî÷íóþ ìåìáðàíó, à òàêaeå N-ãëèêîçèëèðîâàííîãî è Ñ-ïàëüìèòèðîâàííîãî êîíöå-âûõ ôðàãìåíòîâ (ðèñ. 1) [6].…”

“…Îïèîèäíûå k-ðåöåïòîðû ïðåäñòàâëåíû êàê â ÖÍÑ [5,12], òàê è íà ïåðèôåðèè [13,14], è ñïåöèôè÷åñêè àêòèâèðóþòñÿ ýíäîãåííûìè ëèãàíäàìè, îáðàçóþùè-ìèñÿ èç ïðîäèíîðôèíà [15]. Âûñîêèé óðîâåíü k-ðå-öåïòîðîâ ýêñïðåññèðóåòñÿ â âåãåòàòèâíûõ öåíòðàõ ãî-ëîâíîãî, ñïèííîãî ìîçãà, ñòðóêòóðàõ, îòâåòñòâåííûõ çà âîñïðèÿòèå íîöèöåïòèâíîé èíôîðìàöèè [16 -18], êîãíèòèâíûå ôóíêöèè, ýìîöèîíàëüíîå ïîâåäåíèå, ñîç-íàíèå [1,2], à òàêaeå íà ïåðèôåðèè â ãàíãëèÿõ òðîé-íè÷íîãî íåðâà, çàäíèõ êîðåøêîâ ñïèííîãî ìîçãà, êëå-òî÷íûõ òåëàõ òîíêèõ ìèåëèíèçèðîâàííûõ è íåìèåëè-íèçèðîâàííûõ íîöèöåïòèâíûõ àôôåðåíòíûõ îêîí÷àíèé [19 -21], òåðìèíàëÿõ ñåíñîðíûõ íåéðîíîâ êîaeè, ìûøö, ñóñòàâàõ, âíóòðåííèõ îðãàíàõ, ëèìôîöè-òàõ, ëåéêîöèòàõ, êàðäèîìèîöèòàõ [22 -25].…”