Binding of 尾4纬5 by Adenosine A1 and A2A Receptors Determined by Stable Isotope Labeling with Amino Acids in Cell Culture and Mass Spectrometry
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- 作者：Dora Bigler Wang ; Nicholas E. Sherman ; John D. Shannon ; Susan A. Leonhardt ; Linnia H. Mayeenuddin ; Mark Yeager ; William E. McIntire
- 刊名：Biochemistry
- 出版年：2011
- 出版时间：January 18, 2011
- 年：2011
- 卷：50
- 期：2
- 页码：207-220
- 全文大小：1268K
- 年卷期：v.50,no.2(January 18, 2011)
- ISSN：1520-4995
文摘
Characterization of G protein 尾纬 dimer isoform expression in different cellular contexts has been impeded by low levels of protein expression, broad isoform heterogeneity, and antibodies of limited specificity, sensitivity, or availability. As a new approach, we used quantitative mass spectrometry to characterize native 尾纬 dimers associated with adenosine A1:伪i1 and adenosine A2A:伪S receptor fusion proteins expressed in HEK-293 cells. Cells expressing A1:伪i1 were cultured in media containing [13C6]Arg and [13C6]Lys and 尾纬 labeled with heavy isotopes purified. Heavy 尾纬 was combined with either recombinant 尾纬 purified from Sf9 cells, 尾纬 purified from the A2A:伪S expressed in HEK-293 cells cultured in standard media, or an enriched 尾纬 fraction from HEK-293 cells. Samples were separated by SDS鈭扨AGE, protein bands containing 尾 and 纬 were excised, digested with trypsin, and separated by HPLC, and isotope ratios were analyzed by mass spectrometry. Three 尾 isoforms, 尾1, 尾2, and 尾4, and seven 纬 isoforms, 纬2, 纬4, 纬5, 纬7, 纬10, 纬11, and 纬12, were identified in the analysis. 尾1 and 纬5 were most abundant in the enriched 尾纬 fraction, and this 尾纬 profile was generally mirrored in the fusion proteins. However, both A2A:伪S and A1:伪i1 bound more 尾4 and 纬5 compared to the enriched 尾纬 fraction; also, more 尾4 was associated with A2A:伪S than A1:伪i1. Both fusion proteins also contained less 纬2, 纬10, and 纬12 than the enriched 尾纬 fraction. These results suggest that preferences for particular 尾纬 isoforms may be driven in part by structural motifs common to adenosine receptor family members.