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Specific association of β 4 γ 5 to adenosine A 1 and A 2A receptors determined by stable isotope labeling with heavy amino acids in cell culture and mass spectrometry
Author(s) -
McIntire William E,
Wang Dora Bigler,
Sherman Nicholas,
Shan John,
Leonhardt Susan,
Yeager Mark,
Mayeenuddin Linnia H,
Garrison James C
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.25.1_supplement.1011.1
Subject(s) - hek 293 cells , sf9 , gene isoform , fusion protein , chemistry , receptor , microbiology and biotechnology , cell culture , trypsin , recombinant dna , adenosine , tandem mass spectrometry , biochemistry , mass spectrometry , biology , chromatography , gene , enzyme , genetics , spodoptera
Quantitative mass spectrometry was used to characterize native βγ dimers selectively associated with expressed adenosine A 1 :α i1 and A 2A :α S receptor fusion proteins. An A 1 :α i1 fusion protein was expressed in HEK‐293 cells cultured in media containing [ 13 C 6 ] Arg and [ 13 C 6 ] Lys, and the βγ dimers which incorporated heavy isotopes purified. Heavy βγ was combined with either recombinant βγ purified from Sf9 cells, βγ purified from A 2A :αS expressed in HEK‐293 cells cultured in standard media, or βγ enriched from HEK‐293 cells. Samples were separated by SDS‐PAGE, and protein bands containing β and γ were excised, digested with trypsin, separated by HPLC and isotope ratios analyzed by mass spectrometry. Three β isoforms, β 1 , β 2 and β 4 , and seven γ isoforms, γ 2 , γ 4 , γ 5 , γ 7 , γ 10 , γ 11 and γ 12 were identified in the analysis. Compared to cellular HEK‐293 βγ levels, both fusion proteins had increased levels of β 4 and γ 5 . Also, more β 4 associated with A 2A :α S fusion protein than the A 1 :α i1 version. No differences in γ isoforms were observed between the two fusion proteins; however, both contained less γ 2 , γ 10 and γ 12 than the enriched HEK‐293 βγ. These results suggest that preferences for particular βγ isoforms are driven in part by structural motifs common to adenosine receptor family members. Supported by AHA Grant 0535350N, a Pilot Award from the UVa Silvio O. Conte Digestive Disease Research Center and NIH Grant R01‐DK‐19952.