Binding of β4γ5 by adenosine A1 and A2A receptors determined by stable isotope labeling with amino acids in cell culture and mass spectrometry.
Characterization of G protein βγ 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 βγ dimers associated with adenosine A(1):α(i1) and adenosine A(2A):α(S) receptor fusion proteins expressed in HEK-293 cells. Cells expressing A(1):α(i1) were cultured in media containing [(13)C(6)]Arg and [(13)C(6)]Lys and βγ labeled with heavy isotopes purified. Heavy βγ was combined with either recombinant βγ purified from Sf9 cells, βγ purified from the A(2A):α(S) expressed in HEK-293 cells cultured in standard media, or an enriched βγ fraction from HEK-293 cells. Samples were separated by SDS-PAGE, protein bands containing β and γ were excised, digested with trypsin, and separated by HPLC, and isotope ratios were 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. β(1) and γ(5) were most abundant in the enriched βγ fraction, and this βγ profile was generally mirrored in the fusion proteins. However, both A(2A):α(S) and A(1):α(i1) bound more β(4) and γ(5) compared to the enriched βγ fraction; also, more β(4) was associated with A(2A):α(S) than A(1):α(i1). Both fusion proteins also contained less γ(2), γ(10), and γ(12) than the enriched βγ fraction. These results suggest that preferences for particular βγ isoforms may be driven in part by structural motifs common to adenosine receptor family members.
Department of Pharmacology, University of Virginia Health System, Charlottesville, 22908, United States.
Dora
D
Bigler Wang
Nicholas E
NE
Sherman
John D
JD
Shannon
Susan A
SA
Leonhardt
Linnia H
LH
Mayeenuddin
Mark
M
Yeager
William E
WE
McIntire
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D018929:Cell Culture Techniques; D044387:GTP-Binding Protein beta Subunits / Q000032:analysis / Q000378:metabolism*; D044388:GTP-Binding Protein gamma Subunits / Q000032:analysis / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D055503:Protein Multimerization; D043682:Receptor, Adenosine A1 / Q000032:analysis / Q000378:metabolism*; D043705:Receptor, Adenosine A2A / Q000032:analysis / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000378:metabolism; D061987:Sf9 Cells; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D000596:Amino Acids; C118981:G-protein Beta gamma; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D020033:Protein Isoforms; D043682:Receptor, Adenosine A1; D043705:Receptor, Adenosine A2A; D011993:Recombinant Fusion Proteins