Transportation News

Newsletter published by the Texas Department of Transportation for TxDOT employees including information about the organization, projects throughout the state, and other topics related to transportation in Texas

Binding of beta(4)gamma(5) by Adenosine A(1) and A(2A) Receptors Determined by Stable Isotope Labeling with Amino Acids in Cell Culture and Mass Spectrometry

Characterization of G protein beta gamma 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 beta gamma dimers associated with adenosine A(1):alpha(i1) and adenosine A(2A):alpha(S) receptor fusion proteins expressed in HEK-293 cells. Cells expressing A(1):alpha(i1) were cultured in media containing [C-13(6)]Arg and [C-13(6)]Lys and beta gamma labeled with heavy isotopes purified. Heavy beta gamma was combined with either recombinant beta gamma purified from Sf9 cells, beta gamma purified from the A2A:as expressed in HEK-293 cells cultured in standard media, or an enriched beta gamma fraction from HEK-293 cells. Samples were separated by SDS PAGE, protein bands containing beta and gamma were excised, digested with trypsin, and separated by HPLC, and isotope ratios were analyzed by mass spectrometry. Three beta isoforms, beta(1), beta(2), and beta(4), and seven gamma isoforms, gamma(2), gamma(4), gamma(5), gamma(7), gamma(10), gamma(11), and gamma(12), were identified in the analysis. beta(1) and gamma(5) were most abundant in the enriched beta gamma fraction, and this beta gamma profile was generally mirrored in the fusion proteins. However, both A(2A):alpha(S) and A(1):alpha(i1) bound more beta(4) and gamma(5) compared to the enriched beta gamma fraction; also, more beta(4) was associated with A(2A):alpha(S) than A(1):alpha(i1). Both fusion proteins also contained less gamma(2), gamma(10), and gamma(12) than the enriched beta gamma fraction. These results suggest that preferences for particular beta gamma isoforms may be driven in part by structural motifs common to adenosine receptor family members