Binding of β4γ5 by Adenosine A1 and A2A Receptors Determined by Stable Isotope Labeling with Amino Acids in Cell Culture and Mass Spectrometry

• Published in: Biochemistry (Easton) Vol. 50; no. 2; pp. 207 - 220
• Main Authors: Bigler Wang, Dora, Sherman, Nicholas E, Shannon, John D, Leonhardt, Susan A, Mayeenuddin, Linnia H, Yeager, Mark, McIntire, William E
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 18.01.2011
• Subjects: Amino Acid Sequence; Amino Acids - analysis; Cell Culture Techniques; GTP-Binding Protein beta Subunits - analysis; GTP-Binding Protein beta Subunits - metabolism; GTP-Binding Protein gamma Subunits - analysis; GTP-Binding Protein gamma Subunits - metabolism; HEK293 Cells; Humans; Isotope Labeling - methods; Molecular Sequence Data; Protein Isoforms - chemistry; Protein Isoforms - metabolism; Protein Multimerization; Receptor, Adenosine A1 - analysis; Receptor, Adenosine A1 - metabolism; Receptor, Adenosine A2A - analysis; Receptor, Adenosine A2A - metabolism; Recombinant Fusion Proteins - analysis; Recombinant Fusion Proteins - metabolism; Sf9 Cells; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi101227y

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 A1:αi1 and adenosine A2A:αS receptor fusion proteins expressed in HEK-293 cells. Cells expressing A1:αi1 were cultured in media containing [13C6]Arg and [13C6]Lys and βγ labeled with heavy isotopes purified. Heavy βγ was combined with either recombinant βγ purified from Sf9 cells, βγ purified from the A2A:αS 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, β1, β2, and β4, and seven γ isoforms, γ2, γ4, γ5, γ7, γ10, γ11, and γ12, were identified in the analysis. β1 and γ5 were most abundant in the enriched βγ fraction, and this βγ profile was generally mirrored in the fusion proteins. However, both A2A:αS and A1:αi1 bound more β4 and γ5 compared to the enriched βγ fraction; also, more β4 was associated with A2A:αS than A1:αi1. Both fusion proteins also contained less γ2, γ10, and γ12 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.