Gialpha and Gbeta subunits both define selectivity of G protein activation by alpha2-adrenergic receptors.
Previous studies of the specificity of receptor interactions with G protein subunits in living cells have relied on measurements of second messengers or other downstream responses. We have examined the selectivity of interactions between alpha2-adrenergic receptors (alpha2R) and various combinations of Gialpha and Gbeta subunit isoforms by measuring changes in FRET between Gialpha-yellow fluorescent protein and cyan fluorescent protein-Gbeta chimeras in HeLa cells. All combinations of Gialpha1, -2, or -3 with Gbeta1, -2, or -4 were activated to some degree by endogenous alpha2Rs as judged by agonist-dependent decreases in FRET. The degree of G protein activation is determined by the combination of Gialpha and Gbeta subunits rather than by the identity of an individual subunit. RT-PCR analysis and small interfering RNA knockdown of alpha2R subtypes, followed by quantification of radiolabeled antagonist binding, demonstrated that HeLa cells express alpha2a- and alpha2b-adrenergic receptor isoforms in a 2:1 ratio. Increasing receptor number by overexpression of the alpha2aR subtype minimized the differences among coupling preferences for Gialpha and Gbeta isoforms. The molecular properties of each Gialpha, Gbeta, and alpha2-adrenergic receptor subtype influence signaling efficiency for the alpha2-adrenergic receptor-mediated signaling pathway.
Gibson
Scott K
SK
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. scott.gibson@utsouthwestern.edu
Gilman
Alfred G
AG
D004789:Enzyme Activation / Q000502:physiology; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D044387:GTP-Binding Protein beta Subunits / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D034741:RNA, Small Interfering / Q000235:genetics; D018341:Receptors, Adrenergic, alpha-2 / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology*; D013379:Substrate Specificity
D005456:Fluorescent Dyes; D044387:GTP-Binding Protein beta Subunits; D034741:RNA, Small Interfering; D018341:Receptors, Adrenergic, alpha-2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go