Fluorescence analysis of receptor-G protein interactions in cell membranes.
The dynamics of G protein heterotrimer complex formation and disassembly in response to nucleotide binding and receptor activation govern the rate of responses to external stimuli. We use a novel flow cytometry approach to study the effects of lipid modification, isoform specificity, lipid environment, and receptor stimulation on the affinity and kinetics of G protein subunit binding. Fluorescein-labeled myristoylated Galpha(i1) (F-alpha(i1)) was used as the ligand bound to Gbetagamma in competition binding studies with differently modified Galpha subunit isoforms. In detergent solutions, the binding affinity of Galpha(i) to betagamma was 2 orders of magnitude higher than for Galpha(o) and Galpha(s) (IC50 of 0.2 nM vs 17 and 27 nM, respectively), while in reconstituted bovine brain lipid vesicles, binding was slightly weaker. The effects of receptor on the G protein complex were assessed in alpha(2A)AR receptor expressing CHO cell membranes into which purified betagamma subunits and F-alpha(i1) were reconstituted. These cell membrane studies led to the following observations: (1) binding of alpha subunit to the betagamma was not enhanced by receptor in the presence or absence of agonist, indicating that betagamma contributed essentially all of the binding energy for alpha(i1) interaction with the membrane; (2) activation of the receptor facilitated GTPgammaS-stimulated detachment of F-alpha(i1) from betagamma and the membrane. Thus flow cytometry permits quantiatitive and real-time assessments of protein-protein interactions in complex membrane environments.
Sarvazyan
Noune A
NA
Department of Pharmacology, The University of Michigan, Ann Arbor, Michigan 48109-0632, USA.
Lim
William K
WK
Neubig
Richard R
RR
D058647:Adrenergic alpha-2 Receptor Agonists; D000818:Animals; D001710:Biotin / Q000378:metabolism; D016466:CHO Cells; D002417:Cattle; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D006224:Cricetinae; D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000032:analysis* / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D007700:Kinetics; D010743:Phospholipids / Q000378:metabolism; D011485:Protein Binding; D021122:Protein Subunits; D051381:Rats; D018341:Receptors, Adrenergic, alpha-2 / Q000032:analysis* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology
D058647:Adrenergic alpha-2 Receptor Agonists; D005456:Fluorescent Dyes; D010743:Phospholipids; D021122:Protein Subunits; D018341:Receptors, Adrenergic, alpha-2; D011994:Recombinant Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D001710:Biotin; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D016650:Fluorescein-5-isothiocyanate