Evidence for a second, high affinity Gbetagamma binding site on Galphai1(GDP) subunits.
It is well known that Galpha(i1)(GDP) binds strongly to Gbetagamma subunits to form the Galpha(i1)(GDP)-Gbetagamma heterotrimer, and that activation to Galpha(i1)(GTP) results in conformational changes that reduces its affinity for Gbetagamma subunits. Previous studies of G protein subunit interactions have used stoichiometric amounts of the proteins. Here, we have found that Galpha(i1)(GDP) can bind a second Gbetagamma subunit with an affinity only 10-fold weaker than the primary site and close to the affinity between activated Galpha(i1) and Gbetagamma subunits. Also, we find that phospholipase Cbeta2, an effector of Gbetagamma, does not compete with the second binding site implying that effectors can be bound to the Galpha(i1)(GDP)-(Gbetagamma)(2) complex. Biophysical measurements and molecular docking studies suggest that this second site is distant from the primary one. A synthetic peptide having a sequence identical to the putative second binding site on Galpha(i1) competes with binding of the second Gbetagamma subunit. Injection of this peptide into cultured cells expressing eYFP-Galpha(i1)(GDP) and eCFP-Gbetagamma reduces the overall association of the subunits suggesting this site is operative in cells. We propose that this second binding site serves to promote and stabilize G protein subunit interactions in the presence of competing cellular proteins.
Wang
Jingting
J
From the Department of Physiology and Biophysics, Stony Brook University, Stony Brook, New York 11794-8661.
Sengupta
Parijat
P
From the Department of Physiology and Biophysics, Stony Brook University, Stony Brook, New York 11794-8661.
Guo
Yuanjian
Y
From the Department of Physiology and Biophysics, Stony Brook University, Stony Brook, New York 11794-8661.
Golebiewska
Urszula
U
From the Department of Physiology and Biophysics, Stony Brook University, Stony Brook, New York 11794-8661.
Scarlata
Suzanne
S
From the Department of Physiology and Biophysics, Stony Brook University, Stony Brook, New York 11794-8661. Electronic address: Suzanne.Scarlata@sunysb.edu.
D001665:Binding Sites; D002460:Cell Line; D031541:Fluorescence Resonance Energy Transfer; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D044387:GTP-Binding Protein beta Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D044388:GTP-Binding Protein gamma Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D046912:Multiprotein Complexes; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
C118981:G-protein Beta gamma; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D008164:Luminescent Proteins; D046912:Multiprotein Complexes; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go