Abstract
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.
Highlights
The G protein signaling system is initiated when an extracellular agonist binds to its specific G protein-coupled receptor
If G protein subunits do not dissociate in cells, their interaction must change in such a manner as to expose the effector interaction site(s)
We have found that phospholipase C1 (PLC1),4 an important effector of G␣q [23], is bound to G␣q prior to activation and throughout the activation cycle [6] implying that G␣q(GDP) interacts with PLC1 in a non-functional manner
Summary
The G protein signaling system is initiated when an extracellular agonist binds to its specific G protein-coupled receptor (for review see Refs. 9 –12). The ligand-bound receptor will catalyze the exchange of GTP for GDP on the G␣ subunit in the G protein heterotrimer. G␣(GDP) binds strongly to G␥, but in the GTP-bound state this affinity. The interactions between G protein subunits have been studied extensively in vitro, their behavior in cells may differ. If G protein subunits do not dissociate in cells, their interaction must change in such a manner as to expose the effector interaction site(s). To understand the nature of these secondary contacts, we have studied the ability of the G␣i1(GDP)-G␥ heterotrimer to remain complexed through the activation cycle [24]. We present evidence that G␣i1(GDP) has two distinct G␥ binding sites that only differ in affinity by an order of magnitude and may allow for continued association between the subunits upon activation. We find that this site plays an important role in stabilizing G protein associations in cells and provides a mechanism of self-scaffolding
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
