Functional Importance of the Amino Terminus of Gqα

Abstract

Gq alpha is palmitoylated at residues Cys9 and Cys10. Removal of palmitate from purified Gq alpha with palmitoylthioesterase in vitro failed to alter interactions of Gq alpha with phospholipase C-beta 1, the G protein beta gamma subunit complex, or m1 muscarinic cholinergic receptors. Mutants C9A, C10A, C9A/C10A, C9S/C10S, and truncated Gq alpha (removal of residues 1-6) were synthesized in Sf9 cells and purified. Loss of both Cys residues or truncation prevented palmitoylation of Gq alpha. However, truncated Gq alpha and the single Cys mutants activated phospholipase C-beta 1 normally, while the double Cys mutants were poor activators. Loss of both Cys residues impaired but did not abolish interaction of Gq alpha with m1 receptors. These Cys residues are thus important regardless of their state of palmitoylation. When expressed in HEK-293 or Sf9 cells, all of the proteins studied associated entirely or predominantly with membranes, although a minor fraction of nonpalmitoylated Gq alpha proteins accumulated in the cytosol of HEK-293 cells. When subjected to TX-114 phase partitioning, a significant fraction of all of the proteins, including those with no palmitate, was found in the detergent-rich phase. Removal of residues 1-34 of Gq alpha caused a loss of surface hydrophobicity as evidenced by complete partitioning into the aqueous phase. The Cys residues at the amino terminus of Gq alpha are thus important for its interactions with effector and receptor, and the amino terminus conveys a hydrophobic character to the protein distinct from that contributed by palmitate.

The N-terminal Extension of Gαq Is Critical for Constraining the Selectivity of Receptor Coupling

“…Similarly, other functional properties, such as their affinity for ␤␥ subunits and their ability to activate downstream effectors (e.g. phospholipase C␤ (PLC␤)), were also found to be very similar for the two G protein subunits (16,17).…”

“…Similarly, other functional properties, such as their affinity for ␤␥ subunits and their ability to activate downstream effectors (e.g. phospholipase C␤ (PLC␤)), were also found to be very similar for the two G protein subunits (16,17).In this study, we tested the hypothesis that the N-terminal extension in WTq may play a role in maintaining the selectivity of receptor recognition, an issue that had not been addressed yet. Toward this goal, the ability of several different G i/o -and G s -coupled receptors to interact with WTq or Ϫ6q were examined in cotransfected COS-7 cells.…”

“…1). This short sequence is highly conserved among all vertebrate species from which these subunits have been cloned so far (11)(12)(13)(14)(15), suggesting that it may be relevant for some aspect of G␣ q /G␣ 11 function.Previous studies (16,17) analyzing the biochemical properties of a mutant G␣ q subunit lacking the N-terminal extension (hereafter referred to as Ϫ6q) failed to reveal any major functional differences between wild type G␣ q (WTq) and Ϫ6q. Both studies showed that G q/11 -coupled receptors such as the NK2 neurokinin (16) and the m1 muscarinic receptor (17) were able to activate Ϫ6q in a fashion identical to WTq.…”

“…Previous studies (16,17) analyzing the biochemical properties of a mutant G␣ q subunit lacking the N-terminal extension (hereafter referred to as Ϫ6q) failed to reveal any major functional differences between wild type G␣ q (WTq) and Ϫ6q. Both studies showed that G q/11 -coupled receptors such as the NK2 neurokinin (16) and the m1 muscarinic receptor (17) were able to activate Ϫ6q in a fashion identical to WTq.…”

“…Both studies showed that G q/11 -coupled receptors such as the NK2 neurokinin (16) and the m1 muscarinic receptor (17) were able to activate Ϫ6q in a fashion identical to WTq. Similarly, other functional properties, such as their affinity for ␤␥ subunits and their ability to activate downstream effectors (e.g.…”