Analysis of the N-terminal binding domain of Goα

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Abstract
Signalling from membrane receptors through heterotrimeric G-proteins (Gα and Gβγ) to intracellular effectors is a highly regulated process. Receptor activation causes exchange of GTP for GDP on Gα and dissociation of Gα from Gβγ. Both subunits remain membrane-associated and interact with a series of other molecules throughout the cycle of activation. The N-terminal binding domain of Gα subunits interacts with the membrane by several partially defined mechanisms: the anchoring of Gα to the more hydrophobic Gβγ subunits, the interaction of N-terminal lipids (palmitate and/or myristate) with the membrane, and attachment of amino acid regions to the membrane {amino acids 11-14 of Goα (D[11-14]); Busconi, Boutin and Denker (1997) Biochem. J. 323, 239-244}. We characterized N-terminal mutants of Goα with known Gβγ-binding properties for the ability to interact with phospholipid vesicles and membranes prepared from cultured cells (acceptor membranes). In vitro analysis allows membrane interactions that are important to the activated and depalmitoylated state of Gα to be characterized. Subcellular localization was also determined in transiently transfected COS cells. All of the mutant proteins are myristoylated, and differences in myristoylation do not account for changes in membrane binding. Disrupting the N-terminal α-helix of Goα with a proline point mutation at Arg-9 (R9P) does not affect interactions with Gβγ on sucrose-density gradients but significantly reduces acceptor membrane binding. Deletion of amino acids 6-15 (D[6-15]; reduced Gβγ binding) or deletion of amino acids 3-21 (D[3-21]); no detectable Gβγ binding) further reduces acceptor membrane binding. When expressed in COS cells, R9P and D[6-15] are localized in the membrane similar to wild-type Goα as a result of the contribution from palmitoylation. In contrast, D[3-21] is completely soluble in COS cells, and no palmitoylation is detected. The binding of Goα and mutants translated in vitro to liposomes indicates that Goα preferentially binds to neutral phospholipids (phosphatidylcholine). R9P and D[11-14] bind to phosphatidylcholine liposomes like Goα, but D[6-15] exhibits no detectable binding. Taken together, these studies suggest that interactions of the N-terminus of Gα subunits with the membrane may be affected by both membrane proteins and lipids. A detailed understanding of Gα-membrane interactions may reveal unique mechanisms for regulating signal transduction.