Signalling from membrane receptors through heterotrimeric G-proteins (
G alpha and G beta gamma) to intracellular effecters is a highly regul
ated process. Receptor activation causes exchange of GTP for GDP on G
alpha and dissociation of G alpha from G beta gamma. Both subunits rem
ain membrane-associated and interact with a series of other molecules
throughout the cycle of activation. The N-terminal binding domain of G
alpha subunits interacts with the membrane by several partially defin
ed mechanisms: the anchoring of G alpha to the more hydrophobic G beta
gamma subunits, the interaction of N-terminal lipids (palmitate and/o
r myristate) with the membrane, and attachment of amino acid regions t
o the membrane {amino acids 11-14 of G(o)a (D[11-14]); Busconi, Boutin
and Denker (1997) Biochem, J. 323, 239-244}. We characterized N-termi
nal mutants of G(o) alpha with known G beta gamma-binding properties f
or the ability to interact with phospholipid vesicles and membranes pr
epared from cultured cells (acceptor membranes). In vitro analysis all
ows membrane interactions that are important to the activated and depa
lmitoylated state of G alpha to be characterized. Subcellular localiza
tion was also determined in transiently transfected COS cells. All of
the mutant proteins are myristoylated, and differences in myristoylati
on do not account for changes in membrane binding. Disrupting the N-te
rminal alpha-helix of G(o) alpha with a proline point mutation at Arg-
9 (R9P) does not affect interactions with G beta gamma on sucrose-dens
ity gradients but significantly reduces acceptor membrane binding. Del
etion of amino acids 6-15 (D[6-15]; reduced G beta gamma binding) or d
eletion of amino acids 3-21 (D[3-21]); no detectable G beta gamma bind
ing) further reduces acceptor membrane binding. When expressed in COS
cells, R9P and D[6-15] are localized in the membrane similar to wild-t
ype G(o) alpha as a result of the contribution from palmitoylation. In
contrast, D[3-21] is completely soluble in COS cells, and no palmitoy
lation is detected. The binding of G(o) alpha and mutants translated i
n vitro to liposomes indicates that Go alpha preferentially binds to n
eutral phospholipids (phosphatidylcholine). R9P and D[11-14] bind to p
hosphatidylcholine liposomes like G(o) alpha, but D[6-15] exhibits no
detectable binding. Taken together, these studies suggest that interac
tions of the N-terminus of G alpha subunits with the membrane may be a
ffected by both membrane proteins and lipids. A detailed understanding
of G alpha-membrane interactions may reveal unique mechanisms for reg
ulating signal transduction.