L. Busconi et al., N-TERMINAL BINDING DOMAIN OF G-ALPHA SUBUNITS - INVOLVEMENT OF AMINO-ACIDS-11-14 OF G-ALPHA(O) IN MEMBRANE ATTACHMENT, Biochemical journal, 323, 1997, pp. 239-244
Heterotrimeric guanine nucleotide binding proteins (G-proteins) transm
it signals from membrane receptors to a variety of intracellular effec
ters. G-proteins reversibly associate with components of the signal tr
ansduction system, yet remain membrane attached throughout the cycle o
f activation. The G alpha subunits remain attached to the plasma membr
ane through a combination of factors that are only partially defined.
We now demonstrate that amino acids within the N-terminal domain of G
alpha subunits are involved in membrane binding. We used in vitro tran
slation, a technique widely utilized to characterize functional aspect
s of G-proteins, and interactions with donor-acceptor membranes to dem
onstrate that amino acids 11-14 of G alpha(o) contribute to membrane b
inding. The membrane binding of G alpha(o) lacking amino acids 11-14(D
[11-14]) was significantly reduced at all membrane concentrations in c
omparison with wild-type G alpha(o). Several other N-terminal mutants
of G alpha(o) were characterized as controls, and these results indica
te that differences in myristoylation, palmitoylation and beta gamma i
nteractions do not account for the reduced membrane binding of D[11-14
]. Furthermore, when membrane attachment of G alpha(o) and mutants was
characterized in transiently transfected S-35-labelled and [H-3]myris
tate-labelled COS cells, amino acids 11-14 contributed to membrane bin
ding. These studies reveal that membrane binding of G alpha subunits o
ccurs by a combination of factors that include lipids and amino acid s
equences. These regions may provide novel sites for interaction with m
embrane components and allow additional modulation of signal transduct
ion.