From the
Heterotrimeric () G proteins act as molecular
switches to relay information from activated receptors to appropriate
effector proteins (e.g. adenylyl cyclase,
phosphatidylinositol-specific phospholipase C, cGMP phosphodiesterase,
and ion channels). In the inactive state, the G protein exists as a
heterotrimeric complex with GDP bound to the
subunit. Activated
receptors induce exchange of GTP for GDP on the
subunit and
dissociation of
from the
dimer. Both
GTP
and free
can interact with various effector molecules. The
subunit's intrinsic GTP hydrolytic activity converts it
back to a GDP-bound form and results in reassociation of
and
; in some cases this GTPase activity is enhanced by other
proteins.
Although receptor-catalyzed guanine nucleotide exchange
(``turn on'') and subunit GTP hydrolysis (``turn
off'') are the best studied modes of G protein regulation,
covalent modifications of heterotrimeric G proteins represent
additional levels of regulation. This minireview will address recent
advances in understanding how fatty acylation regulates the cellular
localization and function of G proteins. We will present these lipid
modifications within a general framework of all G proteins but also try
to highlight individual differences among G proteins. Other known
covalent modifications of G proteins, phosphorylation (1, 2) and bacterial toxin-catalyzed
ADP-ribosylation(3) , will not be reviewed.
Myristoylation and Palmitoylation of Subunits
All G protein subunits are modified at or near their N
termini by covalent attachment of the fatty acids myristate and/or
palmitate (Fig. 1).
Figure 1:
Sites of G protein lipid modification.
The N-terminal sequences of several G protein subunits and the
C-terminal sequences of two G protein
subunits are shown. Two
subunits of the
family are shown; others in
this family are
,
,
,
, and
. These
proteins contain myristate (M) linked through an amide bond to
an N-terminal glycine (after removal of the initiating methionine), as
indicated by the circled boldface G.
is not
myristoylated, probably because other amino acids, particularly the
asparagine at position 6, reduce the affinity of
for N-myristoyltransferase(7) . All
subunits (except
) contain palmitate (P) attached via a
thioester bond to cysteine residues near the N terminus, as indicated
by the boxed boldface C. The
subunits are prenylated
(
is farnesylated (F) and
is geranylgeranylated (GG)) through a thioether bond to
a cysteine, indicated by C. After prenylation, the C-terminal
three amino acids are removed (
), and the new C terminus is
carboxylmethylated.
Myristoylation, or more specifically N-myristoylation, is the result of co-translational addition
of the saturated 14-carbon fatty acid myristate to a glycine residue at
the extreme N terminus after removal of the initiating methionine. A
stable amide bond links myristate irreversibly to proteins.
subunits of the
family (
,
,
,
,
, and
) are myristoylated (Fig. 1). The
subunit of transducin (
) is
heterogeneously modified at its N terminus by myristate and three other
less hydrophobic fatty acids(4, 5) . This
heterogeneous acylation is apparently not dictated by a unique
structural feature of
but is instead specific to
retinal photoreceptor cells(6) ; the simplest explanation for
such tissue-specific differences in fatty acylation of N-terminal
glycines is that in different cells the fatty acyl-CoA pool contains
different fatty acids(6) .
All G protein subunits so
far examined (except
) contain palmitate (16-carbon,
saturated fatty acid) attached through a labile, reversible thioester
linkage to a cysteine near the N terminus (Fig. 1). In contrast
to myristoylation(7) , the biochemistry of palmitoylation is
not well understood.
Several subunits contain both myristate
and palmitate (Fig. 1). Although the presence of both fatty
acids on the same protein molecule has not been directly shown,
preventing myristoylation of
,
, or
by mutation of glycine to alanine also appears to
prevent palmitoylation of the
subunits(8, 9, 10) . A similar requirement of
prior myristoylation for palmitoylation has been observed for several
non-receptor tyrosine kinases(11, 12) . Thus, the
sequence M-G-C may represent a signal for dual acylation of certain G
protein
subunits(13) . In contrast,
does not require myristoylation for palmitoylation, although it
does have the sequence M-G-C, and other
subunits (e.g.
,
,
) are
palmitoylated on cysteine residues within diverse sequence contexts.
Furthermore, recent evidence indicates that prior myristoylation may
not be an absolute prerequisite for palmitoylation: a G2A mutant of
incorporates palmitate if G protein
subunits are co-expressed(14) . In addition, a G2A mutant of
or
is palmitoylated when
overexpressed in Chinese hamster ovary cells. (
)Taken
together, the available data suggest that myristoylation and/or binding
to
(or other unknown factors) directs the
subunits to
a membrane location where palmitoylation occurs (discussed below).
Although it is often assumed that subunits are uniquely
modified by palmitate on certain cysteine residues, different
subunits may contain other thioester-linked fatty acids in different
cells. In this regard, several
subunits in platelets can
incorporate thioester-linked arachidonate in addition to
palmitate(15) .
G protein subunits are covalently modified by the
20-carbon isoprenoid geranylgeranyl or, in the case of retinal-specific
, the 15-carbon isoprenoid farnesyl (Fig. 1).
As with other prenylated proteins, the geranylgeranyl or farnesyl
moiety is attached via a stable thioether bond to a cysteine residue
located in the C-terminal ``CAAX'' box of
.
This is followed by proteolytic removal of the C-terminal three amino
acids and then by carboxyl methylation at the new C terminus. The
enzymology and substrate requirements of prenylation have been well
reviewed recently (e.g.(16) ).
A recent study (17) addressed the temporal order of dimer formation
and processing of
. Farnesylation or geranylgeranylation of the
appropriate
is not required for
dimerization; however,
a proteolytically truncated isoprenylated
(lacking the C-terminal
three amino acids) was incapable of interacting with a
subunit,
indicating that assembly of an isoprenylated
dimer occurs
prior to proteolysis and carboxyl methylation of the
subunit (17) . Although mutant non-prenylated
can form a stable
dimer with
(reviewed in (18) ), prenylation of
is
necessary for normal function of the
dimer (discussed
below). To date no lipid modification has been identified on G protein
subunits.
Function of Lipid Modifications
For G proteins, as well as other lipid-modified proteins, attached lipids appear to direct interaction with both membrane lipids and other proteins. How can a covalently attached lipid perform such strikingly different roles? This is a conceptually difficult and unanswered question. It is instructive, therefore, to consider distinct, though not mutually exclusive, models to explain how attached lipids may affect both protein-membrane and protein-protein interactions (Fig. 2).
Figure 2: Models for lipid mediated protein-membrane and proteinprotein interactions. A, a lipid attached to a protein may insert directly into the membrane lipid bilayer. B, the attached lipid may bind to a hydrophobic pocket in another protein. C, the attached lipid may remain in the membrane lipid bilayer and specifically interact with another protein. D, the lipid may interact with the protein to which it is covalently attached and thereby stabilize a conformation that is competent to bind another protein.
Studies of subunits that normally contain palmitate but not
myristate provide further evidence for the importance of palmitoylation
in membrane attachment. Non-palmitoylated mutants of
,
, and
exhibited markedly decreased
abilities to associate with membranes(22, 23) .
The
mechanism by which lipid modification enhances the membrane association
of G protein subunits has not been determined. The simplest explanation
is that the fatty acids or prenyl groups insert directly into the
hydrophobic membrane lipids and thus anchor the protein at the
membrane. In this regard, in vitro measurements of the
affinity of acylated peptides for lipid vesicles correlate with
observations of mutant G proteins in cells. Myristate provides barely
enough energy to anchor a protein to membranes, and other factors, such
as positive charges and protein-protein interactions, would be required
to efficiently anchor myristoylated proteins. On the other hand,
palmitate has sufficient binding energy to stably anchor a protein to
membranes(24) . This is exactly what is seen with lipid
modified subunits; where examined, palmitoylated
subunits
have been found in the particulate (microsomal membrane) fraction of
cells, while myristoylated (but not palmitoylated)
subunits vary
in their degree of association with membranes. Direct insertion of the
lipid moieties into membranes remains the simplest model (Fig. 2A) to account for the effects of fatty acylation
on membrane attachment, although no compelling evidence rules out the
possibility that a membrane-bound ``docking protein''
specifically binds fatty acids linked to
subunits.
Other
factors, however, help guide subunits to membranes. The most
obvious example of such a protein is the G protein
dimer(25) , which may even serve as a membrane-bound docking
protein. Co-expression of
with
led to more
membrane-bound and functional
than if
was expressed
alone(26) . A putative palmitoyltransferase is another example
of a protein that may direct
subunits to membranes; that is,
non-palmitoylated
subunits would bind a membrane-bound
palmitoyltransferase, become palmitoylated, and then would be capable
of directly binding to membranes. Thus, binding to other proteins
probably contributes to the appropriate cellular localization of the G
protein
subunits.
Like fatty acylation of subunits,
prenylation of
chains is required for correct membrane targeting
of the
dimer. Expression of mutant non-prenylated
with
in cultured cells produced
dimers that were located in
the cytosolic rather than in the membrane fraction(18) .
Again, peptide studies are relevant in considering mechanisms of
membrane association. For attachment of a peptide to lipid
vesicles in vitro, a farnesyl group provides binding energy
quantitatively similar to that of myristate(27) . To supplement
this relatively low binding energy, the farnesyl group may specifically
interact with an integral membrane protein, as postulated for diverse
prenylated proteins(28) . Indeed, the
subunits may play
such a role, since prenylation of
is required for
binding to
. On the other hand, the more hydrophobic
geranylgeranyl group is sufficient by itself to stably anchor a protein
to membranes(27) . In agreement, farnesylated
dimers, but not geranylgeranylated
dimers, are soluble in the absence of detergents. A high
affinity, heat- and protease-sensitive binding site has been identified
for prenylated peptides in a microsomal membrane
preparation(29) . This binding activity was not found in the
plasma membrane; it may play a role in targeting prenylated proteins to
a membrane compartment where C-terminal proteolysis and carboxyl
methylation occur.
The presence of a methyl group at the C terminus
of is important for membrane attachment of farnesylated retinal
(30) . Similarly, carboxyl
methylation increased the affinity of a farnesylated peptide for lipid
vesicles more than 10-fold(27) . Thus, carboxyl methylation
also contributes to
membrane association, probably by
neutralizing the negatively charged C terminus.
In summary, membrane
association of G protein subunits is a complex process involving
multiple interactions. A combination of covalent lipid modifications
and specific protein-protein interactions is essential for
membrane-attached and functional G proteins. The diverse combinations
of lipid modifications of subunits (Fig. 1) may account
for different subcellular localizations of different
subunits. G
proteins have recently been detected in plasma membrane domains termed
caveolae(31, 32, 33) . Palmitoylation is
required for the caveolar localization of certain members of the Src
family of tyrosine kinases(11) (
);
subunits
may similarly require palmitate for localization to caveolae. In
addition,
subunits are not located only in the plasma membrane (2) but have been detected at other intracellular membrane
sites, including the Golgi apparatus and endoplasmic reticulum. We can
speculate that different attached lipids direct G proteins to unique
cellular membranes.
Recent studies
suggest that palmitoylation is less important than myristoylation for
subunit interaction with
, although the relative
affinity of non-palmitoylated
versus wild type
for
has not been rigorously addressed. A myristoylated but
non-palmitoylated C3S
mutant was a target for
-dependent pertussis toxin-catalyzed ADP-ribosylation in
intact cells(21) , and a myristoylated but non-palmitoylated
C3A
mutant was a better substrate for
-dependent ADP-ribosylation than a non-myristoylated and
non-palmitoylated G2A mutant
in a cell-free
assay(14) .
Besides binding to subunits, lipid
modifications of
may affect its interaction with other proteins (Table 1). One of the best examples is the demonstration that
myristoylation of
is required for its inhibition of
adenylyl cyclase in a cell-free assay (34) . As in the case of
binding to
, the mechanism by which myristate confers
upon purified
the ability to productively interact
with adenylyl cyclase has not been determined.
Mutation of
subunit lipid modification sites and analysis of activity in
transfected cells has provided further evidence that fatty acylation is
important for biological activity. A G2A mutation of a constitutively
active form of
abolished its biological
activity(35) . Since the G2A mutation probably blocked
palmitoylation in addition to myristoylation, the contribution of each
attached fatty acid is not clear. In contrast, non-myristoylated G2A
retains the ability to inhibit adenylyl cyclase in
transfected cells.
In another study(22) , a
non-palmitoylated C9S,C10S
completely lacked the
ability to stimulate its effector, phospholipase C. Whether these
effects are due to a requirement for the attached fatty acid in
protein-protein interactions or are secondary to decreased membrane
association of
subunits remains unknown. Direct reconstitution of
purified palmitoylated versus non-palmitoylated
subunits
will help resolve this issue. Such studies are hampered, however, by
the difficulty of purifying stoichiometrically palmitoylated
subunits because of the lability of this covalent modification.
Although not required for dimer formation, prenylation is
absolutely necessary for productive interactions of
with
subunits, receptors, and effectors (16, 18) (Table 1). In addition, carboxyl
methylation of farnesylated
also
enhances its ability to interact with
and
rhodopsin(30, 36) . Prenylation clearly provides more
than merely a membrane anchor (Table 1). For the G protein
subunit, as with other proteins like the Ras superfamily of
guanine nucleotide-binding proteins(28) , prenylation is
indispensable for many protein-protein interactions.
An important aspect of palmitoylation is its biological
reversibility and consequent potential for regulation. Indeed, the
turnover of palmitate attached to is dramatically
affected by
activation. Activation of
in COS cells by
-adrenergic receptor stimulation (9, 37) or directly with cholera toxin (37) led to an increase in its palmitate labeling. The
increased labeling suggested a faster turnover of palmitate attached to
activated (GTP-bound)
, and indeed,
-adrenergic
receptor activation caused a slightly more rapid depalmitoylation of
in a pulse-chase experiment(9) . This was
confirmed by analyzing the palmitate turnover of
in
S49 cells. In these cells, palmitate attached to
exhibited a half-life of 90 min, whereas activation of the
-adrenergic receptor caused the attached palmitate to turn over
very rapidly (t
2 min)(38) ; in COS cells,
palmitate on a similarly activated
turned over with a
half-life of
30 min(9) . Palmitate attached to a
mutationally activated
in S49 cells also turns over
with a rapidity similar to that of receptor-activated
(38) . This activation-induced rapid
depalmitoylation of
correlates with and is probably
the mechanism for activation-induced translocations of
from membranes to cytosol observed previously (39, 40) (Fig. 3). Moreover, a mutant
non-palmitoylated
could not mediate hormonal
stimulation of its effector, adenylyl cyclase(22) . This raises
the possibility that depalmitoylation of
provides a
physiologically relevant way to damp or turn off G protein signals, in
addition to better established mechanisms (hydrolysis of GTP bound to
, desensitization of receptors, etc.).
Figure 3:
Model of
depalmitoylation and membrane release. In the unactivated state,
GDP (square) associates with
and the plasma membrane. Receptor activation (R*) stimulates
dissociation of GDP from
and formation of active
GTP (diamond).
GTP and
dissociate from each other
but remain at the plasma membrane by virtue of their attached palmitate
and isoprenyl groups, respectively. Palmitate is rapidly cleaved from
activated
GTP by a palmitoyl thioesterase,
however, and
is released from the membrane. Intrinsic
GTP hydrolysis converts both membrane and cytoplasmic
GTP into the inactive GDP-bound form.
Palmitoylation by a palmitoyltransferase facilitates the return of
GDP to the plasma membrane. Although the model
depicts palmitoylation of
as preceding its
association with
, the temporal order of these two events is
unknown.
Thus, regulated
palmitoylation of (and possibly other
subunits)
can control its cellular location and activity (Fig. 3). One
future challenge will be to explore how different cellular locations of
subunits affect their activities. A second major challenge will
be that of explaining the molecular basis of these cycles of
palmitoylation and depalmitoylation (Fig. 3). Little is known
about the enzymes involved. Palmitoyltransferase activity has only been
detected in crude extracts. A palmitoyl thioesterase capable of
removing palmitate from H-ras or G protein
subunits has been
purified to homogeneity (41) but is primarily secreted from
cells (42) and thus unlikely to be responsible for
depalmitoylating G protein
subunits in vivo.
Although
myristoylation is assumed to be a stable, irreversible modification,
two recent reports suggest the possibility of regulated myristoylation.
First, Gpa1, a yeast G protein subunit that functions in the
pheromone response pathway, exists in both a myristoylated and
non-myristoylated form in yeast cells. Pheromone activation leads to an
increase in the fraction of newly synthesized Gpa1 that is
myristoylated(43) . Second, a demyristoylase activity capable
of removing myristate from the protein kinase C substrate MARCKS has
been described(44) .
G protein subunits and
dimers are covalently
modified by lipids. The emerging picture is one in which attached
lipids provide more than just a nonspecific ``glue'' for
sticking G proteins to membranes. We are only beginning to understand
how different lipid modifications of different G protein subunits
affect specific protein-protein interactions and localization to
specific cellular sites. In addition, regulation of these
modifications, particularly palmitoylation, can provide new ways to
regulate signals transmitted by G proteins.