Lipid Modifications of Trimeric G Proteins (*)
- From the (1)Departments of Pharmacology,
- (2)Medicine, and
- (3)Psychiatry, the
- (4)Center for Neurobiology and Psychiatry, and the Cardiovascular Research Institute, University of California, San Francisco, California 94143
- § To whom correspondence should be addressed: S-1212, Box 0450, UC Medical Center, San Francisco, CA 94143. Tel.: 415-476-8161; Fax: 415-476-5292.
INTRODUCTION
Heterotrimeric (αβ
) 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 α subunit. Activated receptors induce exchange
of GTP for GDP on the α subunit and dissociation of α from the β
dimer. Both α•GTP and free β
can interact with various effector molecules. The α subunit's intrinsic GTP hydrolytic activity converts it back to a GDP-bound
form and results in reassociation of α and β
; in some cases this GTPase activity is enhanced by other proteins.
Although receptor-catalyzed guanine nucleotide exchange (“turn on”) and α 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 α Subunits
All G protein α subunits are modified at or near their N termini by covalent attachment of the fatty acids myristate and/or palmitate (Fig. 1).
Sites of G protein lipid modification. The N-terminal sequences of several G protein α subunits and the C-terminal sequences
of two G protein 
subunits are shown. Two α subunits of the αi family are shown; others in this family are α
, α
, α
, α
, and αz. 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. αs is not myristoylated, probably because other amino acids, particularly the asparagine at position 6, reduce the affinity
of αs for N-myristoyltransferase(7). All α subunits (except αt) 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 (
1 is farnesylated (F) and 
2 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. α subunits of the αi family (α
, α
, α
, αo, αz, and αt) are myristoylated (Fig. 1). The α subunit of transducin (αt) 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 αt 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 α subunits so far examined (except αt) 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 α 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 αi, αo, or αz by mutation of glycine to alanine also appears to prevent palmitoylation of the α 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 α subunits(13). In contrast, αs does not require myristoylation for palmitoylation, although it does have the sequence M-G-C, and other α subunits (e.g. αq, α
, α
) 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 α
incorporates palmitate if G protein β
subunits are co-expressed(14). In addition, a G2A mutant of αz or α
is palmitoylated when overexpressed in Chinese hamster ovary cells. (1) Taken together, the available data suggest that myristoylation and/or binding to β
(or other unknown factors) directs the α subunits to a membrane location where palmitoylation occurs (discussed below).
Although it is often assumed that α subunits are uniquely modified by palmitate on certain cysteine residues, different α subunits may contain other thioester-linked fatty acids in different cells. In this regard, several α subunits in platelets can incorporate thioester-linked arachidonate in addition to palmitate(15).
Prenylation of 
Subunits
G protein 
subunits are covalently modified by the 20-carbon isoprenoid geranylgeranyl or, in the case of retinal-specific 
1, 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 β
dimer formation and processing of 
. Farnesylation or geranylgeranylation of the appropriate 
is not required for β
dimerization; however, a proteolytically truncated isoprenylated 
(lacking the C-terminal three amino acids) was incapable of interacting with a β subunit, indicating that assembly of an
isoprenylated β
dimer occurs prior to proteolysis and carboxyl methylation of the 
subunit (17). Although mutant non-prenylated 
can form a stable dimer with β (reviewed in (18)), prenylation of 
is necessary for normal function of the β
dimer (discussed below). To date no lipid modification has been identified on G protein β 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).
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.
Lipid Modifications Affect Membrane Attachment of G Proteins
An obvious function for fatty acylation is to act as a hydrophobic membrane anchor. Recent data suggest that both palmitoylation
and myristoylation contribute to membrane association, with palmitoylation, due to its greater hydrophobicity, providing a
stronger association with membrane lipids. Mutagenesis studies indicated that myristoylation was required for membrane localization
of αi and αo(19, 20); however, those studies predated the realization that αi and αo are also palmitoylated. More recently, expression of myristoylated but not palmitoylated C3A αo(9), C3S α
(8), or C3S αo(21) resulted in significant increases (compared with wild type) in the amounts of each protein in the soluble (or cytosolic)
fraction. αt, probably the only α subunit that does not contain palmitate (Fig. 1), is unique in its ability to be released from membranes in the absence of detergents.
Studies of α subunits that normally contain palmitate but not myristate provide further evidence for the importance of palmitoylation
in membrane attachment. Non-palmitoylated mutants of αs, αq, and α
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 α subunits; where examined, palmitoylated α subunits have been found in the particulate (microsomal membrane) fraction of cells, while myristoylated (but not palmitoylated) α 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 α subunits.
Other factors, however, help guide α subunits to membranes. The most obvious example of such a protein is the G protein β
dimer(25), which may even serve as a membrane-bound docking protein. Co-expression of β
with α led to more membrane-bound and functional α than if α was expressed alone(26). A putative palmitoyltransferase is another example of a protein that may direct α subunits to membranes; that is, non-palmitoylated
α 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
α subunits.
Like fatty acylation of α subunits, prenylation of 
chains is required for correct membrane targeting of the β
dimer. Expression of mutant non-prenylated 
with β in cultured cells produced β
dimers that were located in the cytosolic rather than in the membrane fraction(18).
Again, peptide studies are relevant in considering mechanisms of β
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 α subunits may play such a role, since prenylation of 
is required for β
binding to α. On the other hand, the more hydrophobic geranylgeranyl group is sufficient by itself to stably anchor a protein
to membranes(27). In agreement, farnesylated β1
1 dimers, but not geranylgeranylated β
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 β1
1(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 β
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 α subunits (Fig. 1) may account for different subcellular localizations of different α 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) (2); α subunits may similarly require palmitate for localization to caveolae. In addition, α 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.
Lipid Modifications and G Protein Function
Besides functioning as membrane anchors, what role do attached myristate and palmitate play in the biological activities of
α subunits and their interactions with other proteins? Table 1 summarizes strong evidence that myristoylation greatly increases the affinity of the αi family of subunits for β
. Crystal structures of myristoylated α bound to β
will show whether the attached myristate interacts directly with β
or stabilizes a conformation of α competent to interact with β
(Fig. 2BversusFig. 2D). In either case, the fact that myristate linked to α is required for its interaction with β
in membrane-free systems (Table 1) raises the possibility, noted above, that β
serves as a key docking protein that accounts, at least partially, for the binding of myristoylated α to membranes.
Recent studies suggest that palmitoylation is less important than myristoylation for α subunit interaction with β
, although the relative affinity of non-palmitoylated α versus wild type α for β
has not been rigorously addressed. A myristoylated but non-palmitoylated C3S αo mutant was a target for β
-dependent pertussis toxin-catalyzed ADP-ribosylation in intact cells(21), and a myristoylated but non-palmitoylated C3A α
mutant was a better substrate for β
-dependent ADP-ribosylation than a non-myristoylated and non-palmitoylated G2A mutant α
in a cell-free assay(14).
Besides binding to β
subunits, lipid modifications of α may affect its interaction with other proteins (Table 1). One of the best examples is the demonstration that myristoylation of αi is required for its inhibition of adenylyl cyclase in a cell-free assay (34). As in the case of α binding to β
, the mechanism by which myristate confers upon purified αi the ability to productively interact with adenylyl cyclase has not been determined.
Mutation of α 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 α
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 αz retains the ability to inhibit adenylyl cyclase in transfected cells.1 In another study(22), a non-palmitoylated C9S,C10S αq 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 α subunits
remains unknown. Direct reconstitution of purified palmitoylated versus non-palmitoylated α subunits will help resolve this issue. Such studies are hampered, however, by the difficulty of purifying
stoichiometrically palmitoylated α subunits because of the lability of this covalent modification.
Although not required for β
dimer formation, prenylation is absolutely necessary for productive interactions of β
with α subunits, receptors, and effectors (16, 18) (Table 1). In addition, carboxyl methylation of farnesylated β1
1 also enhances its ability to interact with αt and rhodopsin(30, 36). Prenylation clearly provides more than merely a membrane anchor (Table 1). For the G protein β
subunit, as with other proteins like the Ras superfamily of guanine nucleotide-binding proteins(28), prenylation is indispensable for many protein-protein interactions.
Dynamic Palmitoylation
An important aspect of palmitoylation is its biological reversibility and consequent potential for regulation. Indeed, the
turnover of palmitate attached to αs is dramatically affected by αs activation. Activation of αs in COS cells by β-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) αs, and indeed, β-adrenergic receptor activation caused a slightly more rapid depalmitoylation of αs in a pulse-chase experiment(9). This was confirmed by analyzing the palmitate turnover of αs in S49 cells. In these cells, palmitate attached to αs exhibited a half-life of 90 min, whereas activation of the β-adrenergic receptor caused the attached palmitate to turn over
very rapidly (t ≈ 2 min)(38); in COS cells, palmitate on a similarly activated αs turned over with a half-life of 
30 min(9). Palmitate attached to a mutationally activated αs in S49 cells also turns over with a rapidity similar to that of receptor-activated αs(38). This activation-induced rapid depalmitoylation of αs correlates with and is probably the mechanism for activation-induced translocations of αs from membranes to cytosol observed previously (39, 40) (Fig. 3). Moreover, a mutant non-palmitoylated αs could not mediate hormonal stimulation of its effector, adenylyl cyclase(22). This raises the possibility that depalmitoylation of αs provides a physiologically relevant way to damp or turn off G protein signals, in addition to better established mechanisms
(hydrolysis of GTP bound to α, desensitization of receptors, etc.).
Model of αs depalmitoylation and membrane release. In the unactivated state, αs•GDP (square) associates with β
and the plasma membrane. Receptor activation (R*) stimulates dissociation of GDP from αs and formation of active αs•GTP (diamond). αs•GTP and β
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 αs•GTP by a palmitoyl thioesterase, however, and αs is released from the membrane. Intrinsic GTP hydrolysis converts both membrane and cytoplasmic αs•GTP into the inactive GDP-bound form. Palmitoylation by a palmitoyltransferase facilitates the return of αs•GDP to the plasma membrane. Although the model depicts palmitoylation of αs as preceding its association with β
, the temporal order of these two events is unknown.
Thus, regulated palmitoylation of αs (and possibly other α subunits) can control its cellular location and activity (Fig. 3). One future challenge will be to explore how different cellular locations of α 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 α subunits has been purified to homogeneity (41) but is primarily secreted from cells (42) and thus unlikely to be responsible for depalmitoylating G protein α 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 α 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).
Summary
G protein α subunits and β
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.
Footnotes
-
↵* This minireview will be reprinted in the 1995 Minireview Compendium, which will be available in December, 1995. This work was supported by American Cancer Society Fellowship PF-3776 (to P. B. W.), grants from NARSAD and NIMH (to P. T. W.), NIH Grants GM-27800 and CA-54427 (to H. R. B.), and a grant from the March of Dimes (to H. R. B.).
-
↵1P. T. Wilson, manuscript in preparation.
-
↵2S. Robbins, N. Quintrell, and J. M. Bishop, personal communication.
- © 1995 by The American Society for Biochemistry and Molecular Biology, Inc.
