(Received for publication, August 24, 1995)
From the
Post-translational prenylation of the carboxyl-terminal cysteine
is a characteristic feature of the guanine nucleotide-binding protein
(G protein) subunits. Recent findings show that the farnesylated
COOH-terminal tail of the
1 subunit is a specific determinant of
rhodopsin-transducin coupling. We show here that when synthetic
peptides specific to the COOH-terminal tail of
1 are chemically
modified with geranyl, farnesyl, or geranylgeranyl groups and tested
for their ability to interact with light activated rhodopsin, the
farnesylated peptide is significantly more effective. These results
show that an appropriate isoprenoid on the G protein
subunit
serves not only a membrane anchoring function but in combination with
the COOH-terminal domain specifies receptor-G protein coupling.
Heterotrimeric () G proteins (
)control
key signaling pathways inside a cell by relaying information from
activated transmembrane receptors to specific effector
molecules(1, 2, 3) . All known G protein
subunits are modified by either a 15-carbon farnesyl or by a
20-carbon geranylgeranyl moiety(4, 5) . The
1
subunit, which is associated with the G protein Gt, is farnesylated,
whereas most other
subunits are geranylgeranylated. Prenylation
occurs on the cysteine in the COOH-terminal CAAX motif (C is a
cysteine, A is an aliphatic amino acid, X is any
amino acid), and the nature of the last residue is thought to determine
farnesylation or geranylgeranylation.
Prenylation of the
subunits has been shown to be necessary for G protein membrane
attachment(6, 7, 8) . There is also evidence
that suggests the involvement of the prenyl group in protein-protein
interactions(9, 10, 11, 12, 13, 14) .
However, it is unclear why G protein
subunits are modified by two
different types of isoprenoids. Studies of some prenylated proteins
(rhodopsin kinase(10) , p21(11) , yeast
a-factor(13) ) demonstrate that the biological consequences of
differential prenylation may be more complex than anticipated. It has
also been difficult to examine the role of the different isoprenoids
because recombinant proteins with altered CAAX domains seem to
be prenylated with a mixture of farnesyl and
geranylgeranyl(13, 15) . To circumvent this problem,
we have used peptides chemically modified with various isoprenoids,
since we could confirm appropriate prenylation accurately in each case
by mass spectrometry.
To identify the role of the isoprenoid, we
have examined peptides chemically modified with farnesyl, geranyl
(C-10), and geranylgeranyl, for their relative efficacies at
stabilizing light activated rhodopsin. We have recently shown that the
farnesylated COOH-terminal domain of the 1 subunit directly
interacts with an activated receptor (light-activated rhodopsin) and is
important for the holomeric G protein to effectively couple with
rhodopsin(12, 16) . Both the farnesyl group and the
appropriate primary structure of the COOH-terminal domain of
1 are
essential for effective rhodopsin-Gt interaction. By assaying
farnesylated peptides of varying lengths we have identified here the
portion of the
1 COOH-terminal region that is necessary for
optimal interaction with the receptor. More importantly, we show that a
receptor can distinguish between the
subunit domain modified with
different prenyl groups: farnesyl, geranyl, and geranylgeranyl.
The assay used in these studies was based on the quantitative
analysis of spectrally different intermediates of rhodopsin,
metarhodopsin I (MI), and MII. Absorption of light by dark-adapted
rhodopsin triggers the formation of a relatively stable equilibrium of
two major rhodopsin intermediates, MI (A
= 495 nm) and MII (A
= 380
nm)(18) . Under the experimental conditions used here (4
°C, pH 8.0), Gt stabilizes MII and significantly shifts the ratio
of MII/MI toward MII formation(19, 20) . Synthetic
peptides specific to different domains on Gt, the COOH-terminal regions
of
t (21) and
1(12) , mimic the heterotrimer
by stabilizing MII in a dose-dependent fashion.
To define the
optimal length of the COOH-terminal domain of 1 required for
interaction and stabilization of MII, we synthesized a set of eight
peptides of various lengths (5 amino acid long to 22 amino acid long,
all of them ending with the cysteine, Fig. 1a). These
peptides were chemically farnesylated, FPLC-purified, and assayed for
their ability to stabilize MII. Increasing the length of the peptides
increased their potency, with peptides of 12-14 residues
possessing maximal activity (Fig. 1b). Extending the
amino acid sequence at the NH
terminus farther than 14
amino acids did not produce any significant change in peptide-MII
interaction. These results showed that the COOH-terminal 14 residues
contain all the information for appropriate recognition of the peptide
by rhodopsin. These results also confirmed our original observation
that the farnesylated COOH-terminal 12 amino acids of
1 represent
a distinct domain critical for rhodopsin-Gt interaction. To examine the
role of the isoprenoid attached to this domain, the 12-amino acid-long
peptide,
1(D60-C71), was modified with different isoprenoids.
Figure 1:
Effect of farnesylated peptides of
various lengths on MII stabilization. a, amino acid sequence
of different peptides specific to the 1 subunit. All peptides have
been farnesylated chemically. b, stabilization of MII by the
peptides of indicated length. The numbers in the inset indicate length in amino acids. The data points are the means
± S.E. of three independent
experiments.
The COOH-terminal cysteine in the peptide was modified chemically
with geranyl (C-10), farnesyl (C-15), and geranylgeranyl (C-20)
moieties (1(D60-C71)g,
1(D60-C71)far, and
1(D60-C71)gg) (Fig. 2a). Chromatographic behavior of the three
peptides on a reverse phase FPLC column was as anticipated, based on
their predicted relative hydrophobicities (Fig. 2b). It
was notable, however, that the solubility of all three peptides in
water was the same as observed empirically and as determined by the
quantitative amino acid analysis of the working solutions of these
peptides in comparison with stock solutions.
Figure 2:
Peptides modified with different
isoprenoids. a, a diagrammatic representation of geranylated,
farnesylated, and geranylgeranylated peptides derived from the COOH
terminus of 1. Expected molecular weight (M.W.) of the
peptides and their molecular weight as estimated by electro spray mass
spectrometry (see ``Materials and Methods'') are shown. b, chromatographic traces of peptides separated by reverse
phase FPLC. Peak 1,
1(D60-C71)g; peak 2,
1(D60-C71)far; and peak 3,
1(D60-C71)gg. The dotted line shows the gradient of acetonitrile.
Peptides modified with
different isoprenoids were then compared in the MII stabilization
assay. All three peptides were able to stabilize MII. The relative
efficacies, however, were counter to the hydrophobic properties of the
geranyl, farnesyl, and geranylgeranyl groups. The farnesylated peptide
was most effective, followed by the geranylgeranylated and geranylated
peptides (Fig. 3). The corresponding EC (half-maximal effective concentration) values were 40, 200, and
500 µM, assuming that the maximal extent of MII
stabilization for the three peptides was the same. Since our original
results showed that the interaction of the
1 tail and rhodopsin is
predominantly hydrophobic, the reduced potency of the geranylated
peptide in terms of stabilizing MII is not surprising. However, the
reduced efficacy of the geranylgeranylated peptide in comparison with
the farnesylated peptide was unanticipated, since it is more
hydrophobic. If the efficacy of interaction had been in a fashion
proportional to the hydrophobicity of a prenyl group (geranylgeranyl
> farnesyl > geranyl), it could have been due to the higher
effective concentration of more hydrophobic peptides near rhodopsin. In
fact, behavior correlating with the relative hydrophobicities of
peptides has been noted in the case of geranylgeranylated peptides,
that inhibit the interaction of
t and
t subunits more
potently than when farnesylated(22) . However, the
1(D60-C71)gg peptide is five times less effective at MII
stabilization than the
1(D60-C71)far peptide. This effect cannot
be explained by poor solubility of
1(D60-C71)gg. The amounts of
prenylated peptides added to rhodopsin are the same as confirmed by the
quantitative amino acid analysis of working solutions (see
``Materials and Methods''). Since the effect we observed is
opposite to the hydrophobic nature of the
1(D60-C71)gg, we
conclude that ineffective MII stabilization by the
1(D60-C71)gg
reflects poor interaction of this peptide with rhodopsin. Earlier
results have shown that the appropriate primary structure of the
COOH-terminal tail of
1 is important for interaction with
rhodopsin(12, 16) . In combination with the results
here, it can be inferred that rhodopsin contains a site that
specifically recognizes both the COOH-terminal domain of
1 and the
appropriate isoprenoid. This inference is supported by studies of
rhodopsin kinase interaction with rhodopsin. Rhodopsin kinase is
normally farnesylated and exhibits light dependent translocation to the
membrane, indicating that it binds to the light activated rhodopsin
rather than to the lipid surface. A geranylgeranylated mutant of
rhodopsin kinase was effective at phosphorylating rhodopsin, but it
stayed attached to the rhodopsin-containing membranes independent of
light. The farnesyl group, thus, appears to be recognized specifically
by a site on rhodopsin which binds it with sufficiently low affinity
such that the characteristic cycling of transducin between cytosol and
membrane during visual transduction is not disrupted.
Figure 3:
Stabilization of MII by peptides carrying
three different isoprenoids. The data shown are the means ± S.E.
of independent experiments: 1(D60-C71)g (n = 4),
1(D60-C71)far (n = 3), and
1(D60-C71)gg (n = 3).
The
subunits of G proteins can be divided into two groups based on whether
their COOH-terminal cysteine is modified by farnesyl or geranylgeranyl
moieties. Several subtypes that are geranylgeranylated and at least
three members that are potentially farnesylated have been
identified(23, 24) .
It is possible that
the G protein-linked receptors in general possess sites that
specifically recognize both the
subunit COOH-terminal protein
domain and the appropriate isoprenoid.