Role of Isoprenoid Lipids on the Heterotrimeric G Protein
Subunit in Determining Effector Activation*
Chang-Seon
Myung
,
Hiroshi
Yasuda
§,
Wendy W.
Liu
¶,
T. Kendall
Harden
, and
James C.
Garrison
**
From the
Department of Pharmacology, University of
Virginia Health Sciences Center, Charlottesville, Virginia 22908 and the
Department of Pharmacology, University of North Carolina
School of Medicine, Chapel Hill, North Carolina 27599-7365
 |
ABSTRACT |
Post-translational prenylation of heterotrimeric
G protein
subunits is essential for high affinity
-
and
-
-receptor interactions, suggesting that the prenyl group is
an important domain in the 
dimer. To determine the role of the
prenyl modification in the interaction of 
dimers with effectors,
the CAAX (where A indicates alipathic amino acid) motifs in
the
1,
2, and
11 subunits
were altered to direct modification with different prenyl groups. Six
recombinant 
dimers were overexpressed in baculovirus-infected Sf9 insect cells, purified, and examined for their ability to stimulate three phospholipase C-
isozymes and type II adenylyl cyclase. The native
1
2 dimer (
subunit
modified with geranylgeranyl) is more potent and effective in
activating phospholipase C-
than either the
1
1 (farnesyl) or the
1
11 (farnesyl) dimers. However, farnesyl
modification of the
subunit in the
1
2
dimer (
1
2-L71S) caused a decrement in its
ability to activate phospholipase C-
. In contrast, both the
1
1-S74L (geranylgeranyl) and the
1
11-S73L (geranylgeranyl) dimers were
more active than the native forms. The
1
2
dimer activates type II adenylyl cyclase about 12-fold; however,
neither the
1
1 nor the
1
11 dimers activate the enzyme. As was
the case with phospholipase C-
, the
1
2-L71S dimer was less able to activate
adenylyl cyclase than the native
1
2
dimer. Interestingly, neither the
1
1-S74L
nor the
1
11-S73L dimers stimulated
adenylyl cyclase. The results suggest that both the amino acid sequence
of the
subunit and its prenyl group play a role in determining the
activity of the 
-effector complex.
 |
INTRODUCTION |
Heterotrimeric G
proteins1 are transducers of
numerous extracellular signals from seven transmembrane receptors to
intracellular effectors (1-4). G proteins are composed of
,
,
and
subunits and associate with the inner side of the plasma
membrane. Receptor activation catalyzes the exchange of GDP for GTP on
the
subunit, resulting in dissociation of the GTP-liganded
subunit from the 
dimer (1). Both the GTP-bound form of the
subunit and the released 
dimer regulate a variety of effectors,
including PLC-
(5, 6) and adenylyl cyclases (7, 8). To date, 7
subunits and 11
subunits have been identified in mammalian systems
(9-15). Selective assembly of 
heterodimers from these proteins
may produce a large number of unique complexes that differ in their
interactions with
subunits, receptors, and effectors. While the
first four
subunits identified,
1-
4,
are 85-90% identical in primary amino acid sequence (9), the
subunits are much more divergent. For example, the
1 and
5 subunits are only 25% identical. Thus, the
subunits may impart some specificity to the 
signal.
The G protein
subunits are subject to post-translational
modification by the addition of isoprenoid lipids to an invariant cysteine residue in the CAAX motif at their C terminus
(15-17). The last amino acid (X) of the CAAX
motif is an important determinant for modification by one of two
distinct isoprenoids, the 15-carbon farnesyl group or the 20-carbon
geranylgeranyl group. Among the 11 known
subunits, the
1,
8, and
11 subunits are
thought to be modified with the addition of a 15-carbon farnesyl group, whereas the other
subunits contain a 20-carbon geranylgeranyl group
(10). The
11 subunit was recently identified as a widely expressed
subunit that is 76% identical to the
1
subunit (10, 18). However, the function of 
dimers containing the
11 subunit has not been studied. Moreover, the
significance of farnesyl versus geranylgeranyl modification
of the
subunit in 
subunit-mediated activation of effectors
has not been established.
Lipid modification is important for anchoring the 
subunit to the
membrane (17, 19, 20), but the prenyl group may also play a major role
in determining functional interaction with other proteins (15, 17, 21).
For instance, prenylation of the
subunit is necessary for formation
of an active transducin
-
complex (22, 23), for translocation
of the
-adrenergic receptor kinase to the plasma membrane (24), for
high affinity interactions with either
subunits or adenylyl
cyclases (25), and for stimulation of PLC-
by the
1
1 dimer (26). Since the
subunits may
have different primary amino acid sequences and different prenyl groups
at their C terminus, investigators have attempted to determine whether
the type of prenyl group is an important determinant of the activity of
the 
dimer. Some studies suggest that 
dimers containing
the farnesylated
1 subunit couple rhodopsin to the
Gt
subunit more effectively (27, 28), while others show
a small increase in the activity of 
dimers containing a
geranylgeranylated
1 subunit in receptor-coupling assays
(29). We have determined that 
dimers containing a geranylgeranyl
group on the
subunit couple the Gi
subunit to the
A1 adenosine receptor more effectively than those containing a farnesyl
group (30). Overall, these results suggest that the type of prenyl
group on the
subunit does play an important role in determining the
activity of 
dimers.
Most investigators have considered that the prenyl group is involved in
tethering the 
subunit to the membrane (17, 19, 20). However, the
recent x-ray crystal structure of a phosducin-
complex, which was
crystallized with an intact
subunit containing a farnesyl group,
shows that the isoprenoid group folds into a hydrophobic pocket formed
by blades 6 and 7 of the
propeller (31). Interestingly, the
conformation of the
subunit in this complex is different from the
conformation of the free 
dimer (32). Thus, it is possible that
there is an active conformation of the 
dimer, which occurs upon
binding to effectors, and that the prenyl group participates in
establishing this conformation. These observations provide a compelling
reason to examine the ability of 
dimers in which the
subunit
contains different isoprenoid lipids in regulating effectors.
We have reported previously that the prenyl group on the
1 and
2 subunits can be altered by
mutation of the last amino acid (X) in the CAAX
motif at their C terminus (30, 33). Expression of the mutant
subunits in Sf9 cells with a
subunit allows formation of
functional 
dimers in which the lipid on the
subunit has been
changed from farnesyl to geranylgeranyl or vice versa (30, 33). Using
this approach, we have purified six recombinant 
dimers
containing
subunits with native or altered prenyl groups and
verified that the altered CAAX sequences result in the
expected post-translational modifications using mass spectrometry. We
examined the effects of the
1
2,
1
1, and
1
11
dimers and their prenyl mutants on the activity of PLC-
and type II
adenylyl cyclase. The results indicate that both the primary amino acid sequence of the
subunits and the type of prenyl group on the
subunit are important determinants of the activation of PLC-
and
type II adenylyl cyclase.
 |
EXPERIMENTAL PROCEDURES |
Construction of Recombinant Baculoviruses--
The recombinant
baculoviruses encoding the
S,
1,
1,
2 subunits (34, 35), and the
1 and
2 subunits with altered prenylation sequences in the CAAX motifs,
1-S74L and
2-L71S, were prepared as described (33). A full-length
cDNA encoding the human
11 protein was identified in
the expressed sequence tag data base and obtained from Research
Genetics, Inc. (
11, GenBankTM accession
number H00850). To minimize the length of the construct 5' from the ATG
start codon, the polymerase chain reaction was used to add a
SmaI restriction site to the 5' end of
11.
For the
11 cDNA, the primers used were: sense
primer, 5'-TCCCGGGCGAAAATGCCTGCC-3'; antisense primer,
5'-CCACTTAGGATGCAGTTTCTCCC-3'. The polymerase chain reaction products
were subcloned into the pCNTR shuttle vector, and the
11
coding sequence excised from pCNTR with SmaI and
XbaI was ligated into these sites in the baculovirus
transfer vector, pVL1393. The cDNA encoding a
11
with the altered prenylation sequence in the CAAX motif,
11-S73L, was constructed with the same strategy.
Modification of the C-terminal CAAX motif of
11 was performed using polymerase chain reaction to
introduce a CVIS
CVIL into the CAAX sequence. To produce
the
11-S73L cDNA, the primers used were: sense
primer, 5'-TCCCGGGCGAAAATGCCTGCC-3'; antisense primer,
5'-TTTATAAAATAACACAGCTGCC-3'. The polymerase chain reaction products
were subcloned into the pCNTR shuttle vector, digested with
SmaI and XbaI, and ligated into these sites in
the pVL1393 transfer vector. The completed constructs of both
11 and
11-S73L in the pVL1393 transfer
vector were sequenced to confirm the fidelity of the
sequences.
Recombinant baculoviruses were produced by co-transfecting each
recombinant plasmid DNA with linear wild type BaculoGold® viral DNA
(PharMingen) into Sf9 cells as described (36). The recombinant
baculoviruses were purified by one round of plaque purification
(37).
Expression and Purification of Recombinant G Protein 
Subunits--
G protein
and 
subunits were overexpressed by
infecting Sf9 insect cells with recombinant baculoviruses as
described (34, 35). Sf9 cells were co-infected at a multiplicity
of infection of 3 with the appropriate
and
recombinant
baculoviruses and harvested 48 h after infection. The 
subunits were extracted from frozen cell pellets with 0.1% Genapol
C-100 and purified on a DEAE column followed by affinity chromatography
on a Gi1-
-agarose column as described (35). The
Gs
subunit used in the adenylyl cyclase assays was
prepared from a 0.1% (w/v) CHAPS extract of crude cell lysates as
described (36).
Purification of Phospholipase C-
--
Recombinant turkey
PLC-
and human PLC-
1 and PLC-
2 were purified to homogeneity
following overexpression in baculovirus-infected Sf9 insect
cells using chromatography on Q-Sepharose FF, heparin-Sepharose CL-6B,
HPHT hydroxylapatite, Sephacryl S-300, and FPLC Mono Q HR 5/5 columns
as described previously (38, 39).
Analysis of the Post-translational Processing of
Subunits by
Mass Spectrometry--
Full processing of the
subunit requires the
addition of either a farnesyl or a geranylgeranyl group to the
C-terminal cysteine in the CAAX motif, the removal of the
three C-terminal amino acids (AAX), and the addition of a
carboxymethyl group to the C terminus (16, 33). To determine the extent
of the post-translational lipid modification of the
subunits, the
six purified 
dimers used in this study were analyzed by
electrospray ionization mass spectrometry as described previously (33).
The result of this analysis showed that 96% of the native
1 subunit contained the expected farnesyl group and had
a completely processed C terminus. Similarly, 87% of the prenyl
mutant,
1-S74L, contained the geranylgeranyl group and
was fully processed at its C terminus. Ninety-one percent of the
2 subunit and 70% of the
11 subunit were
found to have the expected geranylgeranyl and farnesyl groups,
respectively, and a completely processed C terminus. The prenyl mutants
of these two
subunits,
2-L71S and
11-S73L, were fully processed with expected isoprenoid
lipids to the extent of 95 and 87%, respectively. Therefore, all
subunits used in this study contain the expected isoprenoid lipid at
the C terminus and are capable of high-affinity interactions with
subunits, receptors and effectors.
Preparation of Large Unilamellar Vesicles--
Phospholipid
vesicles were prepared as described (40). Briefly, phospholipids were
mixed at a molar ratio of 4:1 of phosphatidylethanolamine to
PIP2 with
[inositol-2-3H]PIP2 in a buffer
containing 50 mM HEPES, pH 8.0, 3 mM EGTA, 80 mM KCl, 1 mM dithiothreitol and the mixture
dried under argon. The dried lipid was suspended with 3.0 ml of the
above buffer to give final concentrations of 1 mM
phosphatidylethanolamine, 250 µM PIP2, and
800 cpm/µl [3H]PIP2 and hydrated in the
dark at room temperature. Extruded large unilamellar vesicles were
formed from multilamellar vesicles by vortexing the lipid solution
followed by 10 extrusion cycles through a stack of two polycarbonate
filters using a mini-extruder (Avanti Polar Lipids, Inc.) (41). After
extrusion, the final lipid concentration (phosphatidylethanolamine and
PIP2) was about 1100 µM. Extruded large
unilamellar vesicles were stored under argon at 4 °C until use.
Reconstitution of 
Dimers to Large Unilamellar Vesicles by
Gel Filtration--
CHAPS-solubilized 
subunits (ranging from 10 ng to 10 µg) were mixed with vesicles in a volume of 200 µl. The
final CHAPS concentration in the mixture was held to 0.01%. After
reconstituting the mixture on ice for 30 min, the vesicles were applied
to a 2-ml gel filtration column prepared with Ultrogel AcA34 as
described previously (40, 42). The column was pre-equilibrated at
4 °C with buffer containing 20 mM HEPES, pH 8.0, 2 mM MgCl2, 100 mM NaCl, and 1 mM EDTA (450 µl/min). The vesicles reconstituted with

dimers were eluted at the void volume of the column in a total
volume of about 900 µl. This protocol has been demonstrated to remove
more than 98% of the detergent from the vesicles (42, 43) and to
resolve the vesicles from the free 
dimers (40). The
concentration of lipid in each fraction was monitored by counting the
incorporated [3H]PIP2, and the amount of

protein inserted into the vesicles was quantified by silver
staining as described below. The second fraction from the AcA34 column
contained vesicles with the least amount of free 
dimer and were
used in all experiments (40).
Measurement of Phospholipase C-
Activity--
Reaction buffer
containing 50 mM HEPES, pH 8.0, 17 mM NaCl, 67 mM KCl, 0.83 mM MgCl2, 0.17 mM EDTA, 3 mM EGTA, 1 mM
dithiothreitol, and 1 mg/ml BSA was added to 70 µl of gel-filtered
vesicles (fraction 2) in a total volume of 80 µl. Controls were
performed using column-reconstituted vesicles without 
dimers.
The reactions were initiated by adding 10 ng of PLC-
and 3 µM free Ca2+ and placing the mixture in a
30 °C water bath. After a 15-min incubation, the reactions were
terminated by transferring each assay tube to a 4 °C ice bath and
adding 200 µl of ice-cold 10% trichloroacetic acid followed by the
addition of 100 µl of 10 mg/ml BSA (40). The assay mixtures were
centrifuged to remove precipitated protein and intact PIP2
and the [3H]inositol 1,4,5-trisphosphate released into
the supernatant quantitated by liquid scintillation spectrometry (44,
45).
Measurement of Adenylyl Cyclase Activity--
Adenylyl cyclase
activity was measured as described previously (36, 46). Briefly,
Sf9 insect cells were infected with recombinant baculovirus
encoding rat type II adenylyl cyclase (47) and harvested 48 h
after infection. Sf9 membranes containing type II adenylyl
cyclase (5 µg of protein/assay tube) were reconstituted with
GTP
S-activated Gs
subunit (48) and varying
concentrations of 
dimers on ice for 30 min. The reaction was
initiated by addition of the reconstituted membranes to the reaction
buffer containing 25 mM HEPES, pH 8.0, 10 mM
phosphocreatine, 10 units/ml of creatine phosphokinase, 0.4 mM 3-isobutyl-1-methylxanthine, 10 mM
MgCl2, 0.5 mM ATP, and 0.1 mg/ml BSA and
incubated for 7 min at 30 °C. The assay was stopped by addition of
0.1 N HCl and cyclic AMP measured using an automated
radioimmunoassay (49).
Electrophoresis--
For quantitation of the 
concentration in vesicles, the 
dimers reconstituted into
phospholipid vesicles were electrophoresed on a 12% acrylamide, sodium
dodecyl sulfate-polyacrylamide gel and stained with silver according to
the method of Bloom et al. (50). The 
concentrations
in the gel were estimated using the amount of stained
subunit
protein as compared with ovalbumin standards run in the same gel. The
stained proteins were quantitated using a Bio-Image scanning
densitometer and the Whole Band® software (Bio-Image, Ann Arbor, MI)
as described (36, 40). The accuracy of this procedure was verified by
comparison with the BCA (Pierce) protein assay (40). To better display
the mobility of the
subunits, Tricine/SDS-polyacrylamide gels were
run according to the procedure of Schagger and von Jagow (51). The
separating gel contained 16.5% total acrylamide, 0.4% bisacrylamide,
and 10% (v/v) glycerol. The stacking gel contained 4% total
acrylamide and 0.1% bisacrylamide. Gels were run at constant voltage
(~100 volts) at 10 °C for 4-5 h. Resolved proteins were stained
with silver according to the method of Morrissey (52) with the
modification that the dithiothreitol incubation was reduced to 15 min.
Calculation and Expression of Results--
Experiments presented
under "Results" are representative of three or more similar
experiments. Data expressed as concentration-response curves were fit
to sigmoid curves using the fitting routines in the GraphPad PrismTM
software. Statistical differences between the curves were determined
using all the individual data points from multiple experiments to
calculate the F statistic as described (53).
Materials--
All reagents used in the culture of Sf9
cells and for the expression and purification of G protein 
subunits have been described previously in detail (34, 35). The
baculovirus transfer vector, pVL1393, was purchased from Invitrogen;
the BaculoGold® kit from PharMingen; 10% Genapol C-100 and
phosphatidylinositol 4,5-bisphosphate from Calbiochem®;
phosphatidylethanolamine (bovine heart) from Avanti Polar Lipids, Inc.;
[inositol-2-3H]phosphatidylinositol
4,5-bisphosphate from NEN Life Science Products; CHAPS from Roche
Molecular Biochemicals; BSA (fatty acid-free) from Sigma; the pCNTR
shuttle vector from 5 Prime
3 Prime, Inc. (Boulder, CO);
Q-Sepharose FF, heparin-Sepharose CL-6B, Sephacryl S-300, and FPLC Mono
Q HR 5/5 columns from Amersham Pharmacia Biotech; and HPHT
hydroxylapatite from Bio-Rad. All other reagents were of the highest
purity available.
 |
RESULTS |
To determine the role of the prenyl modification of the
subunit in the interaction of 
dimers with effectors, the
CAAX motifs in the
1,
2, and
11 subunits were altered to direct the addition of
different prenyl isoprenoids. Each
subunit was co-expressed with
the
1 subunit in Sf9 insect cells, and the six

dimers,
1
1,
1
1-S74L,
1
2,
1
2-L71S,
1
11, and
1
11-S73L, were purified using
i1-agarose affinity chromatography and tested for their
ability to activate two effectors, PLC-
and type II adenylyl
cyclase. All six
subunits were analyzed by mass spectrometry to
ensure that their CAAX motifs were appropriately processed. The results of the analysis showed that each
subunit had the expected isoprenoid lipids at its C terminus with the extent of processing ranging from 70 to 95% (see "Experimental Procedures"). Therefore, all 
dimers used in this study are capable of
high-affinity interactions with
subunits, receptors, and effectors.
The purity of these dimers is shown in Fig.
1A. The purity of the three
recombinant PLC-
isoforms used in this study is shown in Fig.
1B.

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Fig. 1.
Sodium dodecyl sulfate-polyacrylamide
electrophoresis of purified G protein 
subunits and three PLC- isoforms.
A, purified, recombinant  subunits of defined subtypes
were electrophoresed on a 16% acrylamide, 10% (v/v) glycerol, sodium
dodecyl sulfate-Tricine gel and stained with silver. The migration of
the and subunits are indicated on the left. B, three
isoforms of purified, recombinant PLC- were electrophoresed on a 8%
acrylamide SDS-PAGE and stained with silver. The mobility of the
molecular weight standards is indicated on the left.
|
|
Differential Effect of Three Different 
Dimers on PLC-
and
Adenylyl Cyclase Activity--
We compared the ability of 
dimers with
subunits containing either a farnesyl or a
geranylgeranyl moiety to stimulate PLC-
. An assay system was used in
which purified 
dimers were reconstituted into extruded
phospholipid vesicles and the vesicles separated from mono-dispersed

dimers and the detergent used to solubilize G proteins on an
Ultrogel AcA34 column (40). The data in Fig.
2A illustrate the ability of
three 
dimers to activate recombinant turkey PLC-
. Avian
PLC-
was used initially, because it has a lower basal activity and
higher 
subunit-stimulated activity than either the mammalian
PLC-
1 or PLC-
2 isozymes (39) (see also Fig.
3). The
1
2
dimer activated turkey PLC-
about 10-fold with an estimated
EC50 value of 0.7 nM (refer to Table II). The
1
11 and
1
1
subunits activated turkey PLC-
with estimated EC50
values of 4.1 and 1.9 nM, respectively. The
Vmax values were 50-60% of those observed with
the
1
2 dimer (refer to Table II). Therefore, the
1
2 dimer is more potent
and effective in activating PLC-
than either
1
1 or
1
11,
and the potency of
1
11 is less than that
of
1
1. The data in Fig. 2B
compare the ability of the three 
dimers to activate type II
adenylyl cyclase. The
1
2 dimer activated
type II adenylyl cyclase about 12-fold with an estimated
EC50 value of 14 nM. However, neither
1
1 (54) nor
1
11 effectively activated type II
adenylyl cyclase. Since the
subunits were combined with the same
1 subunit, these results indicate that differences in
the activation of either PLC-
or type II adenylyl cyclase are due to
differences in the
subunit. The major differences between the known
subunits are their divergent amino acid sequences and their lipid
modifications. Interestingly, the data in Fig. 2A show that

dimers containing the
2 subunit modified with a
geranylgeranyl group are more potent and effective in activating both
effectors than those containing either the
1 or the
11 subunit modified with a farnesyl group. Therefore, to
address the possibility that the identity of the prenyl group on the
subunit is an important determinant of the dimer's activity, the
CAAX motif in the
2 subunit was altered to
direct the addition of the farnesyl group and the ability of the
1
2-L71S dimer to activate three PLC-
isoforms was examined.

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Fig. 2.
Differential abilities of three
 dimers to regulate
PLC- and adenylyl cyclase. A,
the ability of the 1 1 and
1 11 dimers to activate phospholipase
C- as compared with that of the 1 2
dimer. Extruded phospholipid vesicles containing phosphatidylinositol
4,5-bisphosphate were reconstituted with 0.01, 0.03, 0.1, 0.3, 1.0, 3.0, or 10.0 µg of each  dimer on ice for 30 min. The mixture
was applied to a 2-ml Ultrogel AcA34 column, fraction 2 was collected,
and the ability of 1 1 (open
circles), 1 2 (closed
squares), and 1 11 (closed
triangles) in fraction 2 to stimulate turkey PLC- activity was
measured as described under "Experimental Procedures." The
difference between the effect of 1 2 and
either 1 1 or
1 11 was statistically significant
(p < 0.0001). B, comparison of the ability
of three  dimers to stimulate type II adenylyl cyclase.
Sf9 cells were infected with a recombinant baculovirus encoding
type II adenylyl cyclase, membranes prepared, and the cyclase reaction
performed with the indicated concentrations of three recombinant 
dimers as described under "Experimental Procedures." The results
are representative of six similar experiments, each performed in
duplicate. Sigmoid curves were fit to the data and statistical
differences between the curves determined as described under
"Experimental Procedures." The difference between the effect of
1 2 and either
1 1 or 1 11
was statistically significant (p < 0.0001).
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Fig. 3.
Comparison of the activity of the
1 2
and
1 2-L71S
dimers to activate three different isoforms of purified
PLC- . A, the ability of
1 2 (modified with geranylgeranyl;
closed squares) and 1 2-L71S
(modified with farnesyl; open squares) to stimulate
recombinant turkey PLC- (tPLC- ) was measured as described under
"Experimental Procedures." The difference between the effect of
1 2 and
1 2-L71S was statistically significant
(p < 0.001). B, analogous experiments were
performed with recombinant human PLC- 2 (hPLC- 2). The
difference between the effect of 1 2 and
1 2-L71S was statistically significant
(p < 0.001). C, analogous experiments were
performed with human PLC- 1 (hPLC- 1).
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|
Effect of
1
2 and
1
2-L71S on PLC-
Activity--
The
data in Fig. 3 show that the
1
2 dimer
stimulated PLC-
with a subnanomolar potency and that exchanging the
prenyl group on the
2 subunit from geranylgeranyl to
farnesyl (
1
2-L71S) caused a diminution in
the ability of the
1
2 dimer to activate all three isoforms of PLC-
. The farnesyl-modified
1
2-L71S dimer was less potent and
effective in activating turkey PLC-
than the native
1
2 dimer (Fig. 3A). Fitting
the data to sigmoid curves estimated that the maximal stimulation with
the
1
2-L71S dimer decreased by 35% and
that the EC50 value increased from 0.7 to 1.2 nM (refer to Table II). Similarly, the
1
2-L71S dimer was less potent and
effective than the native
1
2 dimer in the
activation of human PLC-
2 (Fig. 3B). The data in Fig.
3C indicate that the 
subunits produced little effect
on the activity of PLC-
1 as demonstrated previously (5, 44, 45, 55,
56). Thus, 
subunits stimulated activity in the order of tPLC-
PLC-
2 >>> PLC-
1. Overall, these results
indicate that the prenyl group on the
subunit is an important
determinant of the interaction between PLC-
and 
dimers.
Partition of Different Recombinant 
Dimers into Phospholipid
Vesicles--
Since most studies of G protein activation of PLC-
have used the reconstitution of pre-aggregated lipid with
or 
subunits to activate PLC-
, a potential problem might arise if 
dimers containing different isoprenoid lipids did not partition equally into pre-aggregated lipid layers. This is especially important considering the different chain lengths of the lipids associated with
the native
1,
2, or
11
subunits. To determine the partitioning into vesicles of the six
different 
dimers used in this study, fractions 2 and 3 from the
AcA34 column were collected and the amount of protein inserted into the
vesicles was measured as described (40). Table
I shows that both native
1
1 and
1
11
dimers containing farnesyl groups partitioned equally with the native
1
2 dimer containing a geranylgeranyl
group. About 27% of the 
dimers added to the reaction mixture
were incorporated into the phospholipid vesicles isolated in fractions
2 and 3. Moreover, the 
dimers with altered CAAX
sequences partitioned equally with native 
dimers. Therefore, the
length of the prenyl chain does not significantly affect the
concentration of 
dimers reconstituted into phospholipid vesicles
containing phosphatidylinositol 4,5-bisphosphate.
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Table I
Partition of different recombinant  dimers into phospholipid
vesicles
Purified  subunits were mixed with phospholipid vesicles,
reconstituted on ice for 30 min, applied to a 2-ml Ultrogel AcA34
column at 4 °C, and fractions 2 and 3 eluting at the void volume of
the column used for measurement of the amount of dimer inserted into
the vesicles. The amount of phospholipid eluted and  dimers bound
to the vesicles were quantitated as described under "Experimental
Procedures." Data are representative of three separate experiments.
|
|
Effect of
1
1,
1
1-S74L,
1
11, and
1
11-S73L on PLC-
Activity--
It was
important to determine whether switching the farnesyl group to
geranylgeranyl improves the ability of either the
1
1 or the
1
11 dimer to activate PLC-
. Thus, the
CAAX motifs in the
1 and
11
subunits were altered to direct the addition of a geranylgeranyl group,
and the ability of both the
1
1-S74L and
1
11-S73L dimers to activate either turkey
PLC-
or human PLC-
2 was examined. The data in Fig.
4A show that the
geranylgeranyl-modified
1
1-S74L dimer
exhibited an increased potency and maximal activity compared with the
native
1
1 dimer. Similarly, the
geranylgeranyl-modified
1
11-S73L dimer
was more potent and effective in activating turkey PLC-
than the
native
1
11 dimer. The
1
1-S74L and
1
11-S73L dimers also exhibited increased
capacity to stimulate human PLC-
2 activity (Fig.
5). These four 
dimers
(
1
1,
1
1-S74L,
1
11, and
1
11-S73L) showed little effect on the
activity of PLC-
1 (data not shown, but see Fig. 3C). The
data in Table II summarize the potency
and efficacy of the six 
dimers used in this study on the
activation of these two PLC-
isoforms. The
1
1-S74L dimer exhibited an about 1.6-fold
increase in maximal activity as compared with the native
1
1 dimer and the EC50 value
decreased from 1.9 to 1.1 nM. The estimated maximal
activity of the
1
11-S73L dimer increased
about 1.3-fold and the EC50 value decreased from 4.1 to 1.6 nM. Similar changes were observed in the ability of the dimers to activate human PLC-
2. Taken together, the data indicate that 
dimers with a
subunit containing a geranylgeranyl group interact with higher affinity with PLC-
than 
dimers with a
subunit containing a farnesyl group.

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|
Fig. 4.
Comparison of the activity of native and
altered  dimers to stimulate
recombinant turkey PLC- . A,
the ability of 1 1 (modified with
farnesyl; open circles) and
1 1-S74L (modified with geranylgeranyl;
closed circles) dimers to stimulate recombinant turkey
PLC- (tPLC- ) was measured as described under "Experimental
Procedures." The difference between the effect of
1 1 and
1 1-S74L was statistically significant
(p < 0.001). B, analogous experiments were
performed with 1 11 (modified with
farnesyl; closed triangles) and
1 11-S73L (modified with geranylgeranyl;
open triangles) dimers. The difference between the effect of
1 11 and
1 11-S73L was statistically significant
(p < 0.001).
|
|

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|
Fig. 5.
Comparison of the activity of native and
altered  dimers to stimulate
recombinant human PLC- 2. A,
phospholipid vesicles reconstituted with
1 1 (modified with farnesyl; open
circles) or 1 1-S74L (modified with
geranylgeranyl; closed circles) dimer were incubated with
recombinant human PLC- 2 (hPLC- 2) and PLC- activity measured as
described under "Experimental Procedures." The data were fit to
sigmoid curves. The difference between the effect of
1 1 and
1 1-S74L was statistically significant
(p < 0.001). B, analogous experiments were
performed with 1 11 (modified with
farnesyl; closed triangles) and
1 11-S73L (modified with geranylgeranyl;
open triangles) dimers. The difference between the effect of
1 11 and
1 11-S73L was statistically significant
(p < 0.001).
|
|
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|
Table II
Comparison of EC50 and Vmax of six 
dimers on the activation of two PLC- isoforms
Phospholipid vesicles containing phosphatidylinositol 4,5-bisphosphate
were reconstituted with different recombinant  dimers on ice for
30 min. After the incubation, the mixture was applied to a 2-ml
Ultrogel AcA34 column at 4 °C and fraction 2 eluting at the void
volume used for the measurement of PLC- activity. Fraction 2 was
incubated for 15 min at 30 °C in the presence of purified
recombinant turkey PLC- (tPLC- ) or human PLC- 2 (hPLC- 2) as
described under "Experimental Procedures." The data are expressed
as mean ± S.E. and the average of three determinations, each
performed in duplicate. The EC50 (nM) and
Vmax (µmol/mg of PLC/min) values were estimated by
fitting of the averaged data to sigmoid curves.
|
|
Effect of the Prenyl Group on the Activity of Type II Adenylyl
Cyclase--
To determine the role of the isoprenoid group in the
interaction with an effector which is membrane-associated, the capacity of the six 
dimers to activate type II adenylyl cyclase was also
examined. The data in Fig. 6A
indicate that switching the prenyl group on the
2
subunit from geranylgeranyl to farnesyl caused a significant decrement
in the ability of the
1
2 dimer to
activate type II adenylyl cyclase. Our previous experiments showed that
about the same amount of the
1
2 and
1
2-L71S (about 25% of the 
subunits added to a reconstitution mixture) are incorporated into
Sf9 cell membrane, indicating regardless of the prenyl group on
the
subunit, the dimers partition equally into the Sf9 cell
membrane (30). Thus, the result from Fig. 6A suggests that
the type of prenyl group is important for the interaction of the 
dimer with adenylyl cyclase. As expected, the
1
1 dimer did not activate type II
adenylyl cyclase (54). Interestingly, the
1
11 dimer was completely ineffective on
type II adenylyl cyclase. Surprisingly, neither the
1
1-S74L nor the
1
11-S73L dimer containing a
geranylgeranyl group on the
subunit were able to significantly
stimulate type II adenylyl cyclase (Fig. 6, B and
C). The data in Table III
summarize the potency and efficacy of the six 
dimers on the
activation of type II adenylyl cyclase. The farnesyl-modified
1
2-L71S dimer was about 60-65% less
active, and its EC50 value was increased from 14 to 76 nM. This change in potency and maximal activity is larger
than that observed with PLC-
(see Fig. 3). Thus, 
dimers with
a
subunit containing a geranylgeranyl group interact with higher affinity with type II adenylyl cyclase than those with a
subunit containing a farnesyl group. While these results are not surprising, the observation that neither the
1 or the
11 subunit modified with a geranylgeranyl group have any
activity on type II cyclase was unexpected. These observations suggest
that the differences in amino acid sequence between
1
and
2 greatly affect the ability of the dimers to
activate adenylyl cyclase.

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|
Fig. 6.
Comparison of the activity of native and
altered  dimers to stimulate
type II adenylyl cyclase. A, Sf9 cells were
infected with a recombinant baculovirus encoding the type II adenylyl
cyclase, membranes prepared, and the cyclase reaction performed with
the indicated concentrations of 1 2
(modified with geranylgeranyl; closed squares) and
1 2-L71S (modified with farnesyl;
open squares) as described under "Experimental
Procedures." The difference between the effect of
1 2 and
1 2-L71S was statistically significant
(p < 0.0001). B, the cyclase reaction was
performed with the indicated concentrations of
1 1 (modified with farnesyl; open
circles) and 1 1-S74L (modified with
geranylgeranyl; closed circles) and compared with the effect
of 1 2 (dotted lines). The
difference between the effect of 1 2 and
1 1 was statistically significant
(p < 0.0001), but the difference between
1 1 and
1 1-S74L was not statistically
significant. C, analogous experiments were performed with
1 11 (modified with farnesyl; closed
triangles) and 1 11-S73L (modified
with geranylgeranyl; open triangles) dimers. The difference
between the effect of 1 2 and
1 11 was statistically significant
(p < 0.0001), but the difference between
1 11 and
1 11-S73L was not significant.
|
|
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|
Table III
Comparison of EC50 and Vmax of six 
dimers on the activation of type II adenylyl cyclase
Sf9 cells were infected with a recombinant baculovirus encoding
the type II adenylyl cyclase, membranes prepared, and the cyclase
reaction performed with the range of  dimer concentrations
described under "Experimental Procedures." The data are expressed
as mean ± S.E. and the average of three determinations, each
performed in duplicate. The EC50 (nM) and
Vmax (nmol/mg of protein/min) values were estimated
by fitting of the averaged data to sigmoid curves.
|
|
 |
DISCUSSION |
This study demonstrates that 
dimers with
subunits
containing different isoprenoids have distinct abilities to activate PLC-
and type II adenylyl cyclase. The two major findings of this
study are (a) the isoprenoid lipid added to the cysteine residue in the CAAX motif of the
subunit plays a role in
determining the activity of the 
dimer at both effectors and
(b) 
dimers with a
subunit containing a
geranylgeranyl group are more potent and effective in activating either
PLC-
or type II adenylyl cyclase than those with a
subunit
containing a farnesyl group. This study also presents the first
demonstration of the functional activity of a 
dimer containing
the
11 subunit. The
1
11
dimer is about six times less potent and about 50% less effective in activating PLC-
than the
1
2 dimer and
has little or no effect on the type II adenylyl cyclase. Overall, the
data presented here indicate that in addition to the primary amino acid
sequence of the
subunits, the prenyl group on the
subunit is an
important determinant of the activation of PLC-
and type II adenylyl cyclase.
The recent x-ray crystal structure of a complex of bovine retinal
phosducin with the transducin 
subunit in which the
subunit
contained a farnesyl group provides a conceptual background for
interpreting the results obtained with PLC-
and adenylyl cyclase
(31). Remarkably, the interaction between phosducin and the 
dimer appears to result in a significant conformational change in the
subunit as compared with the conformation seen in the free 
dimer (32). This event also appears to cause the insertion of the
prenyl group into a hydrophobic pocket in the
subunit formed
between blades 6 and 7 (31). Interestingly, this conformational change
occurs on the outer strands of blades 6 and 7 of the
propeller and
recent mutagenesis studies show that these regions are critical for the
activation of PLC-
(57). If the hydrophobic pocket within the
subunit that binds the prenyl group is able to discriminate between the
different length of the farnesyl and geranylgeranyl lipids, different
conformational changes might be induced by the
1 and
2 subunits (or their prenyl mutants). This possibility
might explain differences in the ability of the various 
dimers
to stimulate the activity of PLC-
and adenylyl cyclase. Overall,
these observations suggest that insertion of a prenyl group into the
hydrophobic pocket formed in the
subunit may be an important part
of the mechanism by which the 
dimers activate effectors when
released from the
subunit.
The interaction of protein prenyltransferases with their isoprenoid
substrates provides another example of a hydrophobic binding pocket
determining isoprenoid specificity. Protein prenyltransferases are
responsible for the addition of isoprenoid lipid to proteins and are
classified by their lipid substrate: protein farnesyltransferase (FTase), protein geranylgeranyltransferase (GGTase-I), and the Rab
geranylgeranyltransferase (GGTase-II) (58). G protein
subunits are
targets for either FTase or GGTase-I. These two prenyltransferases are
heterodimers composed of the common 48-kDa
subunit and distinct
subunits: 46-kDa
subunit for FTase (
F) and 43-kDa
subunit for GGTase-I (
GGI) (59-62). Thus, the
binding sites for different lipid substrates on these two
prenyltransferases are thought to reside in their
subunits (58).
The x-ray crystal structure determined by Park et al. (63)
illustrates that farnesyl diphosphate occupies a hydrophobic pocket in
the center of the
subunit barrel. Recently, it has been
demonstrated that the isoprenoid moiety of farnesyl diphosphate binds
in an extended conformation within the hydrophobic cavity of the
subunit of the FTase, and the diphosphate moiety binds to a positively
charged cleft at the top of this cavity near the subunit interface
(64). These findings suggest that the isoprenoid substrate specificity
of the hydrophobic binding cavity is determined by the depth of the
cavity and that the pocket acts as a ruler discriminating between
isoprenoids of different length (64).
The three classes of PLC isozymes, PLC-
, PLC-
, and PLC-
,
contain two highly conserved catalytic domains, X and Y, but only the
PLC-
isoforms are regulated by G proteins (65). These isoforms are
distinguished from the other families by an extended C terminus. Deletion of the C-terminal amino acids of PLC-
1 and PLC-
2
eliminated Gq
subunit-mediated PLC-
activation
(66-68). Peptides corresponding to a portion of the C-terminal region
of PLC-
1 inhibited activation by Gq
subunit in
vitro (67), suggesting that the C-terminal region might be
important for the
subunit-induced PLC-
activation. Since removal
of the C-terminal region of PLC-
1 and PLC-
2 did not impair 
subunit-induced enzyme activation, the remaining domains of the enzyme
must provide the 
subunit interaction sites. A PLC-
2 fragment
corresponding to a region between the X and Y domains
(Glu435 to Val641) inhibited 
subunit-mediated activation of PLC-
2 in transfected COS-7 cells
(69). Peptides from this sequence expressed as a series of fusion
proteins bound to purified 
subunits in vitro (69).
Overexpression of this region also blocked 
subunit-induced activation of co-transfected PLC-
2 in COS cells (69). It has been
reported recently that two overlapping PLC-
2 fragments corresponding to Asn564-Lys583 and
Glu574-Lys593 of PLC-
2 inhibited activation
of PLC-
2 by 
subunits, inhibited 
subunit-dependent ADP-ribosylation of Gi1
subunit by pertussis toxin, and inhibited 
subunit-dependent activation of phosphoinositide 3-kinase
(70). These observations suggest that the region between Glu574 and Lys583 in PLC-
2 may be the site
that interacts with 
subunits (70).
A number of studies have attempted to determine the sites at which the

dimer interacts with adenylyl cyclase. Type I adenylyl cyclase
is inhibited by Gi
subunits and 
subunits,
whereas type II cyclase is activated by the 
subunits in the
presence of Gs
subunits (7, 71). In an attempt to
identify the sites in adenylyl cyclase that interact with the 
subunits, a chimeric soluble adenylyl cyclase was engineered by linking
the type I adenylyl cyclase C1a domain (the N-terminal part of the
first cytoplasmic domain) with the type II adenylyl cyclase C2 domain (the second cytoplasmic domain) (72, 73). The chimeric soluble enzyme
was unresponsive to the Gi
subunit but was inhibited almost completely by the
1
2 dimer,
suggesting that the C1a domain is critical for 
subunit-induced
inhibition of type I adenylyl cyclase (72, 73). In contrast, a 27-amino
acid peptide (956-982) derived from the C2 domain of type II adenylyl
cyclase blocked the interaction of 
dimers with several
effectors, including PLC-
, type II adenylyl cyclase, and
K+ channels, but did not block 
interaction with
subunits (74). This result suggests that 
dimers can also
interact with a region on the C2 domain of type II adenylyl cyclase
(74).
Whereas the G protein
1-
4 subunits are
highly conserved in primary amino acid sequence, the
subunits are
much more divergent, suggesting that the functional specificity of
different 
dimer combinations may be due to the differences in
subunits. The results presented here support this possibility.
Combination of different
subunits with the same
subunit
resulted in 
dimers exhibiting very different ability to activate
PLC-
and adenylyl cyclase. The differences are due both to the
primary amino acid sequences of the
subunits and to their lipid
modifications. For example, all 
dimers containing a
geranylgeranyl group on the
subunit were more potent and effective
in activating the two effectors than those containing a farnesyl group.
Thus, the type of lipid at the C terminus of the
subunit is
important to the ability of 
dimers to activate effectors. Other
groups have also obtained data suggesting that the isoprenoid lipid is important in the interaction of 
dimers with effectors. Matsuda et al. (29) have shown that the
1
1-S74L dimer containing a mixture of
farnesyl and geranylgeranyl groups had a greater ability to inhibit
Ca2+/calmodulin-stimulated adenylyl cyclase and stimulate
PLC-
than did the native farnesylated
1
1 dimer. However, our data suggest that
the amino acid sequence of the
subunit is also an important determinant of the activity of the 
dimer. For example, neither the
1
1 nor the
1
11 dimer are able to activate type II
adenylyl cyclase. In contrast to the results obtained with PLC-
,
switching the prenyl group in the
1
1 and
1
11 dimers to geranylgeranyl did not
increase their ability to activate type II adenylyl cyclase. Overall,
these results indicate that both the primary amino acid sequence and
the prenyl group of the
subunits are important determinants for the
activation of effectors. For the activation of PLC-
, the type of
prenyl group appears to be a major determinant of the activity of

dimer. In the case of type II adenylyl cyclase, the prenyl group
also appears to be important, but the amino acid sequences of certain
subunits, such as
1 and
11, appear to be incapable of activating the enzyme regardless of prenyl group at
their C terminus.
Our previous data supports the possibility that charge differences in
the
subunit can have a large effect on the ability of 
dimers
to activate type II adenylyl cyclase. Phosphorylation of the
12 subunit in the
1
12
dimer significantly inhibits the ability of the dimer to stimulate type
II adenylyl cyclase (46), and the phosphorylation site has been
determined to be at Ser1 in the N terminus of the molecule
(11). This result suggests that introduction of negative charges at the
N-terminal region of the
subunit inhibits the interaction of the

dimer with the type II adenylyl cyclase. Interestingly,
comparison of the N-terminal 20 amino acids of the
2
with the
1 or
11 subunits shows that
1 and
11 have six negatively charged
amino acids whereas
2 has only one. Thus, the negatively
charged N terminus of the
1 and
11
subunits may explain the inability of dimers containing these
subunits to activate type II adenylyl cyclase. Preliminary experiments
with chimeric
subunits in which the N-terminal 20 amino acids of
2 were replaced with the corresponding amino acids from
1 support this explanation. When expressed with the
1 subunit, this dimer has a greatly reduced ability to
stimulate type II adenylyl
cyclase.2 Thus, multiple
domains of the
subunit may be involved in the interaction with
different effectors.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Ravi Iyengar for the baculovirus
encoding type II adenylyl cyclase, Dr. Anne Hinderliter for advice
about lipid vesicle preparations, the University of Virginia
Biomolecular Research Facility for DNA sequencing and mass
spectrometric analysis, and the University of Virginia Diabetes Core
Facility for the cyclic AMP assays.
 |
FOOTNOTES |
*
This work was supported by the National Institutes of Health
Grants PO1-CA-40042 and RO1-DK-19952 and by United States Public Health
Service Grant GM-29536.The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Present address: Dept. of Medicine, University of Tokyo, Tokyo
112-8688, Japan.
¶
Present address: IDEXX Laboratories, Inc., One IDEXX Drive,
Westbrook, ME 04092.
**
To whom correspondence should be addressed: Dept. of Pharmacology,
Box 448 Health Sciences Center, University of Virginia, Charlottesville, VA 22908. Tel.: 804-924-5618; Fax: 804-982-3878; E-mail: jcg8w{at}virginia.edu.
2
C.-S. Myung and J. C. Garrison, unpublished observation.
 |
ABBREVIATIONS |
The abbreviations used are:
G proteins, guanine
nucleotide-binding regulatory proteins;
Sf9 cells, Spodoptera frugiperda cells (ATCC number CRL 1711);
PLC-
, phosphatidylinositol-specific phospholipase C-
isoform;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate;
BSA, bovine serum albumin;
Genapol C-100, polyoxyethylene (10) dodecyl
ether;
PIP2, phosphatidylinositol 4,5-bisphosphate;
GTP
S, guanosine 5'-3-O-(thio)triphosphate;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine;
FTase, protein farnesyltransferase;
GGTase-I, protein
geranylgeranyltransferase;
GGTase-II, Rab
geranylgeranyltransferase.
 |
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