(Received for publication, August 31, 1995; and in revised form, October 20, 1995)
From the
G is palmitoylated at residues Cys
and Cys
. Removal of palmitate from purified
G
with palmitoylthioesterase in vitro failed
to alter interactions of G
with phospholipase
C-
1, the G protein
subunit complex, or m1 muscarinic
cholinergic receptors. Mutants C9A, C10A, C9A/C10A, C9S/C10S, and
truncated G
(removal of residues 1-6) were
synthesized in Sf9 cells and purified. Loss of both Cys residues or
truncation prevented palmitoylation of G
. However,
truncated G
and the single Cys mutants activated
phospholipase C-
1 normally, while the double Cys mutants were poor
activators. Loss of both Cys residues impaired but did not abolish
interaction of G
with m1 receptors. These Cys residues
are thus important regardless of their state of palmitoylation. When
expressed in HEK-293 or Sf9 cells, all of the proteins studied
associated entirely or predominantly with membranes, although a minor
fraction of nonpalmitoylated G
proteins accumulated in
the cytosol of HEK-293 cells. When subjected to TX-114 phase
partitioning, a significant fraction of all of the proteins, including
those with no palmitate, was found in the detergent-rich phase. Removal
of residues 1-34 of G
caused a loss of surface
hydrophobicity as evidenced by complete partitioning into the aqueous
phase. The Cys residues at the amino terminus of G
are
thus important for its interactions with effector and receptor, and the
amino terminus conveys a hydrophobic character to the protein distinct
from that contributed by palmitate.
Heterotrimeric guanine nucleotide binding proteins (G proteins) ()link cell surface receptors with intracellular
effectors(1, 2, 3) . In the absence of
activators, G protein
,
, and
subunits are associated
with each other and the inner surface of the plasma membrane. Although
the G protein polypeptides are not intrinsically hydrophobic, they are
membrane-bound at least in part because of covalent lipid
modifications. G protein
subunits are prenylated and
carboxymethylated at their carboxyl termini(4, 5) .
These modifications promote association of the
subunit
complex with membranes and interactions of
with
and
effectors(6) . Members of the G
subfamily of
subunits are myristoylated at their amino termini; this modification
also promotes membrane anchorage and interactions of
with
and effectors(7, 8, 9) . Although
members of the G
, G
, and G
subfamilies of
subunits are not myristoylated, they (and
G
family members, excepting transducin) are
palmitoylated on one or more cysteine residues near their amino
termini(10, 11, 12) . Arachidonate may also
be incorporated similarly(13) . In contrast to myristoylation,
palmitoylation of G
subunits is a dynamic and regulated
process. Activation of appropriate receptors appears to stimulate
depalmitoylation of cognate G
subunits(14, 15, 16) .
The lipid
modifications of G subunits are at or near the amino
terminus, and this domain (upstream of the G
nucleotide
binding region; roughly residues 1-35) has been implicated in a
number of functions. The amino terminus is clearly important for
interactions with
(17, 18) , and it is
implicated in interactions with receptors (19) and effectors (9, 18) as well. The amino and carboxyl termini of
G
form an associated subdomain of the GDP-bound form
of the protein that becomes disordered upon
activation(20, 21) . The amino termini of G
and G
differ from those of most other
G
proteins. The site of initiation of translation is
not clear (Met
or Met
in G
;
Met
, Met
, or Met
in
G
). The shorter form (assuming Met
as
initiator) is most similar to other G
subunits(22) . It is not known if only one or both of
these species is expressed naturally, although all currently identified
G
mRNAs (except that from Drosophila)
encode the longer protein. G
and G
also have two amino-terminal Cys residues (positions 9 and 10) that
both serve as sites for
palmitoylation(23, 24, 25) .
The role of G
protein palmitoylation is unclear, and several conflicting reports have
appeared. In the case of G, one study (23) suggests that palmitoylation is essential for membrane
localization, while others (14, 15) indicate that
nonpalmitoylated mutants of G
remain associated with
the plasma membrane. Wedegaertner et al.(23) also
reported that palmitoylation is necessary for association of
G
with membranes and subsequent activation of its
physiological effector, PLC-
1. In contrast, others have indicated
that palmitoylation is not required for association of either G
or G
with membranes(24, 25) , and
Edgerton et al.(25) found that the C9A/C10A mutant of
G
failed to activate PLC-
1. Published data also
suggest that palmitoylation is important for coupling of G
with the NK2 receptor (25) but not the
-adrenergic receptor(23) . All of these
studies have utilized transfected cells expressing G protein
subunits carrying mutations designed to prevent palmitoylation.
We
have investigated the importance of amino-terminal domains of G
proteins using the m1 muscarinic cholinergic
receptor/G/PLC-
1 signaling pathway as a model
system, and we have emphasized studies of purified and reconstituted
proteins. We have found that the amino-terminal domain of G
is clearly important for activation of PLC-
1. Although
palmitoylation of Cys residues 9 and/or 10 is not necessary for
interactions of G
with PLC-
1, receptors, or
, the Cys residues themselves are important for interactions
with PLC-
1 and, to a lesser extent, with receptor. The amino
terminus of G
also appears to confer a hydrophobic
character on the protein, independent of its palmitoylation.
Figure 3:
Palmitoylation and purification of
amino-terminal mutants of G. A,
amino-terminal sequences of wild type G
and mutants.
All constructs were hexahistadine tagged at the carboxyl terminus. B, Sf9 cells were infected with viruses encoding
and
subunits and either wild type
G
(WT-G
), or
amino-terminal mutants of G
(C9A, C10A, and C9A/C10A (C9,10A), or G
-short). Infected cells were
labeled with [
H]palmitate, and G
subunits were recovered from membrane extracts by Ni-NTA affinity
chromatography. Samples were resolved by SDS-PAGE and visualized by
fluorography and by immunoblotting with a specific anti-G
serum (WO82). C, purification of G
CH6
by affinity chromatography and anion exchange chromatography. Sf9 cells
(6-liter culture) were infected with viruses encoding
,
, and G
CH6
subunits. Cholate extracts of cell membranes (L) were bound to
Ni-NTA resin (top left) and eluted with a 150 mM imidazole bump (B). The sample was then bound to and
eluted from Q-Sepharose anion exchange resin (right, top and bottom); individual fractions were analyzed for their
capacity to stimulate phospholipase C-
1 (bottom), and
protein was visualized by Coomassie Blue staining (top).
Phospholipase C-
1 activity is expressed as pmol product/min/ng
phospholipase C-
1.
m1 receptor-catalyzed GTPS binding was measured at 30
°C as described elsewhere(35) , using 100 nM
[
S]GTP
S and 1 mM carbachol; the
final buffer composition was the same as in the GTP hydrolysis assays
(see above). Data describing the time course of GTP
S binding were
fit to a two-component equation: y = A(1
- e
) + (mt + b), where A is the maximum amount of
GTP
S bound in response to carbachol, k is the rate
constant for receptor-stimulated binding, m is the basal rate
of binding (essentially constant over the assay interval), and b is the amount of GTP
S nonspecifically bound at zero time (t). For C9S/C10S G
, the linear component (mt + b) was omitted because there was no
observable binding of nucleotide in the absence of carbachol.
When Sf9 cells are infected with baculovirus encoding
G, G
, or G
along
with viruses encoding
and
subunits,
newly synthesized
subunits accumulate in both membranes and
cytosol; however, only the membrane-associated
subunits
incorporate [(
)H]palmitate (Fig. 1A).
To study the functional
consequences of palmitoylation in greater detail, we generated an
affinity-tagged G
(hexahistadine at the carboxyl
terminus) using the baculovirus expression system. When synthesized in
Sf9 cells, G
CH6 incorporates
[
H]palmitate and can be purified rapidly from
membrane extracts by affinity chromatography with Ni-NTA resin (Fig. 1B).
Figure 1:
Labeling and
isolation of recombinant G protein subunits from Sf9 cells by
immunoprecipitation or affinity chromatography. A, Sf9 cells
were infected with viruses encoding G protein
and
subunits and either G
(r
), G
(r
), or G
(r
). Infected cells were
labeled with [
S]methionine (left) or
[
H]palmitate (right), fractionated into
cytosol (C) and membranes (M), and subjected to
immunoprecipitation using specific antisera (Z811 for
, 584 for
, and P960 for
). Immunoprecipitated, radiolabeled proteins were
resolved by SDS-PAGE and visualized by autoradiography or fluorography.
In this experiment G
is visualized as two bands; see
footnote 3 for explanation. B, Sf9 cells were infected with
viruses encoding
,
, and
hexahistadine-tagged G
(G
CH6).
Infected cells were incubated with [
H]palmitate
and fractionated into cytosol and membranes. Cholate extracts of
membranes (L) were mixed with Ni-NTA resin, the flow through (FT) was collected, and the resin was washed with high salt (W1) and low salt (W2); bound protein was eluted
using 150 mM imidazole (Bump). Fractions were
resolved by SDS-PAGE and visualized by silver staining (left)
or fluorography (right).
Figure 2:
Removal of palmitate from G by treatment with recombinant palmitoylthioesterase (rPTE). A, [
H]Palmitate-labeled
rG
or c-Ha-ras purified by Ni-NTA affinity
chromatography was incubated in the absence or presence of
palmitoylthioesterase. Treated samples were resolved by SDS-PAGE, and
labeled protein was visualized by fluorography. B, HPLC
analysis of a
H-labeled mixture of standards of myristate,
palmitate, and stearate (top), or
H-labeled
products removed from rG
synthesized in the presence
of [
H]palmitate; incubation was performed without (middle) or with (bottom) palmitoylthioesterase. C, purified rG
was incubated with (
) or
without (
) palmitoylthioesterase for 90 min at 30 °C and then
activated for 1 h with 1 mM GTP
S at 30 °C. Treated
rG
was then mixed with purified phospholipase C-
1
and phosphatidylinositol(4,5)-bisphosphate vesicles and assayed for
phospholipase C-
1 activity as described under ``Experimental
Procedures.'' Enzymatic activity is expressed per ng of
phospholipase C-
1. D, purified G
was
treated with (
) or without (
) palmitoylthioesterase for 90
min at 30 °C and then activated for 15 min at 20 °C with 30
µM AlCl
, 10 mM NaF, and 5 mM
MgCl
. Treated rG
(3 nM) was then
mixed with the indicated concentrations of purified brain
G
and held for 10 min at 4 °C. Purified
phospholipase C-
1 (1 ng) and substrate vesicles were added and
phospholipase C activity was measured as described. E,
purified rG
was treated with or without
palmitoylthioesterase and then reconstituted into phospholipid vesicles
with purified G
and m1 muscarinic cholinergic
receptors. Receptor-stimulated [
S]GTP
S (10
nM) binding to G
was measured in the presence
of either atropine (20 µM) or carbachol (100
µM) after a 10 min incubation.
Figure 4:
Activation of phospholipase C-1 by
G
, G
mutants, and
amino-terminally cleaved G
. A,
purified G
CH6 and amino-terminal G
mutants. Each protein (500-750 ng) was resolved by SDS-PAGE
and visualized by Coomassie Blue staining. B, purified
proteins were activated for 1 h with 1 mM GTP
S at 30
°C and activation of added phospholipase C-
1 was assayed as
described under ``Experimental Procedures.'' Data are pooled
results from several experiments: G
CH6 (WT-G
), n = 6;
G
-short, n = 6; C9A, n = 2; C10A, n = 2; C9A/C10A, n = 5; C9S/C10S, n = 3. Mutant proteins were
compared with G
CH6 in each experiment and all values
were normalized to activity stimulated by 100 nM
G
CH6 (100%). Inset, G
CH6,
C9A/C10A, and C9S/C10S were incubated with or without GTP
S as
before and subjected to limited tryptic digestion. Samples were then
resolved by SDS-PAGE and visualized by immunoblotting with
anti-G
serum WO82. C, G
CH6
was activated with GTP
S as before and half of the sample was
subjected to limited tryptic digestion. Undigested
G
CH6, digested G
(NC-G
), and C9S/C10S
G
were then repurified by Ni-NTA chromatography in the
presence of GTP
S. Recovered samples (300-500 ng) were
resolved by SDS-PAGE and visualized (right) by immunoblotting
with anti-G
serum WO82. The indicated concentrations
of each sample were assayed for phospholipase C-
1 stimulation as
described (left). Values are averages of two experiments and
are normalized to the maximal value for G
CH6
(100%).
The capacity of each of these
proteins to stimulate purified PLC-1 is shown in Fig. 4.
The nonpalmitoylated double Cys mutant (C9A/C10A) has a greatly reduced
capacity to activate and apparent affinity for PLC-
1. In contrast,
both of the single Cys
Ala mutants, which are palmitoylated, and
the nonpalmitoylated truncation mutant, G
-short,
retain near full capacity to stimulate PLC-
1. The Cys
Ser
double mutant (C9S/C10S) of G
was indistinguishable
from C9A/C10A. Both C9A/C10A and C9S/C10S G
could be
activated, based on the capacity of bound GTP
S to protect the
proteins from tryptic proteolysis (Fig. 4B, inset). To test the effect of removal of a larger portion of
the amino terminus, we cleaved GTP
S-activated
G
CH6 with trypsin and recovered the product
(NC-G
) by Ni-NTA chromatography; the
hexahistidine-tagged carboxyl terminus was thus intact. Amino acid
sequencing of NC-G
revealed that the first 34 residues
were missing. The capacity of NC-G
to activate
PLC-
1 closely resembles those of C9A/C10A and C9S/C10S
G
(Fig. 4C).
We next studied the
effects of amino-terminal mutations of G on its
interactions with m1 muscarinic receptors and PLC-
1 in
reconstitution assays. Non-His-tagged G
was included
in these experiments to assess the effects of the tag on receptor
coupling. As shown in Fig. 5A, m1 receptors stimulate
nucleotide exchange and GTP
S binding to G
CH6,
G
-short, and C9S/C10S G
. Although
receptor-stimulated GTP
S binding was nearly identical for
G
CH6 and G
-short, the rate of
nucleotide exchange was significantly reduced (5-fold) for the double
Cys
Ser mutant (Table 1; Fig. 5A). In
contrast, the nucleotide binding properties of the single Cys Ala
mutants were largely unchanged (data not shown). Similarly, carbachol
stimulates steady-state GTP hydrolysis by G
CH6,
G
-short, and non-His-tagged G
, but
the effect is reduced significantly with the double Cys
Ser
mutant (Table 1, Fig. 5B).
Figure 5:
m1
Muscarinic cholinergic receptor-stimulated nucleotide exchange and
phospholipase C-1-stimulated GTPase activity of G
and amino-terminal mutants. A, receptor-stimulated
GTP
S binding. Purified G
(not tagged),
carboxyl-terminal hexahistadine-tagged G
(G
CH6), and tagged G
mutants (G
-short, G
C8,10S (C9S/C10S)) were
reconstituted into phospholipid vesicles with purified m1 receptors.
Total carbachol-stimulated [
S]GTP
S binding
to G
was measured in the presence of 100 nM [
S]GTP
S and carbachol (1 mM)
at 30 °C for various times up to 30 min. Data shown are the average
of duplicate determinations from a single experiment, which is
representative of four experiments. B, receptor-stimulated GTP
hydrolysis by G
. G protein heterotrimers were
reconstituted into phospholipid vesicles with m1 receptors and GTPase
activity was assayed in the presence of either 10 µM
atropine (hatched bars; A) or 1 mM carbachol (open bars; C). The assay time was either 8 min
(G
) or 30 min (G
CH6 and mutants). C, phospholipase C-
1-mediated potentiation of
receptor-stimulated GTP hydrolysis by G
. Experiments
were performed as in B, except in the presence of
phospholipase C-
1 (10 nM). Data in B and C are the means ± S.D. of three separate experiments, each
consisting of duplicate determinations.
Phospholipase
C-1 is known to stimulate steady-state GTP hydrolysis by
G
, particularly in the presence of
receptor(34) . Phospholipase C-
1 enhances
receptor-stimulated GTP hydrolysis by greater than 11-fold for both
G
CH6 and G
-short, but only
2-3-fold for C9S/C10S G
(Table 1; Fig. 5C). This is consistent with the relative
inability of C9S/C10S G
to activate phospholipase
C-
1. The effects of phospholipase C-
1 on the single Cys
Ala mutants were similar to those on G
CH6 (data
not shown). A separate point is that carboxyl-terminal hexahistadine
tagging of G
reduces the efficiency of
receptor-G
coupling; this effect is most evident with
phospholipase C-
1-stimulated GTP hydrolysis (
)(Fig. 5C; Table 1).
Figure 6:
Cellular distribution of recombinant wild
type G and amino-terminal mutants of
G
in HEK-293 and Sf9 cells. A,
HEK-293 cells were transiently transfected with DNA (20 µg)
encoding G
, G
-short, or
G
C9S/C10S for 30 h. None of these constructs was
hexahistadine-tagged. Cells were harvested and fractionated as
described under ``Experimental Procedures'' into a low speed
nuclear pellet (NP), high speed membrane pellet (M),
or cytosol (C). Equal volumes of normalized fractions were
resolved by SDS-PAGE, and G
was visualized by
immunoblotting with anti-G
serum WO82. B, Sf9
cells were infected with a virus encoding G
CH6 or each
of the indicated mutants and fractionated as described under
``Experimental Procedures'' into nuclear pellet (NP), membrane pellet (M), or cytosol (C).
Equal volumes of normalized fractions were resolved by SDS-PAGE, and
G
was visualized by immunoblotting with
anti-G
serum WO82. C, same as in B,
except Sf9 cells were infected with viruses encoding each indicated
G
subunit together with G protein
and
subunits.
Cellular distribution of hexahistidine-tagged wild type
and mutant G proteins was also examined in Sf9 cells.
In all cases, some portion of the expressed protein was found in all
three cellular fractions (Fig. 6, B and C),
but the majority was associated with membranes. Concurrent expression
of G
with
and
subunits did not alter the cellular distribution of the proteins
but did decrease (5-fold) the accumulation of G
. The
majority (50-70%) of each wild type and mutant protein associated
with membranes from the above samples could be extracted with sodium
cholate when expressed with
; a much smaller percentage of
each G
subunit (<10%) was extracted when
was not present (data not shown).
Figure 7:
Partitioning of
G, G
, and
amino-terminal mutants of G
into aqueous and
TX-114 rich phases. A, purified proteins including bovine
serum albumin (BSA, 10 µg), E. coli-derived
G
(nonmyristoylated (-myr), 2 µg;
and myristoylated (+myr), 2 µg) were subjected to
TX-114 phase partitioning as described under ``Experimental
Procedures'' (L, applied sample; W, aqueous
phase; and D, the detergent-rich phase). Equal volumes of
normalized (see ``Experimental Procedures''), recovered
fractions were resolved by SDS-PAGE and visualized by either Coomassie
Blue staining (for BSA) or by immunoblotting with anti-G
serum P960. B, Sf9 cells were infected with viruses
encoding G protein
and
subunits and
either G
CH6, G
-short, or C9A/C10A
G
. Infected cells were incubated with
[
H]palmitate, and G
proteins
were recovered using Ni-NTA affinity chromatography and elution in the
presence of TX-114. Samples were then subjected to phase partitioning,
and equal volumes of normalized fractions (see ``Experimental
Procedures'') were resolved by SDS-PAGE and visualized either by
immunoblotting with anti-G
serum WO82 or fluorography. C, purified G
CH6 and C9S/C10S
G
were activated with GTP
S and then incubated
without or with trypsin for limited digestion. Purified
G
CH6 was also treated with palmitoylthioesterase.
Treated samples were recovered by Ni-NTA affinity chromatography in the
presence of TX-114. Samples then were subjected to TX-114 phase
partitioning, and equal volumes of normalized fractions (see
``Experimental Procedures'') were resolved by SDS-PAGE and
visualized by immunoblotting with anti-G
serum
WO82.
Wild type and
nonpalmitoylated G proteins were synthesized in Sf9
cells in the presence of [
H]palmitate, isolated
by Ni-NTA chromatography, and subjected to TX-114 partitioning (Fig. 7B). As before, only the wild type protein
incorporated [
H]palmitate. Wild type and
nonpalmitoylated forms of G
partitioned roughly
equally between the aqueous and detergent phases when analyzed by
immunoblotting. However, nearly all of the
[
H]palmitate-labeled wild type protein was found
in the detergent phase. When compared with the properties of
myristoylated and nonmyristoylated G
, these results
suggest the existence of some factor(s) in addition to palmitate that
confers hydrophobicity on G
. To test this hypothesis
directly, we examined the behavior of purified G
treated with palmitoylthioesterase and amino-terminally truncated
forms of both G
and Cys
Ser G
(Fig. 7C). (
)The majority of wild type
G
was found in the detergent phase. A distinct and
reproducible shift was observed for both C9S/C10S G
and palmitoylthioesterase-treated wild type protein. Although the
majority of either of these preparations was found in the aqueous
phase, presumably due to loss of palmitate, a significant fraction
remained associated with detergent. Of interest, however, the truncated
forms of both G
and C9S/C10S G
were
found exclusively in the aqueous phase, indicating a loss of surface
hydrophobicity associated with removal of residues 1-34.
The amino-terminal domain of G and, more
specifically, residues Cys
and Cys
within this
domain are important determinants of both the cellular localization of
the protein and its interactions with phospholipase C-
1 and, to a
lesser extent, the m1 muscarinic receptor. The cysteine residues are
important per se. Their palmitoylation is not necessary to
observe characteristic interactions between G
and
phospholipase C-
1, although we cannot rule out possible inhibitory
effects of such modification. Both palmitoylation and some other
feature of the amino terminus confer hydrophobicity on G
and influence its cellular distribution.
Two prior reports
describe the failure of Cys and C
mutants of
G
to activate phospholipase C-
. In one case it
was hypothesized that this was due to loss of palmitate(25) ,
while in the other the defect was ascribed to loss of association of
G
with the membrane(23) . We ascribe this
phenomenon to loss of the cysteine residues themselves. Two lines of
evidence indicate that palmitate is not a major direct enhancer of the
interactions between G
and phospholipase C-
1 or
m1 receptors. First, removal of palmitate from G
with
palmitoylthioesterase did not alter its observed interactions with the
effector or receptor. Since we believe that the stoichiometry of
palmitoylation of the purified protein is significant (but not 1 or
greater, see below), we would have observed loss of a substantial
stimulatory effect of palmitate (but not necessarily loss of an
inhibitory one) upon removal of the fatty acid. Second, removal of the
first six residues of G
effectively prevents
palmitoylation of the protein without interfering with its interactions
with phospholipase C-
1 or m1 receptors. However, mutation of the
relevant Cys residues to either Ala or Ser impairs m1 receptor coupling
and causes apparent loss of affinity of G
for
phospholipase C-
1 and substantial loss of capacity to activate the
enzyme as well. Cys residues rather than palmitoylated Cys residues are
thus important. The role of palmitoylation of G
is in
some ways distinctly different from that of myristoylation of members
of the G
subfamily of G proteins; myristate is an
important determinant of the affinity of G
proteins
for both effectors and the
subunit
complex(8, 9) .
There are also similarities in the
effects of myristoylation and palmitoylation, in that both
modifications confer hydrophobic properties on the proteins involved
and facilitate their interactions with
membranes(10, 14, 37, 38) . A
significant fraction of both nonpalmitoylated mutants of G was found in the cytosol of transfected HEK-293 cells; in
contrast, all of the wild type protein was membrane associated. Nearly
all of purified G
labeled with
[
H]palmitate distributed to the detergent-rich
phase in TX-114 partitioning experiments. We have suggested that
amino-terminal acylation of G protein
subunits may not simply
facilitate interactions with lipid bilayers but may regulate
distribution of the proteins to specialized domains of the plasma
membrane such as caveoli(39) .
It has been difficult to
determine the stoichiometry of palmitoylation of G. We
have been unable to observe any alteration of electrophoretic mobility
attributable to palmitate; myristoylated G
subunits can
be distinguished from their nonmyristoylated counterparts in this
fashion. Mass spectrometric analysis has also been unsuccessful,
apparently because of inhibitory effects of residual detergent. The
best clue comes from TX-114 partitioning studies (Fig. 7).
Palmitoylated G
is found almost exclusively in the
detergent phase. Purified G
is distributed in both the
aqueous and detergent-rich phases, and treatment of the protein with
palmitoylthioesterase causes an observable (but not complete) shift of
protein to the aqueous phase. Estimates based on the extent of this
shift suggest a stoichiometry in the approximate range of 20-40%.
The precise role of Cys and/or Cys
is
unclear. Since the loss of either of these residues is well tolerated,
they are not involved in formation of a critical disulfide bond with
each other. They may be involved in direct intermolecular contacts
with, for example, phospholipase C-
or receptors or in
intramolecular interactions that are important determinants of
G
conformation. Higashijima and Ross (19) found that Cys
in G
(the
palmitoylated cysteine analogous to Cys
or Cys
in G
) interacts with Cys-substituted
mastoparans, small amphipathic peptides that mimic the effects of
receptors on G proteins. Similarly, Edgerton et al.(25) reported that Cys
and Cys
mutants
of G
were unable to interact with the NK2 receptor.
However, the same mutant G
protein had a relatively
modest loss of capacity to interact with m1 receptors when tested in
reconstituted systems (above). In all of these scenarios one might
suspect that stoichiometric palmitoylation of both cysteine residues
might inhibit the function in question. This may be true;
alternatively, stoichiometric palmitoylation at both sites may not be
possible.
The exact site of initiation of translation of
G (Met
or Met
) is unknown.
Direct amino-terminal sequencing of purified native protein was
unsuccessful because of an unidentified block. (
)Although
both forms of the protein are capable of the characteristic
interactions of G
with phospholipase C-
1 and m1
receptors, the shorter form of the protein is not palmitoylated. It is
not known if residues 1-6 constitute important sites for
recognition by a hypothetical protein palmitoyltransferase or if they
might be required for access of G
to the enzyme.
Perhaps similarly, myristoylation of G
proteins
greatly facilitates their subsequent
palmitoylation(13, 14, 40) .
Factors other
than palmitate clearly contribute to the hydrophobicity of
G. Nonpalmitoylated forms of the protein are still
associated with membranes to a large extent and, similarly, partition
partially into TX-114. Cleavage of the first 34 amino acid residues of
G
with trypsin eliminates the latter hydrophobic
behavior. Although it is conceivable that this domain might represent a
site of another lipid modification of a G
subunit, we
note that G
synthesized in E. coli (and thus
likely to be free of any such modification) has a strong tendency to
aggregate(29) . However, no clues are found in the
amino-terminal sequence of G
; 16 of the first 34 amino
acid residues are charged.