(Received for publication, December 6, 1994; and in revised form, January 6, 1995)
From the
Monoclonal antibody, LAS-2, directed against the subunit
of transducin (G
), inhibited
G
-dependent, pertussis toxin-catalyzed ADP
ribosylation of G
and was specific for
G
. Immunoblotting studies on proteolytic fragments of
G
were consistent with an amino-terminal epitope. To
define the antibody recognition site, recombinant G
was synthesized in Escherichia coli cotransfected with
or without yeast N-myristoyltransferase. Amino-terminal fatty
acylation of G
, verified by use of radiolabeled fatty
acid, was required for immunoreactivity. LAS-2 did not react with a
chimeric protein consisting of residues 1-9 of G
and the remainder G
, regardless of its
myristoylation. Immunoreactivity was observed when amino acids
1-17 of G
were present in a G
chimera and the protein was amino-terminally myristoylated; there
was no reactivity without myristoylation. It appears that the LAS-2
epitope requires both G
-specific sequence in amino
acids 10-17 and a fatty acyl group in proximity to these
residues. These results are consistent with the hypothesis that the
myristoyl group is essential for protein structure; conceivably it
``folds back'' on and stabilizes the amino-terminal structure
of G
, as opposed to protruding from an amino-terminal
-helix and serving as an amino-terminal membrane anchor.
Heterotrimeric guanine nucleotide-binding proteins (G proteins), ()consisting of
,
, and
subunits, couple
cell surface receptors to effectors(1) . GTP/GDP exchange and
GTP to GDP hydrolysis, which regulate G protein activity, occur on the
subunit (G
) and require the presence of receptor
and the
subunit
(G
)(2, 3, 4, 5) .
The amino-terminal domain of G is important for
interaction with G
. Removal of 21 amino acids from
the amino terminus of the
subunit of transducin
(G
), using Staphylococcal V8 protease, inhibited
-dependent reactions including GTP hydrolysis, GTP/GDP
exchange, and binding of G
to rhodopsin(2) .
Trypsin, which removes the first 18 amino acids of G
,
destroyed the ability of
to stimulate pertussis
toxin-catalyzed ADP-ribosylation of G
(6) as
did amino-terminal deletions in a recombinant
G
(7) . Amino acids 7-10 of recombinant
G
were found to be critical for its interaction with
G
(8) .
Cotranslational acylation of the
amino-terminal glycine with myristate occurs in several of the
G subunits (9, 10, 11, 12) and facilitates
their membrane attachment. Removal of the myristoylated amino terminus
of G
by tryptic digestion produced a soluble protein
from one that was originally membrane bound(13) . G
modified with hydrophilic myristate analogues was less likely
than native G
to associate with membranes (9) . Myristoylation also appears to play a role in
protein-protein interaction(14, 15) . Both
-stimulated ADP-ribosylation and affinity for
-Sepharose were significantly greater with myristoylated than
nonmyristoylated G
(8, 16) . N-Fatty acylation of G
was found important
for its interaction with G
(17) . Based on
these data, it has been postulated that the myristoyl moiety may be
involved in stabilizing the structure of the amino terminus. We
describe here an anti-G
monoclonal antibody, LAS-2,
whose epitope involves the G
-binding domain of
G
and requires amino-terminal fatty acylation as well
as specific G
amino-terminal sequence.
Murine monoclonal antibodies against purified bovine
G were screened for functional effects on G
and G
interaction. One clone, LAS-2,
inhibited G
-stimulated ADP-ribosylation of
G
in a concentration-dependent manner; control
immunoglobulin of the same isotype (IgG
) and LAS-1 did not
inhibit the reaction (Fig. 1).
Figure 1:
Inhibition by LAS-2 of
G-dependent pertussis toxin-catalyzed
ADP-ribosylation of G
. Transducin was incubated with
LAS-1, LAS-2, or mouse IgG
at 4 °C for 1 h before
initiation of pertussis toxin-catalyzed ADP-ribosylation, which was
carried out at 30 °C for 30 min in 50 mM potassium
phosphate (pH 7.5), 10 µM [
P]NAD (2
µCi/assay), 1 mM ATP with 2 µg of activated pertussis
toxin in total volume of 100 µl. Pertussis toxin was activated for
10 min with dithiothreitol immediately before use(6) . Proteins
were precipitated with 7.5% trichloroacetic acid and separated by
SDS-PAGE in 12% polyacrylamide gels followed by autoradiography with
Kodak XAR film and an intensifying screen at -70 °C for 30
min. Lane1, 3 µg of indicated antibody; lane2, 10 µg of indicated antibody; lane3, 25 µg of indicated antibody. Cont.Ig,
control Ig.
To define the LAS-1 and -2
epitopes better, G was cleaved with endoprotease
Arg-C(23) , hydroxylamine at alkaline pH (24) , or
trypsin(22) ; the fragments were separated by SDS-PAGE,
transferred to nitrocellulose, and reacted with antibodies LAS-2 and
AS/7. Endoprotease Arg-C digestion generated LAS-2-reactive fragments
of 34, 23, and 15 kDa, all of which were refractory to microsequencing,
consistent with the presence of a blocked amino terminus (Fig. 2A). Hydroxylamine at alkaline pH cleaves
primarily at Asn-Gly bonds(24) ; cleavage of G
(Asn
-Gly
) produced 32-kDa
amino-terminal and 7-kDa carboxyl-terminal fragments. Both LAS-1 and -2
reacted with the 32-kDa piece, whereas AS/7 did not, confirming that
the LAS-reactive fragment lacked the carboxyl terminus (Fig. 2B, data not shown). Tryptic digestion of
G
initially removes a 2-kDa amino-terminal
fragment(22) ; this modification abolished immunoreactivity
with LAS-2. The presence of the remaining 37-kDa fragment was verified
by reactivity with the carboxyl-directed polyclonal antibody AS/7 (Fig. 2C). These findings all suggested that the LAS-2
epitope was located within the 18 amino-terminal amino acids removed by
trypsin, in agreement with the functional data obtained with
G
(2, 3, 4, 5) .
Figure 2:
Immunoreactivity of LAS antibodies. A, reaction of LAS-2 with submaxillary Arg-C-digested
G. G
(5 µg) in buffer A (20
mM Tris (pH 7.5), 0.5 mM MgCl
, 0.05
mM EGTA, 0.5 mM NaN
, 1 mM dithiothreitol) was incubated with or without 0.625 µg of
Arg-C for 4 h at 37 °C; the reaction was stopped by the addition of
SDS-sample buffer and boiling for 10 min. Proteins and proteolytic
products were separated in 16% polyacrylamide gels and transferred to
nitrocellulose, which was then stained with Ponceau S or reacted with
LAS-2. Lane1, purified G
stained
with Ponceau S; lane2, Arg-C-treated G
stained with Ponceau S; lane3, Arg-C-treated
G
reacted with LAS-2. a-c indicate
LAS-2-reactive peptides subjected to sequencing by Edman degradation
(see text). Positions of protein standards (kDa) are on the left. B, reaction of antibodies with
hydroxylamine-treated transducin. 25 µg of G
in 50 µl of buffer A plus 58 µl of 3.75 M hydroxylamine, pH 9.25, were incubated at 37 °C for 3 h.
15-µl samples of this mixture were subjected to SDS-PAGE in
4-20% NOVEX gradient gels and then were stained with Coomassie
blue or used to transfer separated fragments to nitrocellulose for
reaction with antibodies. Positions of standard proteins are on the left; G
, G
,
G
, and 32 kDa are indicated in the center. Lane-, 2.5 µg of G
; lane+HA, hydroxylamine treatment, both stained
with Coomassie blue; laneLAS1, blot of
hydroxylamine-treated G
reacted with LAS-1; laneAS7, blot of hydroxylamine-treated
G
reacted with AS7. C, reaction of
LAS-2 with G
after tryptic digestion. G
(4 µg) in 40 µl of buffer A was incubated with or without
TPCK-treated trypsin (4 µg/ml) for 45 min at 30 °C,
precipitated with 10% trichloroacetic acid, subjected to
electrophoresis in 10-20% Tricine gradient gel, and transferred
to nitrocellulose. Transferred proteins and fragments were stained with
Amido Black (PRO) or reacted with LAS-2 or AS7 antibodies.
Presence or absence of trypsin is indicated by a (+) or (-),
respectively. Positions of G
, and 37-, 21-, 16-, and
5-kDa fragments are indicated at right.
Further mapping was done using recombinant proteins expressed in E. coli. G cDNA inserted into a pGEX-2T
expression vector (Pharmacia) yielded high levels of rG
in a fusion protein with glutathione S-transferase(31) . Despite reacting with AS/7, this
fusion protein failed to react with LAS-2 both before and after
cleavage of the thrombin-sensitive link between glutathione S-transferase and G
(data not shown). An
amino-terminally myristoylated rG
was synthesized in E. coli by coexpressing G
and yeast N-myristoyltransferase(32) . Myristoylation of
rG
was verified by detection of
H-labeled
G
after addition of [
H]myristic
acid during protein synthesis. This myristoylated rG
reacted with LAS-1 and -2 on immunoblots. rG
synthesized without N-myristoyltransferase was not
labeled with [
H]myristate and did not react with
LAS-2, although it did react with AS/7 and another transducin
monoclonal antibody, 6E4 (27) (Fig. 3). Fidelity of
expression and lack of proteolytic modification of the unmyristoylated
rG
was verified by Edman degradation though five
cycles, yielding the expected Gly-Ala-Gly-Ala-Ser sequence (Harvard
MicroChem).
Figure 3:
Requirement for amino-terminal
myristoylation of G for LAS reactivity. Transducin and
recombinant proteins, prepared as under ``Methods'' were
suspended in sample buffer, subjected to SDS-PAGE in 12%
polyacrylamide, transferred to nitrocellulose, and reacted with LAS or
6E4 antibodies. Amounts of G
(
4 µg) in the
three lanes were similar based on Coomassie blue staining. Lane1, G
; lane2, nonmyristoylated recombinant G
; lane3, myristoylated recombinant
G
.
The LAS-2 antibody was very specific for
G. It reacted with only a single band from a bovine
retinal homogenate (Fig. 4A) and did not react with
G
, G
, or G
on
immunoblots (Fig. 4B). A recombinant
G
, expressed in E. coli, did not react with
LAS-2 either in a nonmyristoylated state or when coexpressed with N-myristoyltransferase and myristoylated (Fig. 4C, lanes1 and 2). A
chimeric protein, G
9-G
, consisting of
G
sequence in amino acids 1-9, and the
remainder G
sequence was constructed by replacing
Cys
, Thr
, and Leu
of G
with Ala
, Gly
, Ala
. This
protein was myristoylated when coexpressed with N-myristoyltransferase but did not react with LAS-2 either
with or without myristoylation (Fig. 4C, lanes3 and 4). A chimeric protein in which the first
21 amino acids of G
were replaced with the first 17
amino acids of G
(G
17-G
) did react with LAS-2,
but only when myristoylated; the nonmyristoylated
G
17-G
did not react (Fig. 4, lanes5 and 6).
Figure 4:
Immunoreactivity of LAS antibodies. A, specificity of LAS-2 for G among bovine
retinal proteins. Bovine retinas were homogenized in 0.25 M sucrose, 10 mM Tris-HCL (pH 7.5), 10 mM EDTA.
The homogenate was centrifuged (30 min/14,000
g).
Supernatant proteins (100 µg) were precipitated with 7.5%
trichloroacetic acid, suspended in 20 mM phosphate buffer (pH
7.5), subjected to SDS-PAGE (16% polyacrylamide), transferred to
nitrocellulose, and stained with Ponceau S (lane1)
or reacted with LAS-2 (lane2). B,
specificity of LAS-2 monoclonal antibody. G
and
G
were prepared as under ``Methods.''
G
and G
were isolated from bovine
brain(21) . G
was a generous gift from Dr.
Victor Rebois of NINDS, National Institutes of Health. 1 µg of each
of the proteins was subjected to SDS-PAGE (12% polyacrylamide),
transferred to nitrocellulose, and reacted with LAS-2. Protein transfer
was verified with 6E4 anti-G
(27) , pcGo
anti-G
(30) , and RM/1 anti-G
(DuPont NEN) antibodies. Lane1, bovine
G
; lane2, bovine
G
; lane3, bovine brain
G
; lane4, bovine brain
G
; lane5, G
. C, reactivity of LAS-2 with rG
and
G
-G
chimeric proteins. rG
and chimeric G
-G
in whole
bacterial lysates were subjected to SDS-PAGE, transferred to
nitrocellulose, and reacted with LAS-2. There was approximately 1
µg of each recombinant protein in each lane. Lane1, myristoylated G
; lane2, rG
; lane3,
myristoylated G
9-G
; lane4, G
9-G
; lane5, myristoylated G
17-G
; lane6, G
17-G
; lane7, protein standards (97, 68, 43, 29, and 18
kDa).
The LAS-1 antibody shown
in Fig. 1reacted in a manner identical to LAS-2 on all
immunoblots including those performed with proteolytic digests of
G, native G
proteins, and recombinant
proteins. It did not, however, interfere functionally with
G
-G
interaction as judged by
inhibition of
-dependent ADP-ribosylation. Why two antibodies
with seemingly identical mapping properties differ functionally is
unclear, but it may have to do with differing affinities for
G
.
It appears that monoclonal antibody LAS-2, a
G-specific monoclonal antibody, which interferes with
G
-G
interaction, has an epitope
that includes specific G
amino acid sequence within
positions 10-17 and requires a fatty acyl group in proximity to
these residues. Just as the first 9 amino acids are not specific for
G
binding(17) , they do not encompass the
LAS-2 epitope, which lies further downstream. The amino terminus of
bovine G
is modified in a heterogeneous manner by
lauroyl (C:12), myristoyl (C:14), (cis-
-5)-tetradecaenoyl
(C:14:1), or (cis,cis-
5,
8)-tetradecadienoyl (C:14:2) fatty
acids(17, 33) . Kokame et al.(17) reported that the length of the amino-terminal fatty
acid was important for the ability of nonapeptides to inhibit the
interaction of G
and G
, although
no requirement for specific amino acid sequence was observed.
Upon
exchange of GDP for GTP, G disassociates from
G
, and a conformational difference in crystal
structures of these two nucleotide-bound states has been documented (34, 35) . The proteins crystallized, however, lacked
the amino-terminal 25 amino acids and thus lacked the
G
-binding domain and the LAS epitope studied
here. It has been postulated that myristate may act to stabilize an
amino-terminal amphipathic helix and participate in protein
function(38) . Recoverin, a retinal protein that is
heterogeneously fatty acylated in a manner similar to
G
, exposed hydrophobic acyl groups in response to
Ca
binding, thus behaving as a
Ca
-myristoyl protein
switch(36, 37) . ADP ribosylation factor, a
myristoylated
20-kDa GTP-binding protein involved in cellular
protein transport may utilize a GTP-myristoyl switch; the myristate may
participate in the reversible GTP-dependent association of ADP
ribosylation factor with membranes(37, 38) . The fatty
acyl group in the N-myristoylated G
proteins
may play a similar role, depending on whether GTP or GDP is bound. Our
findings suggest that within the G
amino-terminal
domain the fatty acid may ``fold back'' on and stabilize
peptide structure, consistent with the hypothesis that the fatty acid
does more than merely project from the protein into the lipid bilayer
to serve as an amino-terminal membrane anchor.