(Received for publication, August 14, 1995; and in revised form, September 12, 1995)
From the
The GTP binding G (transglutaminase II)
mediates the
-adrenoreceptor signal to a 69-kDa
phospholipase C (PLC). Thus, G
possesses both GTPase
and transglutaminase activities with a signal transfer role. The
recognition sites of this unique GTP binding protein for either the
receptor or the effector are completely unknown. A site on human heart
G
(hhG
) has been identified that
interacts with and stimulates PLC. Expressed mutants of hhG
with deleted C-terminal regions lost the response
to(-)-epinephrine and GTP and failed to coimmunoprecipitate PLC
by the specific G
antibody. The interaction regions
were further defined by studies with synthetic peptides of
hhG
and a chimera in which residues
Val
-Lys
of hhG
were
substituted with Ile
-Ser
residues of human
coagulation factor XIIIa. Thus, eight amino acid residues near the C
terminus of hhG
are critical for recognition and
stimulation of PLC.
The G protein, transglutaminase II (TGase II), (
)is unique in that the enzyme exhibits two distinct enzyme
activities, namely guanosine triphosphatase (GTPase) and TGase, with a
signal transfer role ((1) ; see also (2) and (3) ). The GTPase function of G
differs from
other TGases, coagulation factor XIIIa (FXIIIa), keratinocyte, and
epidermal transglutaminases (4) . G
, which is
species specific in molecular mass, directly interacts with
-adrenoreceptor (5, 6) and a 69-kDa
PLC in the activation process(7, 8) . Physiological
TGase role of G
remains unclear(4) . However,
it has been suggested that TGase II is involved in control of cell
growth and differentiation (9, 10, 11) and
activation of cytosolic phospholipase A
(12) .
The amino acid sequences of all TGases including G show high homology in the middle portions of the polypeptides,
which include the TGase active site and a calcium binding
region(13) . However, the N- and C-terminal regions of
G
do not share sequence homology among TGases. This
divergence is particularly greater at the C-terminal domain of
G
, giving rise to the hypothesis that this region may
play a significant role in hormone signaling. In this study, evidence
for a direct interaction between the region of G
and
PLC is demonstrated. This interaction activates PLC.
For the coimmunoprecipitation of the
-adrenoreceptor with hhG
and its
mutants, the membranes that coexpressed hhG
or its
mutants with
-adrenoreceptor or
-receptor alone were incubated with 5
10
M(-)-epinephrine at 4 °C for 3
h. The membranes were then solubilized with 0.2% SM in HSD buffer in
the presence of 5
10
M(-)-epinephrine at 4 °C for 1 h. The recovery of
proteins in the extracts usually reached
40% for the receptor and
40-50% for hhG
and its mutants (see also (5) ). Since the hhG
and its mutants were
overexpressed 3-5-fold as compared to the
-adrenoreceptor, the membrane extracts (100 fmol of
the
-receptor) were incubated with 100 µl of
G
antibody-agarose or nonimmune sera-agarose in 300
µl (final volume) with gentle rotation at 4 °C for 2 h. After
centrifugation at 2000 rpm, the
-receptor density (50
µl of supernatant/tube) was measured using 3 nM
[
H]prazosin (final) in the presence or absence of
10
M phentolamine in 200 µl (final
volume) after removing excess(-)-epinephrine through a dried 3-ml
Sephadex G-25 column. The preimmune sera-agarose-treated samples were
used as controls to calculate the amounts of the receptor
immunoprecipitated for each sample.
The isolated full-length hhG cDNA was an
exact match in the nucleotide and deduced amino acid sequences with the
human endothelial TGase II(14) . To identify interaction sites
of hhG
with the
-adrenoreceptor and
PLC, systematic 10-amino acid-deleted mutants of hhG
cDNA(s) were generated from the C-terminal end (Fig. 1).
The full-length hhG
cDNA and truncated hhG
cDNA(s) were cotransfected into COS-1 cells with
-adrenoreceptor cDNA. The expressed proteins were
recognized by the G
antibody as well as guinea pig
TGase II antibody and were of the expected sizes with
80 kDa for
full-length hhG
and a decrease in size as the length
of nucleotide deletion increased (Fig. 2A). The
-receptor was also expressed, resulting in 2-3
pmol/mg protein of [
H]prazosin binding. The
expressed hhG
proteins exhibited both GTP binding and
TGase activities (Fig. 2B and inset). The
Ca
-stimulated TGase activities of expressed
hhG
and its truncated mutants were completely
inhibited with >100 µM GTP, and the inhibitory potency
(IC
) of GTP was in the range of 20-50 µM for all hhG
proteins, also confirming that GTP is
a negative regulator for the TGase of
G
(1, 3, 4) .
Figure 1:
Map
of the 687-amino acid hhG chain and designation of
truncation mutants and hhG
/human FXIIIa chimera in
hhG
polypeptide.
Figure 2:
A, an autoradiogram of an immunoblot of
the expressed membrane proteins from COS-1 cells transfected with
hhG or mutant hhG
polypeptides using
polyclonal G
antibody. Molecular sizes (in
kilodaltons) are shown on the left. Endogenous G
(TGase II) is seen in the membranes from expressed
-receptor (
-AR) alone and mutants.
The protein expression level was
4-10-fold higher than the
endogenous G
. B, the GTP-mediated inhibition
of TGase activities of the expressed hhG
and mutants.
The inhibition of TGase activity (50 ng/tube) was determined with GTP
in the presence of 0.1 mM [
H]putrescine,
1% N,N`-dimethyl casein, 0.5 mM CaCl
, and 2 mM MgCl
in HSD buffer
at 30 °C for 30 min. The inset shows the stimulation of
TGase (50 ng/tube) in the presence of 0.5 mM CaCl
without GTP under the same conditions. The results are the mean
of three independent experiments performed in
duplicate.
All mutants,
as well as hhG, elevated the basal PLC activity as
compared to that of the
-receptor alone (Fig. 3A), indicating induction of precoupled protein
complexes resulting from overexpression of the
proteins(1, 18) . The(-)-epinephrine-mediated
activation of PLC was increased approximately 2-fold with
hhG
and the
K676 mutant, whereas the
L656
and
E646 mutants lost the agonist-mediated PLC stimulation,
exhibiting a level similar to that of the
-receptor
alone. The
N666 mutant stimulated PLC upon activation of the
-receptor, but to a lesser extent than wild type.
These data suggested that a region comprising 20 amino acids between
His
and Lys
is critical for coupling to the
-receptor or PLC.
Figure 3:
Coupling ability of expressed
hhG and its mutants. A,
epinephrine-stimulated inositol 1,4,5-triphosphate (IP
) accumulation in membranes from COS-1
cells coexpressed with
-receptor and hhG
or its mutants. The
-receptor-mediated PLC
stimulation was determined after normalizing receptor number (100
fmol/tube) in a 100-µl final volume. Receptor number was
normalized, since the receptor number is the determinant in signal
manipulation, not G-protein number(17) , and the expression
level of hhG
and its mutants was also 3-5-fold
higher than the receptor level. The results are the mean ± S.E.
of three independent experiments performed in duplicate.
-AR,
-adrenoreceptor; Ep, (-)-epinephrine; Ph, phentolamine. B, remaining
-adrenoreceptor in the supernatants after
coimmunoprecipitation with hhG
and its mutants using
G
antibody-protein A-agarose. The
-adrenoreceptor absorbed to preimmune protein
A-agarose was less than 5% as compared to the resin-untreated samples.
The preimmune-resin-treated samples were taken as 100% for each sample. C, immunoadsorption of a complex of PLC with hhG
and its mutants by G
antibody-protein
A-agarose. The PLC activity absorbed to preimmune protein A-agarose was
negligible and taken as nonspecific binding. The data shown are the
mean ± S.E. of three independent experiments in
triplicate.
The possibility that the
receptor and/or PLC recognition sites were deleted was examined by
coimmunoprecipitation. The mutants, K676,
N666, and
L656, coexpressed with the
-adrenoreceptor,
coimmunoprecipitated >90%
-receptor as wild type
did, indicating that the receptor interaction site on these mutants was
intact (Fig. 3B). However, the mutant,
E646,
consistently coimmunoprecipitated less receptor (
80%) than other
mutants but more than the receptor alone (
35%). Although less
coimmunoprecipitation of the receptor with this mutant suggested that
this region on hhG
might contain the receptor
interaction site, this point should be further investigated.
Coimmunoprecipitation of the receptor with membrane extract from the
expressed
-receptor alone was probably due to complex
formation between the internal G
and the receptor.
The loss of PLC interaction site was then assessed by
coimmunoprecipitation (Fig. 3C). The results revealed
that the K676 mutant coimmunoprecipitated PLC as effectively as
the wild type, whereas the
L656 and
E646 mutants failed to
coimmunoprecipitate PLC, showing a similar level to that of the
-receptor alone. The
L666 mutant again showed
lower coimmunoprecipitation of PLC than hhG
but higher
than the
L656 and
E646 mutants. The loss of the PLC
interaction site was further confirmed by determining PLC stimulation
in response to GTP (Fig. 4, A and B). As
expected, the basal levels of PLC in membranes expressing
hhG
and mutants were increased 3
6-fold compared
to the
-receptor alone. Within these increases, the
PLC basal activity gradually decreased as the deletion size increased (Fig. 4A). In the presence of GTP, the expressed
hhG
and the mutant
K676 increased PLC stimulation
2-fold (Fig. 4B). Increases in deletion size also
resulted in a gradual decrease of GTP-mediated PLC stimulation. Mutants
L656 and
E646 lost ability to stimulate PLC in response to
GTP. These results were consistent with the finding from the
coimmunoprecipitation studies and strongly suggested that a region
between His
and Lys
on hhG
contained a PLC interaction site.
Figure 4:
The GTP-mediated PLC activation. The
membranes (30 µg/tube) were preincubated with and without 0.1
mM GTP in the presence of 2 mM MgCl at 30
°C for 30 min. Production of IP
was measured with
various concentrations of CaCl
at 30 °C for 10 min. Panels A and B are shown in the absence and presence
of GTP, respectively. The data shown are the mean ± S.E. of
three independent experiments in duplicate.
To further define this
putative PLC interaction site, four overlapping peptides corresponding
to the deleted regions of hhG were synthesized and
tested for their ability to inhibit coimmunoprecipitation of PLC (Fig. 5A). Peptide 4
(Leu
-Lys
), among the four peptides, was
able to inhibit coimmunoprecipitation of PLC (Fig. 5B).
Coimmunoprecipitation of PLC was inhibited in a concentration-dependent
manner, and at 100-200 µM of the peptide, the
inhibition reached
80%, suggesting that other interaction site(s)
probably exist (Fig. 5C). The competition potency
(IC
) of peptide 4 for the interaction between
hhG
and PLC was
20 µM.
Figure 5:
A, map of the synthesized peptides of
hhG. Overlapping amino acids in peptides 3 and 4 are
indicated with asterisks. B, effect of peptides of
hhG
on coimmunoprecipitation of PLC by G
antibody-protein A-agarose. The membrane extracts (100
µg/tube) were preincubated with 100 µM peptide and
subjected to immunoprecipitation. PLC activity was determined as
detailed under ``Experimental Procedures.'' The results are a
mean ± S.E. of three independent experiments performed in
duplicate. C, competition of peptide 4 with hhG
to coimmunoprecipitate PLC by G
antibody-protein A-agarose. The experiments were performed with various
concentrations of peptide 4 under the same conditions as detailed under
``Experimental Procedures.'' The data presented are a mean of
the duplicated experiments using three independently expressed
proteins.
The
findings that a region of 12 amino acids between Leu and
Lys
in hhG
contains a PLC interaction
site were refined by a chimera, hhG
/FXIIIa, in which
eight amino acid residues Val
-Lys
of
hhG
were substituted with the corresponding region
(Ile
-Ser
) of human factor XIIIa (see Fig. 1)(13) . This region of FXIIIa was chosen because
FXIIIa does not interact with or stimulate PLC or bind GTP(4) ,
and this region of FXIIIa is distinct among TGases(13) . The
chimera was expressed
7-fold higher than endogenous G
and to the similar level of hhG
(Fig. 6A). The chimera protein exhibited GTP
binding and TGase activity at the same levels as the wild type (Fig. 6B). In addition, using the partially purified
chimera when the GTP-mediated inhibition of TGase activity was
titrated, the inhibition was similar to
K676 and wild type,
indicating that substitution of this region did not change GTP binding
affinity (data not shown). The chimera also failed to stimulate PLC in
response to GTP (Fig. 6C) and upon activation of the
-receptor (data not shown). The G
antibody did not coimmunoprecipitate PLC but effectively
coimmunoprecipitated the receptor (data not shown). These findings
clearly demonstrate that the C-terminal region of hhG
from Val
to Lys
is a critical site
for interaction and stimulation of PLC.
Figure 6:
Coupling ability of the expressed
hhG/FXIIIa chimera. A, an autoradiogram of an
immunoblot of the expressed membrane proteins from COS-1 cells
transfected with
-adrenoreceptor alone or with
wild-type or hhG
/FXIIIa chimera.
-AR,
-adrenoreceptor; WT, hhG
; Chi, hhG
/FXIIIa chimera. B, TGase
and GTP binding activity of the expressed wild-type or
hhG
/FXIIIa chimera in the membranes. Both enzyme
activities were determined in the presence or absence of 0.1 mM GTP as detailed in Fig. 2B. C,
GTP-mediated PLC stimulation. The PLC activity was measured using the
membranes (30 µg/tube) in the presence of 0.1 mM GTP, 2
mM MgCl
, and 10 µM CaCl
under the conditions detailed under ``Experimental
Procedures.'' The results are the mean ± S.E. of three
independent experiments performed in duplicate. IP
, inositol
1,4,5-triphosphate.
The substituted region of
the chimera has a significant change in properties of the amino acids (Fig. 1). Thus, four charged amino acids (Asn,
Glu
, Asp
, and Lys
) were
substituted for serine, except Asp
. Hydrophobic amino
acids (Val
, Val
, and Phe
)
were also changed to smaller (V666A and F668M) or larger (V665I) amino
acids. Although it has been suggested that replacement of a bulky side
chain of hydrophobic amino acids can result in loss of activity due to
unfavorable van der Waals interactions(19) , overall
hydrophobicity of the substituted amino acids, however, remains
similar. Therefore, it is unlikely that a hydrophobic interaction is
responsible for coupling of hhG
to PLC. Three charged
amino acids in this region, i.e. a hydrophilic interaction,
probably play a critical role in the contact of hhG
with PLC.
In heterotrimeric G-protein-mediated signaling
systems, the near C-terminal domain of the -subunit appears to
contain an effector contact region (adenylyl cyclase with
G
(20, 21, 22) and
cGMP-phosphodiesterase with G
(23) ). Despite
extensive primary structural differences between G
and
-subunits of the heterotrimeric G-proteins, our data indicate that
G
seems to share this common structural feature in
signaling. Our data also suggest that the carboxyl domain of
G
with its primary structure distinct from other
transglutaminases is likely to be involved in signaling functions,
including receptor and GTP binding sites.