(Received for publication, July 13, 1995; and in revised form, December 14, 1995)
From the
The functional organization of G is poorly understood.
Regions of bovine brain G
that interact with a photoaffinity
derivative of an
-adrenergic receptor-derived peptide
from the third intracellular loop (diazopyruvoyl-modified peptide Q
(DAP-Q)) and a hydrophobic membrane probe
(3-trifluoromethyl-3-(m-iodophenyl)diazirine (TID)) were
examined. We previously showed that DAP-Q cross-links to specific,
competable sites on both the
and
subunits of
G
/G
but not on the
subunit and that
subunit was required for stimulation of
G
/G
GTPase activity (Taylor, J. M., Jacob
Mosier, G. G., Lawton, R. G., Remmers, A. E., and Neubig, R. R.(1994) J. Biol. Chem. 269, 27618-27624). Similarly, we show
here that the membrane-associated photoprobe
[
I]TID labels
and
but not
. We
have now mapped the sites of incorporation of DAP-Q and TID into the
subunit. TID labels both the 14-kDa amino-terminal and the 23-kDa
carboxyl-terminal fragments from a partial tryptic digest of
while DAP-Q labels only the carboxyl-terminal fragment. Further mapping
with endopeptidase Lys C reveals substantial labeling of multiple
fragments by TID while DAP-Q labels predominantly a
6-kDa fragment
within the carboxyl-terminal 60 amino acids of
. Thus,
regions within the 7th (or possibly 6th) WD-40 repeat of the
subunit of G protein interact with the receptor-derived peptide while
membrane interaction involves multiple sites throughout the
subunit.
Heterotrimeric G proteins (composed of ,
, and
subunits) transmit intracellular signals from a family of plasma
membrane-associated G protein-coupled receptors (GPCR). (
)This family includes adrenergic receptors, photoreceptors,
and growth factor receptors among others. Binding of ligand causes a
conformational change in the receptor, which activates the associated G
protein. The activated G protein dissociates into an
subunit and
a
subunit complex. Both the
and
subunits
are able to activate intracellular effector enzymes (1) .
The GPCRs have seven transmembrane helices with three cytoplasmic
loops and an intracellular carboxyl terminus(2) . Mutagenesis (3, 4) and competition studies using synthetic
peptides (5, 6, 7, 8, 9) suggest
that the i3 loop and possibly the second cytoplasmic loop and
carboxyl-terminal tail are important for receptor-G protein
interactions. Peptide Q is a tetradecapeptide from the
carboxyl-terminal part of the i3 loop of the AR.
Peptide Q can inhibit
AR-G
coupling and
can also mimic GPCRs by binding to and activating G protein
directly(5, 8, 10) .
We have previously
described a photoaffinity label (DAP-Q) prepared by coupling the
sulfhydryl-reactive Br-DAP to the G protein activator peptide (peptide
Q)(11) . DAP-Q and a radioiodinated derivative
([I]pHBDAP-Q) cross-link at nanomolar
concentrations to specific, competable sites on both the
and
subunits but not on the
subunit of
G
/G
(12) . Also, a functional
interaction between DAP-Q and
is required for
DAP-Q-stimulated GTPase activity. Binding of DAP-Q to the amino
terminus of
(12) is consistent with a number of reports
implicating the closely associated amino and carboxyl termini of
subunits in binding
receptor(9, 13, 14, 15, 16, 17) .
In addition to its role in membrane association of
subunits(18) , much recent evidence supports a direct
interaction of the
subunit complex in the coupling of
receptors to G proteins. Binding of purified
-adrenergic receptors and rhodopsin with their
respective G protein
subunits has been
demonstrated(19, 20) . Kleuss et al.(21, 22) have shown that antisense probes
directed against the
subtype or the
subtype can block muscarinic receptor inhibition of calcium
currents while probes directed against
or
block somatostatin receptor inhibition of calcium
currents(21, 22) . Also, Kisselev and Gautam (23) have shown that rhodopsin binds to a G protein containing
but not
or
subunits. The isoprenoid-modified carboxyl terminus of
subunit is important for coupling to rhodopsin(24) .
The
subunit is composed of seven highly conserved WD-40 repeat
regions characterized by a Gly-His followed by 23-41 core amino
acids and a WD (Trp-Asp)(25) . The function of these WD-40
repeats is not known, but it has been proposed that the repeats are
important for protein-protein interactions(25) . The specific
regions of
subunits participating in either receptor coupling or
membrane association remain to be identified. In this report we have
mapped the major binding site on the
subunit for the G protein
activator peptide (DAP-Q) and have begun to localize labeling sites for
the membrane-associated photoprobe [
I]TID.
Proteolytic cleavage with Lys C was performed using the Cleveland
method(36) . [I]pHBDAP-Q (3
µM) or 1 µM [
I]TID
was photolyzed with purified
(14 µM or 50
µg/lane) in 0.1% Lubrol or azolectin vesicles, respectively. The
labeled
subunit was separated from the
subunit and
non-incorporated label by 12.5% SDS-PAGE. The
subunit was
identified by staining control lanes with Coomassie Blue, and the
corresponding regions of the unstained radiolabeled sample were
excised. The excised bands were then applied to a 16% Tricine gel in
the presence of endoproteinase Lys C (20:1, protein:enzyme), and the
gel was electrophoresed according to the method of
Cleveland(36) .
Several lines of evidence indicate that the
subunit complex binds GPCRs and is important for signal
transduction(19, 20, 21, 22, 23, 24, 35) .
We recently reported that the photoactive
AR-derived
i3 loop peptide, DAP-Q, labels a highly specific site within the
amino-terminal 17 residues of
(12) . DAP-Q
also cross-links to a specific site on the
subunit (12) (see Fig. 1C), and we use this specific
labeling to map a potential receptor-interacting region of the
subunit. In contrast to the specificity of labeling by DAP-Q, labeling
of
and
subunits by the membrane-associated photoprobe
[
I]TID is not blocked by excess unlabeled
compound (Fig. 1A). This lack of competition is
characteristic of membrane-exposed sites(39, 40) .
Interestingly, the
subunit did not label with
[
I]TID (Fig. 1B). While the
isoprenoid tail of
is required for
association with
membranes(41) , it is possible that the lipid tail is not
sufficiently large or reactive to incorporate significant
[
I]TID, or it may be involved in protein
folding or conformation rather than direct lipid interactions.
Figure 1:
Incorporation of
[I]TID or [
I]pHBDAP-Q
into G protein subunits. G
was labeled in azolectin
vesicles with 1 µM [
I]TID (A and B) or in Lubrol with 3 µM [
I]pHBDAP-Q (
73 Ci/mmol) (C)
as described under ``Materials and Methods.''
[
I]TID-labeled samples (A and B) were separated on a 16% Tricine gel, and
[
I]pHBDAP-Q-labeled samples (C) were
separated on a 10% Laemmli gel. Radioactivity was detected with a
PhosphorImager. Excess non-radioactive TID (100 µM) or
peptide Q (750 µM) was added where indicated to test
specificity. A and B are from the same exposure of a
single gel indicating that labeling of
subunit is minimal
compared with that of
and
.
The
functional significance of the specific DAP-Q binding site(s) on
subunit is supported by the absolute requirement of the
subunit for stimulation of
subunit GTPase
activity (12) . Thus, to begin to map the major binding sites
on
for DAP-Q and TID we examined their cross-linking to
trypsin-treated
subunit. Trypsin treatment of native
subunit results in cleavage of
at Arg
,
which generates a 14-kDa amino-terminal fragment and a 23-kDa
carboxyl-terminal fragment. Thomas et al.(35) have
shown trypsin treatment of native
subunits does not disrupt
tertiary structures or the ability of the complex to associate
functionally with the
subunit. For this experiment, the ability
of DAP-Q to induce a gel shift of the G protein
subunit was
utilized. Fig. 2shows that DAP-Q photolabels the 23-kDa
carboxyl-terminal trypsin fragment (A) but not the 14-kDa
fragment of the
subunit (B). It is also evident from Fig. 2A that DAP-Q cross-links equally efficiently to
the 23-kDa fragment of trypsin-treated
and to the uncleaved
subunit. (
)In contrast, [
I]TID
labels both the 14- and 23-kDa fragments (Fig. 2C).
Quantitation reveals 23 ± 4% (n = 2) as much
labeling of the 14-kDa fragment as the 23-kDa fragment, consistent with
significant degrees of membrane contact for both fragments.
Figure 2:
Photolabeling trypsin-digested
subunit with DAP-Q. Purified
(100 nM or 0.85
µg/lane) in Lubrol (A, B) or azolectin (C) was digested with TPCK-treated trypsin (1:100, w/w) as
described under ``Materials and Methods.'' After 30 min, the
reaction was stopped by adding soybean trypsin inhibitor (10:1,
inhibitor:trypsin). The trypsin digest was then photolyzed with DAP-Q
(1 µM) or 1 µM [
I]TID. The products were separated by 16%
Tricine gel electrophoresis, transferred to Immobilon, and analyzed by
Western blot using a carboxyl-terminal anti-
antibody (A), stained with colloidal gold (B), or scanned with
a Molecular Dynamics PhosphorImager (C). The data are
representative of four (A and B) or two (C)
separate experiments.
indicates the position of the
native (37 kDa)
subunit, whereas
and
indicate the positions of the carboxyl-terminal
23-kDa fragment and the amino-terminal 14-kDa fragment. * indicates the
position of a DAP-Q cross-linked product. T.I. indicates the
position of soybean trypsin inhibitor.
To
further localize the major binding sites on the subunit we
labeled purified
with [
I]pHBDAP-Q in
Lubrol and digested the products with endoproteinase Lys C (20:1,
protein:enzyme) according to Cleveland et al.(36) . Fig. 3shows the results of a partial endoproteinase Lys C
digestion of [
I]pHBDAP-Q-labeled
subunit.
The majority of the radioactivity migrates as a
6-kDa fragment of
the
subunit (lane A, solid arrow). The silver
stain of this gel (lane C) reveals a number of
fragments
ranging from 28 to 1 kDa. Complete digestion of the
subunit
should result in fragments ranging from 10.4 to 0.4 kDa. Thus the high
molecular weight fragments in lane C show that the
subunit does not digest to completion under these conditions. Labeling
of
by [
I]TID showed a much more extensive
distribution of label (Fig. 3, lane B). The
6-kDa
fragment incorporated a significant amount of
[
I]TID, but there were 5 additional fragments
clearly labeled.
Figure 3:
Partial Lys C digest of purified
labeled with [
I]pHBDAP-Q. Purified
subunit (14 µM) was photolyzed in the presence
of [
I]pHBDAP-Q (A and C, 3
µM,
73 Ci/mmol) or [
I]TID (B, 1 µM), and the products were electrophoresed
on 12% SDS-PAGE. The cross-linked
subunit was excised, and the
gel slices were loaded onto a 16% Tricine gel in the presence of Lys C
as described under ``Materials and Methods.'' The
polyacrylamide gel was either fixed and exposed to a PhosphorImage
screen (A and B) or silver-stained (C). The filled arrow indicates the
6-kDa fragment labeled with
DAP-Q, and the open arrows indicate two large fragments, which
were not labeled with DAP-Q (see text). The data are representative of
three separate experiments.
To identify the position of the 6-kDa fragment
within the sequence of the
subunit, we examined the size of
predicted Lys C fragments that overlap the 23-kDa COOH-terminal tryptic
fragment of the
subunit. Only labeling within residues
281-341 in
or 302-341 in
predicts labeled fragments smaller than 8.9 kDa (Fig. 4). (
)Since labeling within 281-301 (light gray in Fig. 4) would generate a radiolabeled fragment of
approximately 11.5 kDa from the
subunit, which is not
observed, it is likely that both subunits are labeled within residues
302-341 (black in Fig. 4). However, the
6-kDa apparent molecular mass of the radiolabeled fragment is
consistent also with cross-linking to
in the region
of 281-301 provided that cleavage at Lys
does not
occur.
Figure 4:
Linear model of the subunit showing cleavage sites and the location of the DAP-Q
labeling site. A linear model of
subunit is shown
indicating the 23-kDa carboxyl-terminal trypsin digestion fragment.
Within this fragment the Lys C cut sites are shown by residue number
following the cut. WD-40 repeat regions are indicated by the numbered boxes. The location of the DAP-Q labeling site is
highlighted with the black region being the most likely site
while the gray region cannot be absolutely
excluded.
The absence of labeling within
-(128-281) or
-(128-301)
is further supported by the major non-labeled fragments of
approximately 16.5 and 19 kDa on the silver stain (Fig. 3C, open arrows). These are similar to
the expected masses of 16,671 of
-(128-280) and
19,077 of
-(128-301). These partial Lys C digest
fragments would be generated if cleavage did not occur at
Lys
, which may be buried in the fourth WD-40 domain.
In summary, trypsin digest data indicate that the only sites of
subunit labeling by DAP-Q are within the carboxyl-terminal region
of the
subunit. Lys C digestion shows that the majority of label
migrated at
6 kDa. Therefore, the radiolabeled Lys C fragment
corresponds to a site within residues 302-341 of either
or
or possibly 281-301 of
(Fig. 4). The region of DAP-Q labeling
includes WD-40 repeat 7 and the connecting loop to 6 and possibly
repeat 6 itself (Fig. 4). Interestingly, the DAP-Q binding site
on
overlaps with the
binding site on
, which has been
shown to include portions in the carboxyl-terminal half of
(42, 43, 44) .
In contrast to the
limited region of contact with the receptor peptide, the membrane
contact sites on subunit appear to be much more extensive. They
encompass both the amino- and carboxyl-terminal tryptic digest
fragments, and within these major fragments several small Lys C
fragments are labeled. Further mapping will be required to define the
details of membrane association as has been done for the Torpedo
nicotinic acetylcholine receptor(40) .
Functional data
support DAP-Q as an appropriate tool for identifying receptor
interaction sites on the subunit. Our previous results showed
that
was required for peptide stimulation of
GTPase activity(12) , just as is true for receptor
activation of purified
subunits of G proteins(45) . In
addition to the site on
described in this report, we have shown
that DAP-Q labels the amino terminus of
(12) .
This same NH
-terminal region on
has been shown to
bind to mastoparan (46) and to disrupt rhodopsin-transducin
interactions(9) . Although the regions on the
subunit
that bind to
are not well defined, Neer and colleagues (47, 48) have shown that residues 204 and 271 in the
carboxyl-terminal 23-kDa fragment of
can be
chemically cross-linked to the
subunit.
The sequences of
subunits are highly conserved and display approximately 83% amino acid
identity among the known subtypes(49) . In fact, within the
region 281-341 only 8 amino acids are different among
,
, and
(the most
abundant subtypes). Are these few differences sufficient for
somatostatin and muscarinic receptors to recognize different
subunits(21) ? As noted above, DAP-Q and the
subunit bind
to overlapping regions on the
subunit. Thus, it is possible that
the
subunit could provide an additional level of specificity to
the
AR-G protein interaction. In accordance with this
hypothesis, somatostatin receptors, muscarinic receptors, and rhodopsin
all distinguish between G proteins composed of different
subunits (22) . Unlike the
subunits,
subunits display great
sequence diversity with only 30% homology between the known
subtypes(49) .
This report is the first to define regions of
the subunit that interact with a receptor-derived G protein
activator and a membrane-associated photolabel. Based on these data and
that of many other groups, it is likely that the binding site for
receptor involves interactions with
,
, and
subunits
with the carboxyl terminus of
playing a significant role.