(Received for publication, August 2, 1994; and in revised form, November 2, 1994)
From the
The existence of multiple ,
, and
subunits
raises questions regarding the assembly of particular G proteins.
Based on the results of a previous study (Rahmatullah, M., and
Robishaw, J. D.(1994) J. Biol. Chem. 269, 3574-3580), we
hypothesized that the assembly of G proteins may be determined by the
interactions of the more structurally diverse
and
subunits.
This hypothesis was confirmed in the present study by showing striking
differences in the abilities of the
and
subunits to interact with the the
subunit.
Chimeras of the
and
subunits were
used to delineate which region is responsible. Support for the
importance of the N-terminal region of the
subunit comes from our
observations that 1) the
subunit and the
chimera bound strongly to the
-agarose matrix, but the
subunit and
the
chimera bound weakly, if at all; 2) an
N-terminal peptide made to the
subunit blocked the
binding of the
chimera to the
-agarose matrix; 3) both the
chimera and the N-terminal peptide were able to partially protect
the
subunit against tryptic cleavage; and 4) the
chimera, but not the
chimera,
supported ADP-ribosylation of the
subunit.
The ability of cells to process a large number of extracellular
signals into the appropriate array of intracellular responses depends
on the types of signaling pathways that are expressed in the plasma
membranes of these cells. The most common type of signaling pathway
relies on the interaction of three kinds of proteins: receptors,
heterotrimeric G proteins, ()and effectors. The recent
identification of more than 100 different types of receptors, more than
320 possible combinations of G protein heterotrimers, and an unknown
number of effectors (1, 2) raises important questions
as to how these signaling pathways are assembled to ensure the correct
transmission of a signal from a particular receptor to a specific
effector. Given its position between the receptor and the effector, it
seems likely that much of the fidelity of signal transmission must
reside in the
subunit structure of the G protein.
While it has generally been assumed that a G protein should be
defined on the basis of the subunit, the rapidly growing number
of different
and
subunits suggests that a G protein should
more properly be defined on the basis of its unique combination of
subunits. This idea is supported by antisense
``knockout'' studies by Kleuss et
al.(3, 4, 5) , demonstrating different
combinations of G protein
subunits couple somatostatin
and muscarinic receptors to inhibition of the same Ca
channel. However, since the
and the
subunits
undergo an appreciable rate of dissociation in the course of detergent
extraction and purification, it has not been possible to confirm the
composition of a particular G protein by biochemical
studies. Furthermore, the tight association of the
subunits
has not permitted the regions of the individual
and
subunits that interact with the
subunits to be examined.
In a
previous paper(6) , we expressed the individual and
subunits in the baculovirus system and showed the direct association of
the
subunit with the
subunit of G
(
subunit). This result suggested the
possibility that selective association of the
and
subunits
might provide the mechanistic basis for the assembly of particular G
protein
subunit combinations. With the recent
identification of at least 10 different
subunits (
)(7) and with their expression in the baculovirus
system, it is now possible to examine the specificity of
and
subunit interactions in a systematic fashion. In the present
paper, we show differential association of the
and
subunits with the
subunit.
Furthermore, we identify the N-terminal 15 amino acids of the
subunit as the region responsible for conferring the specificity of
association with the
subunit, using a combination of peptide and
chimeric approaches.
Previously, we reported that the subunit
can stably interact with the
subunit of G
proteins(6) . This interaction was markedly enhanced by
prenylation of the
subunit but was not dependent on
the presence of the
subunit. This result raised the possibility
that selective interaction of particular
and
subunits may
provide the mechanistic basis for the assembly of specific
combinations of the G proteins. This possibility was
confirmed in the present paper by showing marked differences in the
abilities of the
and
subunits,
which are only 36% identical at the amino acid level(19) , to
interact with the
subunit (Fig. 1).
Figure 1:
Elution profile of the
and
subunits from the
-agarose column. A portion of the 1% cholate extract (LYS) from Sf-9 cells infected with either
or
was loaded onto the
-agarose column. The flow through (F) was
collected. Following successive washes with buffer D + 0.5% LPX
(high detergent wash, lane3) and buffer D +
0.05% LPX (lanes4-7), the
G
-agarose column was eluted with buffer E + 0.05%
LPX containing AMF (lanes 8-18). The elution fractions
(AMF) represent a pool of every three fractions after the addition of
AMF (lanes 8-11) and thereafter a pool of every five
fractions.
Figure 2:
Construction of chimeric subunits.
The regions of the
and
subunits
that were switched to generate the chimeric
and
subunits are shown. To monitor the expression and
purification of the
,
, and
subunits, an antibody raised to a peptide common to
these subunits was used, as shown by the stippledareas.
The elution
profiles of the cytosolic and particulate fractions from
-infected cells are shown in Fig. 3. Both the
cytosolic and particulate forms of the
subunit were
detected in the flow through and 0.5% Lubrol wash fractions (lanes2 and 3) but not in the subsequent 0.05% Lubrol
wash fractions (lanes4-7) or AMF elution
fractions (8-18) (panelsA and B, topinsert). The elution profiles of the
cytosolic and particulate fractions from
-infected
cells were similar. Using the same immunoblotting conditions that were
used for the detection of the
subunit, both the
cytosolic and particulate forms of the
chimera were
detected in the flow through and 0.5% Lubrol wash fractions (lanes2 and 3) but not in the AMF elution fractions (lanes8-18) (panelsA and B, middleinserts). However, after prolonged
exposure of the autoradiogram for 5 days, we observed a low but
detectable amount of the particulate form of the
chimera in the AMF elution fractions (data not shown). The
presence of the
chimera was further confirmed by
concentration of the AMF elution fractions followed by detection with
antisera. The same procedure failed to detect either the cytosolic or
particulate form of the
subunit in the AMF elution
fractions (data not shown). Taken together, these results suggest that
the
subunit shows no detectable binding to the
-agarose matrix, while the
chimera
shows very low binding.
Figure 3:
Elution profile of the chimeric
subunits from the
-agarose column. A portion of the 1%
cholate extract (LYS) from cytoplasmic (panelA) or membrane (panelB) fractions from
Sf-9 cells infected with
(top),
(middle), or
(bottom) was loaded onto the
-agarose
column. The flow through (F) was collected. Following
successive washes with buffer D + 0.5% LPX (high detergent wash, lane3) and buffer D + 0.05% LPX (lanes
4-7), the G
-agarose column was eluted with
buffer E + 0.05% LPX containing AMF (lanes 8-18).
The elution fractions (AMF) represent a pool of every three fractions
after the addition of AMF (lanes 8-11) and thereafter a
pool of every five fractions.
In contrast, the elution profiles of the
cytosolic and particulate fractions from chimera-infected cells were markedly different (Fig. 3).
Using the same immunoblotting conditions that were used for the
detection of the
and
subunits, a
significant portion of the cytosolic form of the
chimera was stably retained on the
-agarose
matrix and specifically eluted with buffer containing AMF (panelA, bottominsert). Interestingly,
elution with AMF appeared to result in the resolution of the cytosolic
form of the
chimera into two peaks (lanes8-9 and 13-16, respectively).
Although the reason for this is not known, the two peaks may be related
to the nature of the prenyl group. In support of this possibility, we
showed that the cytosolic form of the
subunit is
modified by both farnesyl and geranylgeranyl moieties when expressed in
Sf9 cells.
Since the
chimera contains
the prenylation recognition sequence of the
subunit,
it is likely that the
chimera is similarly modified
by both farnesyl and geranylgeranyl groups. Thus, the two peaks may
represent forms of the
chimera with different
prenyl moieties. Likewise, a significant portion of the particulate
form of the
chimera was also selectively retained
and specifically eluted with AMF (panelB, bottominsert). However, unlike the cytosolic form, the
particulate form of the
chimera was eluted in a
single peak (lanes8-10). Again, this may be
related to the nature of the prenyl group and/or the extent of carboxyl
methylation.
Nevertheless, it is clear from these results
that replacement of the N-terminal 18 amino acids of the
subunit with the corresponding 15 amino acids of the
subunit is sufficient to confer the specificity of interaction
with the
-agarose matrix.
Figure 4:
Elution profile of the subunit from the
-agarose column in the presence
of N-terminal or mid-region
peptides. The
-agarose column was loaded with 8 ml of a 0.15 mM solution (in buffer D + 0.05% LPX) of peptide 1 (panelB) or peptide 2 (panelC) and allowed
to sit for 1.5 h prior to loading with the cytoplasmic form of the
chimera. PanelA shows retention
and elution of the
subunit in the absence of
peptides, while panelsB and C show
retention and elution of the
subunit in the
presence of the N-terminal and internal
peptides,
respectively.
Figure 5:
Effect of varying molar ratios of
or
on tryptic cleavage of
G
and G
. Purified G
(panelA) or G
(panelB) was mixed with varying concentrations of the
affinity-purified
or
subunit
for 30 min at 4 °C and 10 min at room temperature prior to addition
of trypsin at a constant protein:trypsin ratio of 50:1. Digestions were
carried out for 60 min at 30 °C. The stoichiometries of
:
or
:
were 1:0.25 (lane3), 1:0.5 (lane4), 1:1 (lane5), and 1:2 (lane6). Lanes1 and 2 represent
or
in the absence or presence of trypsin, respectively,
in the absence of any
subunit.
We also
decided to examine whether the addition of the chimera was equally able to protect the
subunit of G
(
subunit) against tryptic cleavage. As shown in panelB of Fig. 5, the
subunit (lane1) was digested rapidly by
trypsin (lane2). Moreover, even at the highest
stoichiometry of
to
of 2:1 (lane6), the
chimera showed no
protection of the
subunit against tryptic
proteolysis. Taken together, these results eliminate the possibility of
the
subunit acting as an inhibitor of trypsin, and as such,
protecting the
subunit. More importantly, these results suggest
that structural differences in the
subunits as well as the
subunits are critical in specifying their interactions.
Figure 6:
Effect of N-terminal and
peptides on tryptic cleavage of G
.
Purified G
(3.3 µM) was incubated with
peptide 1 (N-terminal
peptide) or peptide 3
(N-terminal
peptide) for 1.5 h at 4 °C and 10 min
at room temperature prior to addition of trypsin at a constant
:trypsin ratio of 50:1. Digestions were carried out
for 60 min at 30 °C. From left to right:
in absence of trypsin (lane1) and
presence of trypsin (lane2); trypsin digestion of
in presence of 13.4 µM (lane3) and 84 µM (lane4)
peptide 1; and trypsin digestion of
in presence of
13.4 µM (lane5) and 84 µM (lane6) of peptide
3.
Figure 7:
Support
of ADP-ribosylation of the subunit by
and
subunits. Purified G
(0.55 µg) was
ADP-ribosylated in the presence of
,
,
, or
subunits for 20 min at varying molar ratios of
to
subunits. Assay conditions were described
under ``Experimental Procedures.'' From left to right:
alone (lane 1) and in the
presence of
(lanes
2-4) or
(lanes
5-7) at molar ratios of 0.2, 0.5, and 1, respectively, to
or in the presence of
(lanes
8-11) or
(lanes 12-15) at
molar ratios of 0.3, 0.9, 1.8, and 3, respectively, to
.
The existence of multiple ,
, and
subunits,
many of which are expressed in the same tissue, raises questions
regarding the assembly of G proteins with distinct roles in
receptor-effector coupling. Hypothetically, the assembly of particular
combinations of
,
, and
subunits might be regulated by
differential expression of these subunits in various cell types and/or
differential affinities of these subunits to associate with each other.
If we extrapolate from previous findings on
-
(21, 22) and
-
(6) interactions, it
seems likely that the assembly of particular combinations of
,
, and
subunits may be determined by their differential
affinities. It further seems plausible that functional diversity of the
G proteins may be conferred by the specific assembly of the more
structurally diverse
and
subunits rather than the less
structurally diverse
subunits. This hypothesis was confirmed in
the present study by showing striking differences in the abilities of
the structurally diverse
and
subunits to interact with the
subunit.
In an earlier report(6) , we showed that
prenylation of the subunit is important for
-
interaction. The present results extend that observation. Following
expression in Sf9 insect cells, the
subunit is
modified exclusively by a geranylgeranyl moiety, whereas the
subunit is modified by farnesyl and geranylgeranyl
moieties in roughly equal proportions.
Nevertheless, even
though a significant proportion of the
subunit is
modified by a geranylgeranyl group, the
subunit
cannot interact with
subunit within the limits of
detection. Thus, the N-terminal region of the
subunit rather than
prenylation of the C-terminal region plays the primary role in
determining the specificity of the
-
interaction. However, it
is possible that the nature of prenyl group may play a secondary role
in determining the specificity and/or stability of the
-
interaction. Nevertheless, it is clear from these studies (6) that both a favorable N-terminal region and a prenylated
C-terminal region of the
subunit are needed for optimal
-
association. Interestingly, a recent study by Neer and
Spring (20) has implicated the middle region of the
subunit in determining the specificity of
-
interaction.
Taken together, these results would predict a model in which the N- and
C-terminal regions of the
subunit come together to form the face
for
interaction while the middle region forms the opposing face
for
interaction.
The results of the present study support the
computer-based prediction of the N-terminal regions of both and
subunits as their possible sites of interaction. In this regard,
a computer-based analysis of coiled coil forming probabilities has led
to the hypothesis that G protein heterotrimers form via triple-stranded
coiled coil interactions involving the N-terminal regions of the
,
, and
subunits(23) . However, this is the first
experimental evidence to support the involvement of the N-terminal
region of the
subunit in this interaction.