From the
The identification of multiple G protein
GTP binding regulatory proteins (G proteins) are heterotrimers
consisting of three subunits,
Findings from several laboratories have demonstrated
selectivity in
Soluble
The first set of nonisoprenylated
In order to analyze the individual contribution of residues within
this triplet of amino acids, a series of point mutants were made
exchanging individual residues in this region between
The substitution of progressively smaller linear segments of
While it
seems likely that this region of
A model of
The three-residue
domain of
Because this
study of
An alignment of the amino acid sequences predicted
by cDNAs for bovine
We thank Dr. J. Hurley for the generous gifts of
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
and
subunit
subtypes suggests a potential diversity of
heterodimers,
which may contribute to the specificity of signal transduction between
receptors and effectors. The assembly of
and
subtypes is
selective. For example,
can assemble with
but not with
, whereas
assembles with both
isoforms. To identify the structural
features of the
and
subunits governing selectivity in
heterodimer assembly, a series of nonisoprenylated chimeras of
and
was constructed, and their
interaction with
and
was assessed
by their ability to direct
expression to the cytosol in
cotransfected COS cells. All of the
/
chimeras were capable of interacting with
as
judged by the cotransfection assay. Chimeras containing
sequence near the middle of the molecule between two conserved
sequence motifs were capable of interacting as well with
. Among 12 divergent residues in this region, it was
found that replacement of three consecutive amino acids in
, Glu-Glu-Phe (residues 38-40), with the three
corresponding amino acids of
, Ala-Asp-Leu (residues
35-37), conferred the ability to assemble with
.
The reciprocal chimera containing Glu-Glu-Phe in the context of
failed to assemble with
. The last
residue of this triplet is occupied by a leucine in all known mammalian
subunits except
and appears to be a key
determinant of the ability of a
subunit to assemble with
. This locus maps to a region of predicted
-helical structure in the
subunit, likely to represent a
point of physical contact with the
subunit.
,
, and
(1) . The
and
subunits exist as a tightly bound complex that can only
be dissociated under denaturing conditions and that functions as an
entity throughout the signaling cycle
(2, 3) . The
formation of
heterodimers from particular combinations of
and
subtypes may contribute to signaling specificity as the
complex interacts with G
, effectors, and receptors
(4, 5) . We developed a transient transfection paradigm
that allows us to assess the ability of different
and
subunits to dimerize by monitoring the cytosolic expression of
subunits upon cotransfection with nonisoprenylated
constructs
(6) . This assay was recently used to study truncated and
point-mutated
subunits
(7) , leading to the development of
a model for
interaction in which the N terminus of
forms a coiled coil-like structure with
. Pronin and Gautam
(8) used a similar paradigm to study subunit specificity in
heterodimer formation. Using this and other approaches,
several laboratories have demonstrated that
interacts
with
but not with
, whereas both
subunits interact with
and
(8, 9, 10) . This paradigm was recently
used to map the domains of the
subunit responsible for
discrimination between
and
using a
series of
/
chimeras
(11) .
With respect to the
subunits, there is only 38% sequence identity
at the amino acid level between
(12) and
(13, 14) , and little is known about
the domain or domains that are responsible for their differential
ability to assemble with
. We report here the
construction of a series of
/
chimeras and point mutants that map the region responsible for
discrimination to a short stretch of three amino
acids in a predicted
-helical domain. This putative
-contact
site lies just C-terminal to a proposed coiled coil region of the
subunit.
Construction of Expression Vectors
Chimeric and
mutant cDNA constructs were made by the polymerase chain reaction
(15) using Pyrococcus furiosus DNA polymerase
(Stratagene, La Jolla, CA) and wild-type bovine (16) , human
(17) , bovine
(12) , or
(13) as
the original templates. The 3` primers for the
constructs
contained mutations in the codon for C AAX
(
)
domain cysteine resulting in prenylation-deficient mutants
(C71S in
and C68S in
(6) ).
The mutagenic oligos used to generate chimeric
components created
junctions as follows: ``QLK'' junctions, after Lys codon AAG
(residue 23 in
, 20 in
);
``EDPL'' junctions, after Leu codon TTA (residue 53 in
, 50 in
) in chimeras
112*,
212*, and
2
(21) 2*; ``EDPL'' junctions,
before Glu codon GAG (residue 50 in
, 47 in
) in chimeras
121*,
221*, and
1
(12) 1*; ``VSK'' junctions, before Val codon GTG
(residue 33 in
, 30 in
). Cluster and
point mutants of
subunits utilized the corresponding
codons of
and vice versa. Polymerase chain
reaction amplification of the
cDNAs was used to eliminate the
untranslated flanking sequences. Polymerase chain reaction products
were purified, digested, and ligated into pCDM8.1
(18) according to standard procedures
(19) . Plasmids
were amplified in Escherichia coli MC1061/P3 (InVitrogen, San
Diego, CA) and purified by a commercially available column adsorption
method (Qiagen, Chatsworth, CA). The DNA sequences of the inserts were
verified by the method of chain termination
(20) using
Sequenase 2.0 (U. S. Biochemical Corp.).
Cell Culture and Transfection
COS-7 cells were
cultured in Dulbecco's modified Eagle's medium with 10%
fetal bovine serum and 100 units/ml penicillin and 100 units/ml
streptomycin. Cells were transfected at approximately 80% confluency
with DEAE-dextran (500 µg/ml) in phosphate-buffered saline (PBS)
without Caand Mg
(Biofluids,
Rockville) according to standard procedures
(21) . Typically 5
µg of each
construct and 15 µg of each
construct
was used per 75-cm
culture flask except where indicated.
The total amount of DNA added was kept constant by the addition of
vector DNA as needed. Cells were harvested after 2 days, washed in
phosphate-buffered saline, pelleted, and kept at -80 °C prior
to analysis.
Protein Analysis
Cells were lysed and fractionated
into a particulate and soluble fraction as described
(6) .
Protein concentrations were determined according to Lowry with bovine
serum albumin as standard
(22) . Limited tryptic digestion of
soluble proteins was accomplished using
L1-tosylamido-2-phenylethyl chloromethylketone-treated trypsin
(Sigma T-8642) at a ratio of 1:20 or 1:25 (w/w) by incubation at 37
°C for 30 min, at which time the reaction was terminated by the
addition of denaturing sample buffer and boiling
(23, 24, 25) . Proteins were separated on
polyacrylamide gels under denaturing conditions and transferred to
polyvinylidene difluoride membranes by electrotransfer for 12 h at 150
mA in Dunn's buffer
(26) . Analysis of subunits was
performed on 10 or 11% polyacrylamide gels
(27) , whereas
analysis of
subunits employed tricine gels according to the
method of Schägger and von Jagow
(28) . Immunoblotting was
performed as described previously
(29) . The primary antibodies
used were directed against residues 330-340 of
(the sequence is identical in
) (SW), residues
58-68 of
(PE), residues 53-70 of
(LVKG), or residues 47-64 of
(EDPL). Primary antibodies were detected by autoradiography of
blots following incubation with
I-labeled protein A as
described
(7) .
heterodimer assembly so that while both
and
can assemble with
or
-C68S (
2*),
but not
can form heterodimers with
or
-C71S (
1*)
(8, 9, 10) . To
determine which residues in
were responsible for its
inability to interact with
, we made chimeras between
1* and
2* with the aim of identifying the minimal sequence of
, which might confer
interaction in
the context of a
1* polypeptide. For convenience in this paper,
chimeras are identified by subscripts in a three- or four-digit binary
code in which 1 indicates that
sequence was used, 2
that
sequence was used. A schematic overview of the
chimeras employed in this study is presented in Fig. 1, A and B. Three-part chimeras were initially constructed
using the conserved sequence motifs Gln-Leu-Lys (QLK, residues
21-23 in
and 18-20 in
)
and Glu-Asp-Pro-Leu (EDPL, residues 50-53 in
and 47-50 in
) as junctions between
chimeric components (Fig. 1 A). Thus chimera
121*
refers to the chimera containing
sequence from the N
terminus to the QLK motif and from the EDPL motif to the C terminus,
with the
sequence in between. A second set of
chimeras further divided the middle segment into two regions with a
junction at the Val-Ser-Lys sequence motif (VSK, residues 33-35
in
and 30-32 in
)
(Fig. 1, B and C). These chimeras are described
by a four-digit subscript such that chimera
2
(12) 2* refers
to the nonisoprenylated chimera containing
sequence
from the N terminus to the QLK motif and from the VSK motif to the C
terminus, with the
sequence between.
Figure 1:
Schematic overview of the
chimeric constructs employed in this study and comparison of
and
sequence homology around a
putative
contact site. A and B, schema
depicting the structure of the
/
chimeras employed in this study. Open bars indicate polypeptide sequence derived from
, and
closed bars indicate that derived from
. Junctions between chimeric segments indicated by
vertical lines correspond to sequence motifs
conserved in both
and
as follows:
Gln-Leu-Lys (QLK, residues 21-23 in
and
18-20 in
), Glu-Asp-Pro-Leu (EDPL, residues
50-53 in
and 47-50 in
),
and Val-Ser-Lys (VSK, residues 33-35 in
and
30-32 in
). Numbers to the right of each construct correspond to its descriptive subscript,
employing a 3- or 4-digit binary code as explained in the text. Before
C-terminal processing,
contains 74 and
contains 71 residues. C, sequence homologies between the
conserved VSK and EDPL motifs of
and
as indicated by the algorithm GAP of the Genetics Computer Group
(39). Boxed residues represent the region containing
a putative
contact site identified in this study. Numbers in the margins indicate residue
position.
To study the
subunit selectivity of the chimeras, they were cotransfected into COS-7
cells with either or
.
assembly was determined by assessing the ability of these
nonisoprenylated mutant
subunits to direct expression of
subunit to the soluble protein fraction
(6, 7) .
Recombinant
and nonisoprenylated
subunits associate in a
stable soluble complex
(10, 30) , and trypsin treatment
of a COS cell cytosolic fraction containing
* produces a
stable 26-27-kDa C-terminal
fragment characteristic of
native
complexes (Ref. 31 and see below). Transfection of
or
cDNA alone produced an increase
in
immunoreactivity over vector-transfected controls in the crude
particulate fraction (Fig. 2, A and B,
lanes 3 and 5, cf. lane 1) but not in the soluble fraction (Fig. 2, A and B, lanes 4 and 6). The
increase in
immunoreactivity in the crude membrane fraction due
to transfection of
cDNA alone was more marked than observed
previously
(6) but is consistent with work from other
laboratories
(31) . While these differences in the crude
membrane fraction may result from variations in the efficiency of
separation of the low speed nuclear pellet from the postnuclear
fractions
(6) , they were not explored further in this study.
Figure 2:
Selectivity in assembly
demonstrated by the cytosolic shift assay. COS cells were transfected
with vector alone ( Con) or with
or
cDNAs alone or in combination with
-C71S (
1*) ( A) or
-C68S
(
2*) ( B) cDNAs as indicated. After fractionation into
particulate ( P) and soluble ( S) components, aliquots
of 50 µg were analyzed by SDS-polyacrylamide gel electrophoresis
and immunoblotting with the
/
C-terminal antibody SW ( upper panels, on 10%
polyacrylamide gels) or the
-directed C-terminal antibodies PE
(
C-terminal) or EDPL (
C-terminal)
( lower panels, on tricine gels (28)) as described
under ``Experimental Procedures.'' Shown are 24-h
autoradiograms after incubation with
I-labeled protein A.
To the left, the relative mobility of marker proteins is shown in kDa
(36/35 kDa, the SW-reactive
doublet from bovine brain; 6.5 kDa,
aprotinin). The data shown are representative of five such experiments
with similar results.
Cotransfection in COS cells of with either
1*
or
2* resulted in the expression of both
and
subunits
in the cytosol (Fig. 2, A and B, lanes 8). In contrast,
cotransfection with
2* but not
1* resulted in the cytosolic expression of both
subunits (Fig. 2, A and B, lanes 10), confirming the previously described incompatibility
of
with
and
1*
(8, 9, 10) . It should be noted that in
cotransfected cells, the appearance of significant
immunoreactivity in the cytosol correlates with the presence of a
signal there (Fig. 2, A and B, cf.
lanes 8 and 10). This results presumably
from subunit stabilization engendered by cotransfection of compatible
and
subunits as recently described
(32) . Subsequent
screening of nonprenylated
chimeras for their ability to assemble
with
or
was therefore performed by
analysis of cytosolic fractions only.
heterodimers containing nonisoprenylated
subunits show no
functional interaction with effector molecules such as adenylyl cyclase
(10) and phospholipase C-
(33, 34) nor
with G
(10) , although reports to the contrary have
appeared
(35) . To verify that the global folding of the
subunits in the soluble nonprenylated
complex mimics that of
functional
complexes, analysis by limited tryptic digestion
was performed (Fig. 3)
(23, 24, 25) . A
26-kDa C-terminal
fragment was protected from trypsin in the
cytosolic fraction of COS cells cotransfected with
1*,
2*, and
2*, which comigrated with the fragment generated
by identical treatment of authentic brain
(Fig. 3)
consistent with previous reports
(31) . Prior heat denaturation
abolished the trypsin resistance of the C-terminal fragments
(Fig. 3). These findings support the contention that soluble,
nonprenylated
complexes resemble native
heterodimers in the global conformation of their subunits.
Figure 3:
Limited tryptic digestion of COS cell
cytosolic fractions. Soluble fractions (25 µg of protein) of COS
cells transfected with vector alone (-) or with cDNAs for
or
in combination with
-C71S (
1*) or
-C68S (
2*)
were either left on ice or incubated with trypsin (1 µg) for 37
°C for 30 min either without or with prior heat treatment at 80
°C (5 min) as indicated ( lanes 1-10).
Samples were subsequently analyzed by SDS-polyacrylamide gel
electrophoresis (11% polyacrylamide) and immunoblotting with the
C-terminal antibody SW as described under ``Experimental
Procedures.'' Lanes 11-13 show the results
of an identical analysis of purified bovine brain
(
) performed in parallel (200 ng combined with 25
µg of control COS cytosol/lane). Autoradiograms were exposed for 14
h. To the left are indicated the relative mobility of
untreated
subunit and the major
26 kDa
tryptic
fragment. The experiment was repeated three times with similar
results.
Because
our strategy was to estimate the ability of chimeras to assemble
with
subunits by monitoring cytosolic
immunoreactivity, we
sought experimental conditions that would minimize variability because
of unequal distribution of
and
cDNAs during cotransfection
or due to small variations in the quality of chimeric
cDNA
plasmid preparations. We wanted conditions therefore in which the
amount of
but not
cDNA would be limiting with respect to
the expression of cytosolic
subunit. Under these conditions, any
observed differences in soluble
expression among cotransfectants
employing different mutant
constructs would be more likely to
reflect differences in subunit expression and protein-protein
interaction. To establish these conditions, a constant amount of
cDNA was cotransfected with increasing amounts of
2* cDNA and the level of cytosolic
expression monitored.
was chosen because the gain of
interaction by
1* mutants substituted with
-specific residues was the principal end point of
mutagenesis. The
signal generated from 5 µg of
cDNA became saturated at 10-15 µg of
2* cDNA (not
shown). For subsequent screening of
/
chimeras, therefore, a 3:1 ratio of
to
cDNA (w/w) was
employed.
chimeras was
constructed using the conserved QLK and EDPL sequence motifs as
junctions dividing the
subunit into three regions
(Fig. 1 A). Transfection of vector or
1 cDNA alone
produced no
immunoreactivity in the cytosol (Fig. 4,
A, lanes 1 and 2) as previously
shown. In contrast cotransfection of
1 with
1* (
111*),
2* (
222*), and each of six three-part chimeras,
121*,
211*,
221*,
112*,
122*, and
212*, all promoted
the appearance of cytosolic
-immunoreactivity
(Fig. 4 A, lanes 3-10 upper panel). Immunoblotting with
-specific C-terminal
antibodies confirmed the expression of all of the chimeric constructs
(Fig. 4 A, lanes 3-10, lower panels). In contrast to the results with
, only chimeric
constructs
121*,
221*,
and
122* along with
2* (
222*) supported cytosolic
expression when
was cotransfected (Fig. 4 B,
lanes 4, 6, 8, and 10).
These results focussed our attention on the region of the
subunit
between the QLK and EDPL sequence motifs as chimeras containing
but not
sequence here proved
capable of assembling with
.
Figure 4:
Selective cytosolic expression of
subunit isoforms cotransfected with different
1*/
2* chimeras.
COS cells were transfected with vector ( Con) or
cDNA
alone or
cDNA in combination with
1* (
-111*),
one of six
1*/
2* chimeras, or
2* (
-222*).
The nomenclature and composition of the
1*/
2* chimeras are
explained in the text and the legend to Fig. 1 A. Shown are
24-h autoradiograms of immunoblots of the cytosolic fractions of
experiments employing
( A) and
( B) cDNAs. The antibodies employed are as explained in
the legend to Fig. 2. Three other experiments produced an identical
expression pattern.
A second set of
chimeras derived from
121* and
212* was created by
subdividing the middle region around a conserved Val-Ser-Lys (VSK)
sequence motif (Fig. 1 B). Like the parental chimera
121* (
1
(22) 1*), chimera
1
(12) 1* was
capable of directing the expression of both
and
to the cytosol (Fig. 5 A, upper panel, lanes 5, 6, 8,
and 9). Similarly, expression of both chimeras
1
(22) 1* and
1
(12) 1* was demonstrable in
immunoblots of cells cotransfected with either
or
(Fig. 5 A, lower panel, lanes 5, 6, 8,
and 9). In contrast chimera
1
(21) 1* displayed a
1*-like phenotype, interacting with
but not with
(Fig. 5 A, lanes 11 and
12). These results suggested that residues within the region
between the VSK and EDPL motifs of
and
determine the ability to dimerize with
(Fig. 1 C). Consistent with this interpretation was
the finding that chimera
2
(12) 2* but not
2
(21) 2* (Fig. 1 B) interacted with
in the cytosolic shift assay (data not shown). These results
confirm a recent report by Spring and Neer
(36) who utilized
subunit chimeras synthesized by in vitro translation to
map the
discrimination region to this same 14-amino acid region
(Fig. 1 C). As noted previously with nonchimeric
*
subunits
(7, 11) , when transfected alone, none of the
three
* chimeras produced significant cytosolic
or
immunoreactivity (Fig. 5 A, lanes 4,
7, and 10).
Figure 5:
Selective cytosolic expression of
subunit isoforms cotransfected with different
*/
* chimeras or
*-derived cluster mutants. COS cells were transfected
with vector (-),
,
, or
cDNA alone or the combination of
or
cDNA with
121* (1(22)1) or one of the
1*/
2* chimeras
1(12)1* or
1(21)1* ( A) or one of the
1*-derived cluster mutants
1*-ADL,
1*-MAYC, or
1*-AHAK ( B) as indicated. The nomenclature and
composition of the
1*/
2* chimeras and cluster mutants is
explained in the text. Shown are 24-h ( A) or 14-h ( B)
autoradiograms of immunoblots of cytosolic fractions (50
µg/ lane) employing antibodies SW (
) or LVKG
(
C-terminal) as indicated. Similar results were
obtained in three additional experiments.
Among the 14 amino acids that lie
between the conserved VSK and EDPL sequence motifs, 12 residues differ
between and
(Fig. 1 C). A pair of cysteine residues at
positions 36 and 37 in
, represented by twin alanine
residues in
, can be linked by disulfide bond
formation to Cys
of the
subunit in the
presence of cupric phenanthroline
(37) . This pair of
cysteines lies closely apposed to the N-terminal
-helical region of
in a recent model of
interaction based on a coiled coil interaction
(7) . In a study
of the effect on
assembly of point mutations in the putative
coiled coil region of
, it was found that mutation of
both Cys
and Cys
to Ala failed to confer the
ability to assemble with
.
(
)
The
10 remaining divergent residues in
were therefore
replaced with
sequence in three mutant constructs,
each containing a cluster of three or four point mutations. In mutant
2*-ADL, residues 38-40 of
1*, Glu-Glu-Phe, were
replaced with Ala-Asp-Leu. Mutant
1*-MAYC was made by replacing
residues 41-44 of
1* with the sequence Met-Ala-Tyr-Cys (the
Tyr is conserved in both
and
). In
mutant
1*-AHAK residues 46-49 were replaced by the
sequence Ala-His-Ala-Lys. Fig. 5 B shows the
results of cotransfection of the three
2* cluster mutants with
and
. When transfected alone, none
of the three
2* cluster mutants produced significant cytosolic
or
immunoreactivity (Fig. 5 B, lanes 4, 7, and 10). While cotransfection of
all three cluster mutants with
resulted in the
cytosolic expression of
immunoreactivity, only
2*-ADL
produced a significant cytosolic
signal when the cotransfection
employed
(Fig. 5 B, upper panel, lane 6, cf. lanes 9 and 12). As expected, steady-state
chimera immunoreactivity in the cytosol of
-cotransfected cells correlated with the strength of
the
signal and was greatest in the case of
2*-ADL (Fig.
5 B, lower panel, lane 6,
cf. lanes 9 and 12). These
observations identify this triplet of amino acids as the dominant locus
between the conserved VSK and EDPL motifs of
and
determining the ability to interact with
(Fig. 1 C). This region forms part of an
-helix in both
and
according
to secondary structure predictive algorithms
(38, 39) .
1* and
2*. Point mutants E38A, E39D, and F40L of
1* were made and
transfected alone or in combination with either
or
(Fig. 6 A). When transfected alone, none of
the three point mutants of
1* produced significant
or
immunoreactivity in the cytosol (Fig. 6 A, lanes 4, 7, and 10). Cotransfection of all
three
1*-derived point mutants with
, however,
resulted in a significant cytosolic shift of
(Fig. 6 A, upper panel, lanes 5, 8, and 11) and definite
-immunoreactivity (Fig. 6 A, lower panel, lanes 5, 8, and
11). Upon cotransfection with
, only
1*-F40L interacted significantly as seen in both
and
blots (Fig. 6 A, lane 12, cf.
lanes 6 and 9). When residues from this
region of
were substituted into
2* to create
2*-EEF and
2*-L37F, these mutants, like the parental
construct
2*, were expressed and directed
-immunoreactivity
to the cytosol when cotransfected with
but not when
transfected alone (Fig. 6 B, lanes 4,
7, and 10 cf. lanes 5,
8, and 11). Both
2*-EEF and
2*-L37F,
however, demonstrated a greatly reduced ability to interact with
by comparison with parental
2*
(Fig. 6 B, lanes 6 and 9 cf. lane 12). Construct
2*-EEF was
somewhat more selective against
than the L37F point
mutant as evidenced by both
and
immunoblots
(Fig. 6 B, lane 6 cf. lane 9). The
2*-derived point mutants A35E and D36E
interacted significantly with
in similar experiments
(not shown).
Figure 6:
Selective cytosolic expression of
subunit isoforms cotransfected with point mutants of
1* or
2*. COS cells were transfected with vector (-),
,
, or
cDNA alone or the
combination of
or
cDNA with the
1*-derived point mutants E38A, E39D, or F40L ( A) or the
2*-derived mutants
2*-EEF or L37F or the parental construct
2* ( B) as indicated. Shown are 7-h ( A) or 17-h
( B) autoradiograms of SW (
), LVKG (
C-terminal) or EDPL (
C-terminal) immunoblots of
cytosolic fractions (50 µg/ lane) as indicated. Two
additional experiments yielded essentially the same
results.
The iterative process of 1* mutagenesis employed
in this study identified the chimera or mutant best able to interact
with
at any given stage by comparison with
``sibling'' mutants containing similar amounts of
sequence. A side-by-side longitudinal comparison of the entire
series of
-interacting
1* chimeras and mutants
revealed however that the
1*-F40L point mutant did not
consistently support the cytosolic expression of
to
the same extent as
2*,
121*,
1
(12) 1*, or
1*-ADL (Fig. 7 A, lanes 3, 5,
7, and 9 cf. lane 11). To
exclude the possibility that the chimeric and mutant derivatives of
1*, which directed
expression to the cytosol,
did so by virtue of an anomalous interaction with the
polypeptide, a limited trypsin digestion of cytosolic fractions from
cells cotransfected with
and
121*,
1
(12) 1*,
1*-ADL, or
1*-F40L was performed
(Fig. 7 A). In all cases, a
26 kDa C-terminal
fragment of
was protected from further degradation,
which comigrated with the C-terminal
fragments derived from
coexpressed
2* treated in parallel
(Fig. 7 A, lanes 6, 8,
10, and 12 cf. lane 4).
Tryptic analysis of cytosolic fractions from cells cotransfected with
and
2*-EEF or
2*-L37F similarly
demonstrated preservation of a
26-kDa C-terminal
fragment (Fig. 7 B). These results confirm the
native folding of the heterodimers formed between
subunits and
these mutant derivatives of
1* or
2*.
Figure 7:
Limited tryptic digestion of COS cell
cytosolic fractions cotransfected with or
and mutant derivatives of
1* or
2*. COS
cells were transfected with vector alone (-) or with cDNAs for
or
in combination with
1*,
2*,
121*,
1(12)1*,
1*-ADL, or
1*-F40L
( A) or
2*,
2*-EEF, or
2*-L37F ( B) as
indicated. Soluble fractions (25 µg of protein) were either left on
ice or incubated with trypsin (1 µg) at 37 °C for 30 min where
indicated. Shown are 12-h ( A) or 17-h ( B)
autoradiograms of SW (
C-terminal) immunoblots employing different
batches of [
I]-protein A. Similar results were
obtained in two other experiments.
into the context of a nonprenylated mutant of
has allowed the identification of a short segment of
predicted
-helix three residues in length, which confers the
ability to form heterodimers with
. This locus is
contained within the 14-amino acid region of the
subunit between
the conserved VSK and EDPL sequence motifs first identified as a
discrimination region in a report by Spring and Neer
(36) ,
which appeared while this manuscript was in preparation. The present
study identifies a conserved hydrophobic residue, Phe
in
and Leu
in
, which
appears to play a major role in determining the
phenotype with
respect to
selectivity. Interestingly, all known mammalian
subunits except
contain a leucine in this position
(), and
,
,
, and
can all form stable
heterodimers with
(8, 9, 10, 40, 41, 42, 43) .
Because the leucine-containing
is currently available
only as a partial cDNA clone
(44) , its ability to assemble with
remains an open question. Since the
interaction of the
1*-F40L and
1*-ADL mutants was
weaker than that of chimeras containing additional
sequence, it is quite likely that residues neighboring the
conserved hydrophobic amino acid contribute to the proper conformation
of this locus. The domain of
identified here may not be the only
region determining selectivity in
assembly, however, since
neither
,
, nor
assemble with
(8, 9) . The novel
component of
dimers purified from retina
has not yet been identified
(45) , and whether its sequence in
this region resembles the known
subunits is unknown.
may represent a point of
physical contact with the
subunit, it is also possible that the
effect of the
mutations described here results from a more
general perturbation of the conformation of the
chain affecting a
distant
contact site (or sites). The ability of the
mutants
employed in this study to protect the 26-kDa C-terminal fragment of
from further tryptic digestion would make a grossly abnormal
conformation unlikely, however. In either case, the deleterious effect
of Phe on
assembly relative to Leu might result from
steric hindrance: the inability of
to accommodate the
aromatic side chain at a point of
contact or an unfavorable
effect of the bulky Phe side chain imparted to a remote
contact
site on
. Because the discrete domain of
described here was
identified by screening for selectivity in
/
interaction, these results in no
way exclude the possibility of other nonselective contact sites between
and
( e.g. through possible coiled coil formation
(7, 46) , see below).
interaction involving the parallel arrangement of
-helical
segments in a coiled coil was recently proposed based on mutagenesis of
the
subunit
(7) and molecular modelling
(7, 46) . This model accounted for the proximity of
residues Cys
in
and Cys
in
inferred from earlier cross-linking studies
(37) . The favored
model (of two that differed in the domain of the
subunit
involved) would position the three-amino acid region of
identified in the current study just C-terminal to the putative
coiled coil domain
(7) ().
identified in this study also lies
immediately C-terminal to the pair of cysteine residues implicated in
the
cross-linking study
(37) , placing residue Phe
approximately 4.5-6 Å in axial distance away from
Cys
if this region of
is indeed
-helical.
One might therefore expect the corresponding domain in
, which contacts
and discriminates between
and
, to map close to
residue
Cys
. To the contrary, results from two laboratories
exclude the N-terminal region of
as the site of
discrimination
(8, 11) . A recent study of
/
chimeras that mapped the domains
responsible for selective assembly with
found that
multiple residues dispersed in the linear sequence contributed to this
function
(11) . In simple two-component chimeras, the shortest
linear segment of
or
that conferred
the parental phenotype spanned residues 215 to the C terminus
(11) . This region of the
subunit consists of internally
homologous repeat segments of approximately 40 amino acids in length,
termed GH-WD repeats, or WD-40 repeats, which have been also identified
in a phylogenetically and functionally diverse array of proteins
(47, 48, 49) . A concentration of residues in
the
subunit important for discrimination between
1* and
2* was found in the variable connecting segments joining conserved
portions of the 5th and 6th GH-WD repeats
(11) .
/
chimeras and the preceding
one employing
/
chimeras
(11) used the same paradigm to address directly complementary
questions, the most economical interpretation would be that the
discrete domain on
identified here interacts with multiple
residues of the
subunit, including most likely residues in the
variable connecting segments within and adjacent to the 5th and 6th
GH-WD repeats
(47) . Predictive algorithms suggest the secondary
structure of these connecting segments may include turns or coils
(38, 39) . Thus the compact folding of the
subunit
inferred from previous biochemical studies
(25) might position
the residues of
subserving
discrimination into a single
hydrophobic surface or pocket sufficient to accommodate the residue
corresponding to Phe
in
.
Table:
Homologies among G protein subunits around
a putative
contact site identified in
and
(12),
(13),
(44), murine
(44), bovine
(50), and
(51) flanking a putative
contact site is shown. Residues aligning with the three-amino
acid locus identified in this study are shown in boldface. An overlying
``c'' indicates involvement in a putative
coiled coil structure according to model 1 of Ref. 7. A similar
coiled coil model of
by Lupas et al. (46)
suggests that Ser
may be the last involved residue. An
asterisk identifies a conserved hydrophobic residue identified in the
current study, which helps define the
specificity of this
proposed contact site. The two Cys residues immediately preceding this
region in
(Cys
) are those that can be
cross-linked to Cys
of
by cupric
phenanthroline (37). Numbers indicate the residue positions predicted
by the cDNAs, except in the case of the sequence shown for
, which derives from a partial clone (44). It should
be noted that human
differs from the bovine sequence
by the substitution of Val for Phe at residue 40 (52).
and
cDNAs and Dr. N. Gautam for the
generous gifts of
and
cDNAs. We
also thank Dr. Regina Collins for help with cell culture, George Poy
for oligonucleotide synthesis, and Dr. Andrew Shenker for critical
review of the manuscript. Finally, we thank Dr. Allen Spiegel for
continued support and encouragement.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.