G
Affinity for Bovine Rhodopsin Is Determined by the
Carboxyl-terminal Sequences of the Subunit*
Xiaoying
Jian
§,
William A.
Clark§¶,
Jeffrey
Kowalak
,
Sanford P.
Markey,
William F.
Simonds**, and
John K.
Northup
From the Laboratory of Cellular Biology, NIDCD, the
Laboratory of Neurotoxicology, National Institute of Mental
Health, and the ** Metabolic Diseases Branch, NIDDK, National
Institutes of Health, Bethesda, Maryland 20892
Received for publication, July 27, 2001, and in revised form, October 12, 2001
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ABSTRACT |
Two native 
dimers,
11 and
12, display very different affinities for
receptors. Since these subunits differ in both primary structure
and isoprenoid modification, we examined the relative contributions of
each to G interaction with receptors. We constructed
baculoviruses encoding 1 and 2 subunits
with altered CAAX (where A is an aliphatic
amino acid) motifs to direct alternate or no prenylation of the chains and a set of 1 and 2 chimeras with
the 2 CAAX motif at the carboxyl terminus.
All the constructs coexpressed with 1 in Sf9
cells yielded 1 dimers, which were purified to near
homogeneity, and their affinities for receptors and G
were
quantitatively determined. Whereas alteration of the isoprenoid of
1 from farnesyl to geranylgeranyl and of 2 from geranylgeranyl to farnesyl had no impact on the
affinities of 1 dimers for Gt,
the non-prenylated 12 dimer had
significantly diminished affinity. Altered prenylation resulted in a
<2-fold decrease in affinity of the 12
dimer for rhodopsin and a <3-fold change for the
11 dimer. In each case with identical
isoprenylation, the 12 dimer displayed
significantly greater affinity for rhodopsin compared with the
11 dimer. Furthermore, dimers containing
chimeric G chains with identical geranylgeranyl modification
displayed rhodopsin affinities largely determined by the
carboxyl-terminal one-third of the protein. These results indicate that
isoprenoid modification of the G subunit is essential for binding to
both G and receptors. The isoprenoid type influences the binding
affinity for receptors, but not for G. Finally, the primary
structure of the G subunit provides a major contribution to receptor
binding of G, with the carboxyl-terminal sequence conferring
receptor selectivity.
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INTRODUCTION |
Heterotrimeric G
proteins1 transduce a wide
variety of extracellular signals recognized by seven-transmembrane
receptors, initiating signaling through a diverse array of
intracellular effectors (1). G proteins are composed of three
polypeptides: a GTP-binding subunit and a dimer of and subunits that functions as a monomer. Ligand-activated G
protein-coupled receptors catalyze the exchange of GTP for GDP tightly
bound to the inactive G subunit, resulting in dissociation of the
GTP-activated subunit from both its cognate G dimer and the
receptor. The GTP-activated subunit and the dissociated G
dimer in turn regulate intracellular effectors (1, 2). At least 20 different subunits, 5 subunits, and 12 subunits (3) have
been identified to date. Such a diversity of structures is believed to
contribute importantly to the specificity of signaling through these pathways.
It is well established that the selective interaction between receptors
and the G subunits provides a major determinant of signaling
specificity. There are numerous examples of the selectivity of
receptors for G subunits. For example, the -adrenergic receptor couples primarily to members of the Gs family (4-6),
whereas the 2A-adrenergic receptor couples to members of
the Gi family (7-10). G protein-coupled receptors can
even display remarkable selectivity among closely related G
structures. The bombesin receptor subtypes selectively couple with
different subtypes of Gq (11). A growing body of
evidence also points to the contribution of subunits in
determining receptor-G protein coupling selectivity. The role of
diversity in the specificity of G protein signaling is supported by
both in vivo and in vitro studies. Antisense RNA constructs for G, G, and G selectively disrupt receptor
signaling in rat pituitary GH3 cells (12, 13). These
studies have demonstrated a specific requirement of 3
and 4 subunits for muscarinic receptor signaling,
whereas 1 and 3 subunits mediate
somatostatin receptor signaling. Also, ribozyme-mediated suppression of
7 subunit expression has led to a specific attenuation
of -adrenergic receptor signaling in HEK293 cells (14). Moreover,
in vitro reconstitution with dimers of differing
composition demonstrates that both the subunit (15-17) and the subunit (16, 18, 19) provide coupling specificity for various
receptors. We initially interpreted the synergy and selectivity
of rhodopsin-catalyzed GTP binding to Gt to mean that
rhodopsin made independent binding contacts with the and
subunits (20). Direct interaction of transducin with rhodopsin
has been demonstrated separately with fluorescence spectroscopy (21)
and with surface plasmon resonance measurements (22). A receptor
contact site on the subunit was identified in cross-linking studies
in which a synthetic peptide derived from the third intracellular loop
of the 2A-adrenergic receptor could be cross-linked to
the carboxyl-terminal region of the subunit (23).
A series of post-translational modifications are required for the
functions of G proteins. In the case of G subunits, these modifications include the thioether linkage of an isoprenoid group to
the conserved cysteine side chain within a carboxyl-terminal CAAX motif (where C is cysteine, A is an
aliphatic amino acid, and X is any amino acid), proteolytic
cleavage of the AAX tripeptide, and methylation of the
resulting carboxyl terminus. The terminating residue in the
CAAX motif appears to determine the identity of the
isoprenoid. G protein subunits 1, 8
(cone) and 11 with serine at this position are modified
with a 15-carbon farnesyl, whereas the other subunits terminating
in leucine are modified with a 20-carbon geranylgeranyl. The lipid
modification of the subunits has been shown to be responsible for
attaching G proteins to membranes (24, 25). There is also ample
evidence suggesting the involvement of the prenyl groups in
protein-protein interactions (26-29). Two dimers,
11 and 12,
display a >10-fold difference in their affinity for bovine rhodopsin
(20, 30), but no apparent difference in interaction with retinal
Gt. Since these two subunits differ in both primary
amino acid sequence and isoprenoid modification, we designed this study
to examine the quantitative contributions of isoprenoid modification
and protein structure to the dimer affinity for rhodopsin. Our
data indicate that although the lipid modification is essential for a
competent dimer, the type of prenyl group on the subunits
influences the binding affinity for rhodopsin, but not for
Gt. In addition, the primary structure of the subunit provides a major contribution to rhodopsin binding of
dimers, with the carboxyl-terminal sequences conferring receptor selectivity.
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EXPERIMENTAL PROCEDURES |
Construction of Prenylation Mutants and Chimeras--
Mutant
and chimeric cDNA clones were made by PCR amplification. Bovine
1 and 2 were the original templates. A
recombinant baculovirus encoding 1-CVIL (31) was kindly
provided by Dr. James Garrison (University of Virginia), and
recombinant baculoviruses encoding 2-CVIS and the
cysteine-terminated construct "2-A" were gifts from
Dr. Nick Ryba (NIDCR, National Institutes of Health). Plasmids encoding
chimeric 1/2 structures as previously
reported (32) were used as PCR templates for generating the current
chimeric cDNA constructs, in which all the 3'-reverse primers
contained nucleotides encoding CAIL prior to the stop codon. The
resulting chimeric structures therefore all contain a
carboxyl-terminal sequence that is known to direct geranylgeranylation.
The PCR products encoding the chimeric structures were cloned into
the transfer vector pBacPAK8 (CLONTECH) for
production of recombinant baculoviruses. Insect cell culture,
transfection, plaque purification, and virus amplification were carried
out according to the manufacturer's recommended procedures
(CLONTECH).
Purification of G Protein Subunits--
G proteins were isolated
from bovine retina and baculovirus-infected Sf9 cells expressing
recombinant 1 dimers. Bovine retinal transducin was
isolated from rod outer segment discs prepared by discontinuous
gradient sedimentation (33). Gt and
11 were purified using previously
published procedures (20, 34, 35). Sf9 cell-expressed
1 dimers were purified as described for
12 (30) with minor modifications.
Sf9 cells were co-infected with 1-encoding and
the appropriate -encoding baculoviruses at multiplicities of
infection of 1 for the 1 virus and 3 for the virus. The cells were harvested 60 h after infection; the cells
were lysed; and a P2 membrane fraction was isolated. All the dimers
were purified from Sf9 cell membranes, except
12-A, which was purified from the
cytosolic fraction. After sequential chromatography over DEAE-Sephacel (Amersham Pharmacia Biotech) and Ultrogel AcA44 (IBF), the
fractions containing the 1 dimers were pooled and
concentrated and then further purified on a Superdex HR-75 FPLC column
(Amersham Pharmacia Biotech) with the final storage solution (10 mM MOPS, pH 7.5, 100 mM NaCl, and 8 mM CHAPS). Two of the samples,
1221 and
12-A, required an additional step of
purification through a Mono-Q FPLC column (Amersham Pharmacia Biotech)
before the final Superdex HR-75 FPLC size-exclusion chromatography.
Electrophoretic Methods--
Pre-cast Tricine-16%
acrylamide gels (Novex) were run according to the
manufacturer's protocol. Samples were electrophoresed at 125 V for
~90 min at room temperature. Protein bands were visualized by
Coomassie Blue staining.
Mass Spectrometry--
Liquid chromatography/mass spectrometry
was performed by directly coupling a Shimadzu LC10VP HPLC system to the
electrospray ion source of an LCQ ion trap mass spectrometer.
Reversed-phase HPLC was performed at a flow rate of 10 µl/min
using a 0.3 × 100-mm Betabasic 4 column (Keystone Scientific
Inc., Bellefonte, PA). Solution A was H2O/acetonitrile
(19:1) and 0.1% formic acid; Solution B was acetonitrile/1-propanol
(4:1) and 0.1% formic acid. The column was equilibrated at 80%
Solution A and 20% Solution B, and the chromatograph was developed
using a linear gradient to 20% Solution A and 80% Solution B in 20 min. Full scan mass spectra over the m/z 300-2000 range
were acquired continuously for the duration of the chromatography.
dimers were prepared for mass spectrometry by acetone
precipitation. Samples (60-201 pmol in 20 µl) in 8 mM
CHAPS were mixed with 9 volumes of ice-cold acetone in a Vortex mixer
and incubated for 20 min on ice, and precipitates were collected by sedimentation at 12,000 × g for 15 min at 4 °C.
After aspirating the supernatant liquid, pellets were allowed to
air-dry and then were suspended in 25 µl of Solution A, and 5 µl of
the sample was injected for liquid chromatography/mass spectrometry analysis.
Protein Concentration Determination--
Gt
concentration was determined by rhodopsin-catalyzed GTPS binding
(20). G11 was determined by the Amido
Black binding assay (36) using bovine serum albumin as a standard. The
protein concentrations of all other 1 dimers were
determined from the staining intensity of the chain calibrated with
different amounts of 11 on a Coomassie
Blue-stained gel by densitometry, with all the samples and
11 standards within linear range.
Reconstitution Assays--
The activities of the
1 dimers were quantified by rhodopsin-catalyzed
GTPS binding to Gt and pertussis toxin-catalyzed ADP-ribosylation of Gt as described previously (20). For
both assays, the detergent CHAPS was adjusted to a final concentration of 1.6 mM. Urea-washed rod outer segment disc membranes,
the source of rhodopsin, were prepared as described (37). Initial rate estimates for the catalyzed GTPS binding to Gt and
pertussis toxin-catalyzed ADP-ribosylation of Gt were
determined by single time point reactions, which consumed <20% of the
Gt substrate.
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RESULTS |
The goal of this study was to delineate the relative contributions
of isoprenoid modification and protein structure of the G subunit to
the interactions of the dimer with G subunits and receptors.
We have examined these questions using the retinal G protein and
rhodopsin because affinity differences between
11 and 12
dimers are well characterized for this receptor, and these two dimers
differ in both the primary structure and isoprenoid modification of the
chain. Data from several laboratories have suggested that the
isoprenoid modification provides the major determinant of differences
between these two dimers, so we initially tested the effects of
altering the prenylation. To examine this question, we tested three
mutant constructs: the 1 mutant
1-CVIL was expected to change 1 from
farnesyl to geranylgeranyl; the 2 mutant
2-CVIS was expected to change 2 from
geranylgeranyl to farnesyl; and 2-A, a truncated form of
2 that terminates at the cysteine residue of the
CAAX motif, was expected to result in a mature protein
lacking any prenyl group. Co-infection of Sf9 cells with viruses
encoding these mutant constructs and a 1-encoding
virus resulted in the expression of a 1 dimer for all
constructs. Fig. 1 shows the
1 dimers resulting from such co-infections purified
to near homogeneity and separated on a Tricine gel stained with
Coomassie Blue. The electrophoretic mobility differences among the chains are noticeable in this gel system. None of the samples contained
any detectable G contamination measured in the rhodopsin-catalyzed
GTPS exchange assay or pertussis toxin-catalyzed ADP-ribosylation
assay (data not shown).
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Fig. 1.
SDS-PAGE of purified
G1
subunits with altered isoprenylation. Purified recombinant
subunits of defined subtypes were electrophoresed on a
Tricine-16% acrylamide gel and stained with Coomassie Blue. The
positions of the 1 and various subunits are
indicated on the left.
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To confirm the nature of the post-translational modifications, we
analyzed purified 1 dimers using electrospray
ionization mass spectrometry. Fig. 2
shows the deconvoluted mass spectra of 1,
1-CVIL, 2, and 2-CVIS. The
observed molecular mass for each of the constructs is summarized in
Table I. As predicted, the
1 subunit showed a molecular mass of 8332 Da, in
agreement with the calculated molecular mass of the polypeptide chain
deduced from the DNA sequence plus all known post-translational
modifications. They include removal of the amino-terminal methionine,
attachment of a farnesyl group to cysteine 71, removal of the
carboxyl-terminal tripeptide, and methylation of the new carboxyl
terminus. The 1-CVIL sequence is predicted to direct
geranylgeranyl modification. The observed mass of 8401 Da is consistent
with exchange of the farnesyl group of wild-type 1 for a
geranylgeranyl. Consistent with the findings of Lindorfer et
al. (31), the mass spectra also showed a set of signals
corresponding to the fully processed molecule minus the prenyl group.
These authors have demonstrated that the loss of the farnesyl group
occurs during the ionization process and is not due to heterogeneity of
modification. Our data are consistent with their findings since the
HPLC elution resolves 1 chains bearing differing
isoprenoid modification prior to the ionization for the mass
spectrometry. The 2 and 2-CVIS subunits also showed the observed masses consistent with the amino acid composition deduced from the DNA sequence plus the predicted isoprenoid modifications (farnesyl for 2-CVIS and geranylgeranyl
for 2), endoproteolysis, carboxyl methylation, the
removal of the amino-terminal methionine, and amino-terminal
acetylation. The 2-A chain showed an observed mass of
7463 Da, consistent with that of the predicted amino acid sequence of
2 terminating at cysteine of the CAIL carboxyl terminus,
lacking prenylation or methylation, with its amino-terminal
methionine removed and the new amino terminus acetylated. Minor amounts
of inappropriately prenylated species were observed for the
2 products (Fig. 2, C and D). The
relative abundance of farnesylated versus geranylgeranylated
subunits is summarized in Table I as well.
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Fig. 2.
Electrospray ionization mass spectra of
recombinant 1
subunits with altered isoprenylation. Samples of purified
dimers containing prenylation mutant and wild-type chains were
acetone-precipitated and analyzed by liquid chromatography/mass
spectrometry as described under "Experimental Procedures." The
sample amounts were as follows: retinal
11, 40 pmol;
11-CVIL, 40 pmol; wild-type
12, 19 pmol;
12-CVIS, 40 pmol. The spectra are labeled
for the masses corresponding to chains modified with farnesyl
(C15) and geranylgeranyl (C20) or
non-prenylated with and without methyl (CH3).
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To assess the effects of altering prenylation of G on
interactions with receptors, we tested -dependent,
rhodopsin-catalyzed GTPS binding to Gt in the
presence of different 1 samples. Fig.
3 shows results from one such experiment.
The K1/2 values obtained are summarized in Table
II. The 11
subunit showed a >12-fold lower affinity compared with the
12 subunit for rhodopsin (K1/2 = 227 versus 18 nM).
When 1 prenylation was altered from farnesyl to
geranylgeranyl, the affinity of 11-CVIL
for rhodopsin increased almost 3-fold (Fig. 3A), but still
did not equal that of 12 for rhodopsin
(K1/2 = 78 versus 18 nM).
When 2 prenylation was altered from geranylgeranyl to
farnesyl, the affinity of 12-CVIS for
rhodopsin decreased <2-fold (Fig. 3B), distinct from the
lower affinity of 11 for rhodopsin
(K1/2 = 31 versus 227 nM). However, although the alteration of 2 prenylation from
geranylgeranyl to farnesyl diminished affinity for rhodopsin only
modestly, the absence of a prenyl group diminished the interaction of
the 12-A dimer to undetectable levels,
with no enhancement of GTPS binding at all (Fig. 3A).
These data suggest an absolute requirement for the isoprenoid
modification of the subunit for the interaction with
receptors.
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Fig. 3.
Saturation of rhodopsin-catalyzed
GTPS binding to
Gt by
1 subunits
with altered isoprenylation. The indicated concentrations of
11 ( ),
11-CVIL ( ), and
12-A ( ) in A and
12 ( ) and
12-CVIS ( ) in B were
incubated in reactions containing 500 nM Gt
and 30 nM rhodopsin as described under "Experimental
Procedures." The curves drawn are the best fit for a single site
binding model using GraphPAD Prism.
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To determine whether the differences in affinity observed among
subunit forms were due to differing affinities for interaction with
Gt, we analyzed the dependence of pertussis
toxin-catalyzed ADP-ribosylation of Gt. Fig.
4 shows the saturation of
ADP-ribosylation of Gt by increasing concentrations of
different forms of 1 dimers. In contrast with the
rhodopsin-catalyzed GTPS binding to Gt, the pertussis
toxin-catalyzed ADP-ribosylation of Gt displayed no distinction between 11 and
12 with either form of prenylation (Table
II). The non-prenylated 2 dimer
(12-A) displayed a measurable enhancement
of the ADP-ribosylation of Gt, but with an affinity for
Gt that was >30-fold lower than that for the
prenylated 11 or
12 dimer. These data suggest that for the
interaction with the G subunit, the isoprenoid modification
of the subunit, not the exact identity of the prenyl group, is
important.
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Fig. 4.
Saturation of pertussis toxin-catalyzed
ADP-ribosylation of Gt by
1 subunits
with altered isoprenylation. The indicated concentrations of
11 (),
11-CVIL (),
12 (),
12-CVIS (), and
12-A () were incubated in reactions
containing 500 nM Gt and 5 µg/ml pertussis
toxin as described under "Experimental Procedures." The curves
drawn are the best fit for a single site binding model using GraphPAD
Prism.
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Since the difference in prenyl group did not account for the majority
of the affinity difference between 11 and
12 for rhodopsin, we investigated the
contribution of primary structures. Fig.
5 depicts the set of chimeras we employed
containing heterologous sequence at approximately one-third intervals
based upon regions of sequence identity between the 1
and 2 proteins. These constructs were based upon the
previously published chimeras (32), except that all of the chimeric
constructs were altered to code for CAIL at the carboxyl terminus so
that they would all have the same geranylgeranyl modification.
Co-infection of Sf9 cells with baculoviruses encoding these
chimeric chains and a 1-encoding virus led to the
expression of 1 dimers for all constructs. Because
the yields of 1111 protein were quite
low, we used the 11-CVIL product for this
protein structure. 1222 is the wild-type
12 construct. Fig.
6 shows the 1 chimeras
along with the parent structure 11-CVIL
and 12 purified to near homogeneity and
separated on a Tricine-16% acrylamide gel stained with Coomassie Blue.
As found for the prenylation mutant chains, this gel system clearly resolves the chimeric chains. HPLC electrospray ionization mass spectrometry confirmed that the isoprenoid modification of the chains was predominantly the geranylgeranyl directed by the CAIL
sequence. Geranylgeranyl-modified chain accounted for >74% of the
mass of each purified subunit (Table
III).
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Fig. 5.
Construction of chimeric subunits. A, the amino acid sequences of the
1 and 2 subunits served as parent
structures for the chimeric subunits as previously described (32).
Numbers on the right indicate the amino acid residue positions. Before
C-terminal processing, 1 contains 74 residues, and
2 contains 71 residues. In the current constructs, the
C-terminal CAAX motif in 1 has been changed
from its native CVIS to CAIL so that all the resulting chimeric subunits contain the same C-terminal CAIL as 2 that
directs geranylgeranylation. Boldface residues
(QLK and EDPL) correspond to sequence motifs used
as junctions between chimeric segments. B, shown is a
schematic overview of the 1/2 chimeras.
Open bars indicate polypeptide sequence derived from
1, and closed bars indicate that derived from
2. Vertical lines indicate junctions between
chimeric segments.
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Fig. 6.
SDS-PAGE of purified
G1
subunits with chimeric chains.
Purified recombinant subunits of defined subtypes were
electrophoresed on a Tricine-16% acrylamide gel and stained with
Coomassie Blue. The positions of the 1 and various subunits are indicated on the left.
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Fig. 7 shows the saturation of
rhodopsin-catalyzed GTPS binding to Gt for the
purified 1 chimeras. They displayed clear differences
in both affinity and maximum catalytic rate, as seen for the dimers
containing the parent 1 or 2 protein.
These differences could be sorted based upon the identity of the
sequence for the carboxyl-terminal one-third of the chimera. As
summarized in Table IV,
1122 and
1112 had similar high affinity for
rhodopsin compared with 12
(K1/2 = 24, 23, and 18 nM,
respectively). The other two chimeric dimers,
1221 and
1211, had lower apparent affinity
(K1/2 = 39 and 68 nM, respectively),
with the value for the latter close to the value for
1111 (i.e.
11-CVIL, 78 nM). This result
suggests that the primary structure in the carboxyl-terminal region of the subunit determines the affinity of the interaction with rhodopsin. The amino acid sequence in the middle and amino-terminal regions of the 2 subunit also contributed to the high
affinity interaction, although to a lesser degree.
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Fig. 7.
Saturation of rhodopsin-catalyzed
GTPS binding to
Gt by
1 subunits
with chimeric chains. The indicated
concentrations of 1122 (),
1112 (),
1221 (), and
1211 () were incubated in reactions
containing 500 nM Gt and 30 nM
rhodopsin as described under "Experimental Procedures." The curves
drawn are the best fit for a single site binding model using GraphPAD
Prism.
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As expected from the similar affinities of wild-type
11 and 12,
the 1 chimeras,
11-CVIL, and
12 showed essentially identical affinity
for Gt in the pertussis toxin-catalyzed ADP-ribosylation assay (Fig. 8 and Table IV). These data
imply that the affinity differences found for dimers with distinct subunit primary structures are due to differences in rhodopsin-
interaction rather than differences in -Gt
interaction.
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Fig. 8.
Saturation of pertussis toxin-catalyzed
ADP-ribosylation of Gt by
1 subunits
with chimeric chains. The indicated
concentrations of 1111 (i.e.
11-CVIL; ),
1122 (),
1211 (),
1112 (),
1221 (), and
1222 (i.e. wild-type
12;  ) were incubated in reactions
containing 500 nM Gt and 5 µg/ml pertussis
toxin as described under "Experimental Procedures." The curves
drawn are the best fit for a single site binding model using GraphPAD
Prism.
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DISCUSSION |
With the emerging evidence suggesting that in addition to the G
subunit, both the and subunits of G proteins also affect receptor-G protein interactions (15-19), it has become an increasingly accepted notion that dimers contribute to the selectivity of the
receptor interaction. This study examined quantitatively the role of
the prenyl modification and amino acid sequence of the G subunit in
determining signaling specificity. We confirmed that the apparent
affinity for rhodopsin was >12 times higher for
12 than for
11 for the wild-type structures, whereas
the two 1 dimers displayed no difference in affinity
for retinal Gt. Although one study using rhodopsin to
catalyze GDP/GTP exchange on the Gt subunit showed that
the 11 dimer supported a faster rate of
exchange than did 12 or
13 (38), others have found a much lower
apparent affinity for 11 than for other
dimers (20, 27, 30, 39). The direct measurement of rhodopsin-G protein interactions using surface plasmon resonance also revealed a
higher affinity of 12 for rhodopsin
compared with 11 and showed that this
difference was due to a lower dissociation rate for
12 (22). We have capitalized upon this
significant quantitative difference in the binding interaction with
rhodopsin to assess the independent contributions of the subunit
primary structure and post-translational modification to this
difference. Our results clearly implicate the primary structure of the
chain as a major determinant of the affinity of 1
dimers for rhodopsin. The set of chimeras with uniform
geranylgeranyl modification further pinpointed the carboxyl-terminal
sequence of the subunit as the region responsible for receptor
selectivity. Underscoring the importance of the amino acid sequence of
the subunit in receptor-G protein interactions, a recent study
showed that 11 supported only a very low
level of coupling to the 2A-adrenergic receptors,
whereas the 111 dimer produced a high
level of coupling (16). Since both the 1 and
11 subtypes are farnesylated, the difference in coupling
level observed for these dimers must also be attributed to differences
in the amino acid sequences.
Consistent with previous findings on 5-hydroxytryptamine type 1A
receptors (40), adenosine receptors (28), and bombesin receptors (11),
we found that bovine rhodopsin prefers G complexes containing
the geranylgeranylated subunit. Our results obtained with
prenylation mutants confirmed the findings of several laboratories that
dimers containing geranylgeranylated subunits have higher
affinities for receptors than those with farnesylated chains (11,
27, 28, 40). In our study, given the same amino acid sequence of the
subunit, the 1 dimer was more effective in
coupling the Gt subunit to rhodopsin when the subunit was modified with geranylgeranyl compared with farnesyl.
Furthermore, a prenylation-deficient construct was found to be
incompetent to participate in these protein-protein interactions.
Although this has suggested that the affinity differences found between retinal 11 and other dimers could
be attributed solely to the isoprenylation difference (27, 28), we
found that identically isoprenylated 11
and 12 retained significant affinity
differences for bovine rhodopsin for both geranylgeranylated and
farnesylated chains. Moreover, the effect of the prenyl type was
more profound with the 1 primary structure than with the
2 structure, which implies the importance of the amino
acid sequence of the 2 subunit in conferring a high
affinity rhodopsin interaction.
The apparent affinity differences that we measured for bovine rhodopsin
for this set of 1 dimers with varying compositions clearly do not originate from distinct
Gt interaction since all of the dimers displayed
essentially identical apparent affinity for Gt,
as others have also noted for the retinal G protein subunits (20, 41).
This may not be the case for dimers interacting with other G
proteins. A significant difference in apparent affinity for
Gs, but not for Go or Gi,
has been reported for dimers of differing compositions (42).
Whereas ADP-ribosylation by pertussis toxin did not reveal a difference
among brain dimers for Go, inhibition of GDP
release did (41). Since the primary contacts between G and G
occur within the amino-terminal sequences of subunits, and these
are distinct for the various G species, it is likely that subunit selectivity may contribute importantly to differences among
dimers in receptor signaling. This will be important to clarify
in future studies.
The differences that we observed among the dimers in rhodopsin
interaction are also not likely to originate from the different purities of the samples. We have previously shown that substances interfering with the quantitative assay of dimers in cholate extracts from Y1 adrenal cortical tumor cells were completely removed
by chromatography over Ultrogel AcA44 (43). Similarly, we found
that chromatography over DEAE-Sephacel was sufficient to produce
partially purified recombinant 12 dimers
expressed in Sf9 cells using baculoviral vectors that showed
quantitatively identical apparent rhodopsin affinity as homogenous
preparations of the dimer.2
All dimers utilized in the present study were purified by both
DEAE-Sephacel and Ultrogel AcA44 chromatography with additional FPLC
size-exclusion chromatography to assure hydrodynamic dimers with
defined detergent concentration, and they were assessed to contain no
measurable contaminating GTP-binding proteins or pertussis toxin
substrates. We also note that the 1211
dimer, which had equal apparent purity compared with the
12 dimer sample, displayed reduced
apparent affinity for rhodopsin, but not for Gt.
Furthermore, although the measured affinity of the dimers
containing chimeric chains varied systematically with the
carboxyl-terminal sequence of the chain, it did not vary
systematically with sample purity, and the affinity of all samples for
Gt was indistinguishable. We think it unlikely, then,
that either specific contaminants or sample purity accounts for the
differences in activities found in our recombinant dimers.
Similar to the previously reported results from Sf9 expression
systems (27, 31, 44), we found heterogeneity in isoprenoid modification
of several of the G subunit constructs. There is no doubt that in
the presence of a high concentration of virally encoded
substrate, both farnesyl- and geranylgeranyltransferases in Sf9
cells misprenylate a fraction of the protein. Whether such
misprenylation is caused by excessive substrate, as suggested by
Lindorfer et al. (31), or prenyltransferase specificity is also encoded by primary structures other than the CAAX
motif, as suggested by Kalman et al. (44), remains to be
tested. Neither our nor the previous data have directly assessed the
prenyltransferase activities; rather, we have measured the long-term
accumulation of recombinant protein products. Our data appear to
suggest the lack of a consensus sequence upstream of the
CAAX motif that directs farnesylation or geranylgeranylation
since the proportion of prenylation did not vary systematically in our
1/2 chimeras with the presence of
1 or 2 sequence. In addition to the lipid
modification, G subunits are also methylated at the
carboxyl-terminal cysteine after the proteolytic cleavage of the
AAX tripeptide. The importance of the subunit carboxyl
methylation to the interaction with G subunits has been
reported (45, 46); its effect on interaction with receptors has
yet to be fully examined. Whatever the effect is, it should not
complicate the interpretation of our data for the following reasons.
First, our mass spectra showed that except for retinal
11, which showed >50% methylation, all
the Sf9-expressed 1 dimers were predominantly
methylated (>90%). Second, we did not observe any difference among
all the tested 1 dimers in their interaction with
Gt, including retinal 11.
Moreover, fully methylated 11 purified
from heterologously expressed Sf9 cells showed low receptor
coupling with adenosine receptors (28, 31), just as we have observed
for retinal 11 with rhodopsin.
The data presented in this study demonstrate that the prenyl group on
t
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ABSTRACT
INTRODUCTION
