 |
INTRODUCTION |
Consistent with the steadily increasing number of G
protein1
and
subtypes
that has been revealed in recent years (1), in vivo studies
have indicated a role for this structural diversity in the specificity
of signaling. In this regard, antisense studies by Kleuss et
al. (2, 3) have demonstrated a specific requirement for the
1 and
3 subunits in the somatostatin
receptor signaling pathway in rat pituitary GH3 cells, with
a similarly specific requirement for the
3 and
4 subunits in the muscarinic receptor signaling pathway.
Also, a ribozyme study by Wang et al. (4) has shown a
specific involvement of the
7 subunit in the
-adrenergic receptor signaling pathway in human kidney 293 cells.
Taken together, these in vivo studies indicate that the
composition of the 
dimer has important ramifications for the
fidelity of signaling that is probably manifested at the level of the receptor.
A growing body of in vitro evidence supports a direct
interaction between the receptor and the 
dimer (5). In
particular, direct interaction of transducin 
with rhodopsin has
been shown with a fluorescence energy transfer technique (6). This
association was blocked by a synthetic peptide derived from the
carboxyl-terminal tail of rhodopsin, suggesting a site of direct
contact between 
and rhodopsin. Moreover, cross-linking studies
have confirmed a receptor contact site on the
subunit. A synthetic
peptide derived from the carboxyl-terminal portion of the putative
third cytoplasmic loop of the
2A-AR could be
cross-linked to the carboxyl-terminal region of the
subunit
(7).
To date, in vitro studies examining the contribution of a
limited number of 
dimers to the specificity of receptor coupling have not yielded the same high degree of discrimination shown in the
in vivo studies cited above (8, 9). The present study extended this analysis to the
2A-adrenergic receptor and
to 
dimers that represent the most extensive degree of structural diversity examined to date. Since baculovirus expression has been shown
to be an effective means for producing functional G protein subunits
(10-13) as well as G protein-coupled receptors (8, 9, 14, 15), this
system was used to measure the level of interaction between the
recombinant
2A-adrenergic receptor expressed in
Sf9 cell membranes and reconstituted in the presence or absence of purified Gi proteins of varying
or
subtype
composition. Among the two
subtypes or eight
subtypes tested,
30-fold differences were observed in their relative abilities to
support coupling of the same
subunit to the recombinant
2A-adrenergic receptor. These data demonstrate that the
specificity of
2-adrenergic receptor-G protein
interactions is affected by the 
dimer composition.
 |
EXPERIMENTAL PROCEDURES |
Expression of
2A-Adrenergic
Receptor--
A pVL1392 transfer vector containing
2A-adrenergic receptor cDNA was generously provided
by Dr. H. Kurose and R. Lefkowitz (Duke University, Durham, NC).
Recombinant baculovirus encoding the
2A-adrenergic
receptor was generated by co-transfection of
2A-pVL1392
with a linearized lethal deletion mutant of Autographa californica as directed by the supplier (BaculoGold, PharMingen Corp.). Expression by recombinant baculovirus was identified by specific binding of the
2-adrenergic radioligand,
[3H]yohimbine (described below). A positive recombinant
was isolated through four rounds of plaque purification. Receptors were
expressed by inoculating Sf9 insect cells at an m.o.i. of 1 in
IPL-41 medium, 1× lipid concentrate, and 1% heat-inactivated fetal
bovine serum (Life Technologies, Inc.) at a density of 2 × 106 cells/ml. After 72 h, cell pellets were lysed by
nitrogen cavitation (500 pounds/square inch for 30 min at 4 °C) in
100 ml of ice-cold lysis buffer (25 mM Tris, pH 7.4, 1 mM EDTA, 10 mM MgCl2, 100 mM NaCl, 0.02 mg/ml phenylmethylsulfonyl fluoride, 0.03 mg/ml leupeptin, and 1 mM benzamidine) and centrifuged at
4 °C for 10 min at 600 × g. The supernatant was
centrifuged at 40,000 × g for 40 min at 4 °C. The
pellets were resuspended, washed once in lysis buffer (40,000 × g, 40 min), and resuspended in 10 ml of lysis buffer.
Protein concentration was determined by Coomassie assay (Pierce).
Particulate fraction protein was snap-frozen with liquid N2
in aliquots of 300 µg each and stored at
80 °C. Receptor expression was quantitated by saturation binding of
[3H]yohimbine, as described under "Experimental
Procedures." A single 500-ml expression culture yielded adequate
material to carry out all of the reconstitution experiments.
Expression and Purification of G Protein
Subunits--
Recombinant baculoviruses directing the expression of
1,
1,
2,
3,
5, and
7 recombinant baculovirus were
described previously (16, 17). Isolation of human cDNAs encoding
3 (18) and
4,
10, and
11 (19) was described previously. In these cases, recombinant baculoviruses were obtained by co-transfection of Sf9 insect cells with pVL1393 transfer vectors containing
3,
4,
10, or
11 and a linearized lethal deletion mutant of A. californica nuclear polyhedrosis virus as directed by the supplier (BaculoGold, PharMingen). Recombinant viruses were identified by
immunoblotting Sf9 cell lysates for expression of the
appropriate subunits. Subtype-specific antibodies were generated as
described previously (20) using the following synthetic peptides:
4, CKEGMSNNSTTSIS (amino acids 2-14);
10, CKDALLVGVPAGSNPFREPR (amino acids 45-63); and
11, CPALHIEDLPEK (amino acids 2-12). Other subtype-specific antibodies have been described previously (20, 21).
Recombinant virus encoding G
i1 containing a
hexahistidine tag at amino acid position 121 was kindly provided by Dr.
T. Kozasa (Southwestern Medical Center, Dallas, TX). One-liter cultures of Sf9 insect cells in IPL-41 medium, 1% heat-inactivated fetal bovine serum, and 1× lipid mix (Life Technologies, Inc.) were inoculated at a density of 2 × 106 cells/ml
simultaneously with recombinant baculoviruses encoding
,
, and
subunits as follows: 6his
i1 at m.o.i. = 2,
1 or
3 at m.o.i. = 3, and each of the
subtypes at m.o.i. = 3. Under this condition, those
subtypes
predicted to contain a C-20 geranylgeranyl group are appropriately
modified. However, those few
subtypes predicted to contain a C-15
farnesyl group are variably modified at high levels of protein
expression (10). Therefore, to optimize the addition of a C-15 farnesyl moiety, cultures of Sf9 cells expressing the
1
and
11 subtypes were also infected with recombinant
baculovirus encoding both subunits of the mammalian farnesyltransferase
at m.o.i. = 0.2. This virus was kindly provided by Dr. Thomas Kost
(Glaxo Corp.). Cultures of Sf9 cells infected with the
farnesyltransferase virus displayed greater than 15-fold higher
activity toward the Ha-Ras fusion protein substrate than cultures not
so infected. Moreover, cultures of Sf9 cells infected with the
farnesyltransferase virus resulted in the majority of the
1 and
11 subtypes being modified with the
C-15 farnesyl moiety, as shown previously (10). Expression of G protein
and
subunits in particulate fractions was confirmed 72 h later by immunoblotting with subtype-specific antibodies (22).
Recombinant 
heterodimers were purified to apparent homogeneity
using the procedure described by Kozasa and Gilman (11) for
purification of
1
2. Following cholate
extraction of particulate fractions, the cholate-soluble protein was
diluted to 0.2% sodium cholate with 20 mM Hepes, pH 8.0, 100 mM NaCl, 1 mM MgCl2, 10 mM
-mercaptoethanol, 10 µM GDP, and 0.5%
polyoxyethylene 10-lauryl ether. The cholate-soluble extract was loaded
onto a 4-ml Ni-NTA resin bed at 3-4-bed volumes/h (4 °C) and washed
with 100 ml of the same buffer containing 300 mM NaCl and 5 mM imidazole. 
dimers were eluted from the column by
activation of 6his

with AMF (30 µM
AlCl3, 50 mM MgCl2 and 10 mM NaF)-containing buffer: 20 mM Hepes, pH 8.0, 50 mM NaCl, 10 mM
-mercaptoethanol, 10 µM GDP, 1% sodium cholate, 5 mM imidazole,
50 mM MgCl2, 10 mM NaF, and 30 µM AlCl3. The peak 
-containing
fractions were identified by immunoblotting for both
and
subunits and then pooled and diluted to less than 10 mM
NaCl using 20 mM Hepes, pH 8.0, 1 mM EDTA, 3 mM DTT, 3 mM MgCl2, and 0.7%
CHAPS. 
subunits were further purified by fast protein liquid
chromatography on a Mono Q column (Amersham Pharmacia Biotech, HR 5/5)
eluted with a linear NaCl gradient from 0 to 400 mM. Peak
fractions were again confirmed by immunoblotting. The elution peaks
were pooled and dialyzed overnight (3 buffer changes) against 20 mM Hepes, pH 8.0, 1 mM EDTA, 3 mM
DTT, 3 mM MgCl2, 100 mM NaCl, and
0.7% CHAPS (Spectra/Por tubing, 6000-8000 molecular weight cut-off,
Spectrum Medical Industries, Houston, TX). A mixture of 
subunits
purified from bovine brain by a previously described method (23) was also further purified on a Mono Q column by the same procedure. Following dialysis, purified 
subunits were concentrated to approximately 0.1 mg/ml in an Amicon ultrafiltration device (PM10 membrane), and the final protein concentrations were determined by
staining with Amido Black. Purified 
subunits were snap-frozen in
small aliquots with liquid N2 and stored at
80 °C.
The 6his
i1 subunit was expressed alone
(m.o.i. = 3) in a 1-liter culture of Sf9 insect cells for
subsequent purification. Protein extraction, loading, and washing of
the Ni-NTA column were identical to that described for 
subunits.
The 6his
i1 was eluted from the Ni-NTA column
with the following buffer, 20 mM HEPES, pH 8.0, 100 mM NaCl, 10 mM
-mercaptoethanol, 1 mM MgCl2, 0.5% polyoxyethylene 10-lauryl
ether, 10 µM GDP, and 150 mM imidazole, and
was subsequently purified further on a Mono Q column (fast protein
liquid chromatography) using the same procedure described for 
subunits with the exception that collection tubes contained an aliquot
of GDP to yield a final concentration of 10 µM GDP in
each of the column fractions. Subsequent handling of
6his
i1 was identical to 
subunits.
Reconstitution of Receptor-G Protein Coupling--
Purified
and 
subunits were combined in 20 mM Hepes, pH 8.0, 1 mM EDTA, 3 mM DTT, 10 µM GDP,
0.02% sodium cholate to allow formation of G protein heterotrimers of
defined subtype composition. The mixture was incubated in a total
volume of 30 µl on ice prior to reconstituting the G proteins into
Sf9 cell plasma membranes. An aliquot of the Sf9 membrane
preparation expressing
2A-adrenergic receptor was thawed
and diluted to approximately 0.5 mg of protein/ml in 25 mM
Tris, pH 7.6, 1 mM EDTA, 10 mM
MgCl2, 1 mM benzamidine, 1 µg/ml pepstatin A,
and 1 µg/ml aprotinin; then Gi heterotrimers were added
to the membranes at the desired ratio of G protein to receptor and
incubated on ice for 30 min prior to receptor binding assays. The CHAPS
concentration during this incubation was
0.04%.
ADP-ribosylation Assay--
To assess the relative affinities of
the various 
dimers for the
subunit, 330 ng of the various
purified
1
dimers were combined with 500 ng of
purified 6his
i1 at a final ratio of 50:75
(mol:mol) of 
:
. At this ratio, differences in the relative
affinities of certain 
dimers for the receptor were detected. As
described previously (24), incubation of the resulting Gi
heterotrimers was carried out in the presence of 200 ng of islet
activating protein and [32P]NAD at 30 °C for 20 min
and then terminated by precipitation with 30% trichloroacetic acid
followed by rapid filtration over BA85 nitrocellulose filters.
ADP-ribosylation of 6his
i1 was measured by
scintillation counting to detect [32P] bound to filters.
Radioligand Binding Assays--
[3H]UK-14,304 and
[3H]yohimbine binding incubations were carried out in a
total volume of 250 µl at 24 °C for 60 min in a reaction buffer
consisting of 25 mM Tris, pH 7.6, 1 mM EDTA, 10 mM MgCl2 (plus 100 mM NaCl in
assays of [3H]yohimbine binding). Radioligand binding was
initiated by addition of 2-10 µg of Sf9 membrane protein and
terminated by addition of 3 ml of the same ice-cold reaction buffer
followed by rapid filtration over Whatman GF/C glass fiber filters on a
Millipore filtration apparatus. Filters were rinsed twice more with the same buffer. Bound radioligand was quantitated by liquid scintillation counting (Beckman LS6500). Nonspecific binding was determined in the
presence of 100 µM yohimbine. Statistical analysis of
coupling (high affinity [3H]UK-14,304 binding) supported
by different
subtypes was done by a two-tailed, Student's
t test for comparison of the variation between two means,
0.05 indicated statistical significance.
Materials--
[imidazoylyl-4,5-3H]UK-14,304,
[methyl-3H]yohimbine,
[32P]nicotinamide adenine dinucleotide, and G protein
-common antiserum (SW/1) were obtained from NEN Life Science
Products; Ni-NTA-agarose was obtained from Qiagen (Chatsworth, CA);
oxymetazoline was obtained from Research Biochemicals International
(Natick, MA); ECL reagent and horseradish-linked anti-rabbit IgG were
obtained from Amersham Pharmacia Biotech (Buckinghamshire, UK); and
other chemicals were obtained from Sigma.
 |
RESULTS |
Characterization of
2A-Adrenergic Receptor
Expression in Sf9 Insect Cell Plasma Membranes--
Expression
of the
2A-adrenergic receptor in Sf9 insect cells
was initially assessed using the antagonist
[3H]yohimbine. Uninfected cells did not express the
2A-adrenergic receptor (open circles, Fig.
1A). However, a significant
level of the
2A-adrenergic receptor was detected by
specific [3H]yohimbine binding at 48 h after
infection of cells with the
2A-AR baculovirus
(filled squares, Fig. 1A). The
KD values for the recombinant receptor calculated by
Scatchard analysis of the saturation binding curve ranged from 3.5 to 6 nM with Bmax values ranging from 2.7 to 13.3 pmol/mg protein depending on the cell preparation
(inset of Fig. 1A). This KD
range of the recombinant receptor was consistent with that of the
native
2A-adrenergic receptor (for review, see Ref. 25).
Moreover, this moderate Bmax value was optimal
for the purpose of this study since this level of recombinant receptor
expression was readily measurable but required minimal amounts of
purified G proteins for reconstitution. Finally, the properties of the
recombinant
2A-adrenergic receptor were characteristic
of those of the native receptor. The specific
[3H]yohimbine binding showed a NaCl sensitivity that is
typical of the native receptor (25), i.e. the
Bmax binding plateau was 45-55% lower in the
presence of 100 mM NaCl (data not shown). Prazosin and
oxymetazoline displaced the specific [3H]yohimbine
binding from the recombinant receptor with the same order of potency of
the native receptor (KD values of 4 and 0.022 µM, respectively). Taken together, these data confirmed the suitability of Sf9 cells for the expression of recombinant
2A-adrenergic receptor that is functionally similar to
the native receptor (26).

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Fig. 1.
Expression of
2A-AR in Sf9 insect cell
membranes. Sf9 insect cell plasma membranes were prepared
following receptor expression, and [3H]yohimbine binding
was assayed as described under "Experimental Procedures."
Triplicate receptor binding incubations contained 5 µg of membrane
protein each; data points are mean values ± S.E. A,
, specific [3H]yohimbine binding (total minus binding
in the presence of 100 µM unlabeled yohimbine) in
membranes following 2A-AR expression; , specific
[3H]yohimbine binding in control membranes (no
recombinant baculovirus); B, binding of 1.5 nM
[3H]yohimbine in the presence of the indicated
concentrations of competing ligand; triplicate incubations containing
10 µg of Sf9 plasma membrane and 10 µM GTP S
were handled as described under "Experimental Procedures."
Curves were fit to the data by non-weighted, non-linear
regression analysis using a one-site competition formula.
Symbols are defined as follows: , displacement of
[3H]yohimbine from 2A-AR by prazosin
(KD = 4 µM); , displacement by
oxymetazoline (KD = 0.022 µM).
KD values were derived from the EC50
using the relationship described by Cheng and Prusoff (38).
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Reconstitution of
2A-Adrenergic
Receptor-G Protein Coupling in Sf9 Cell Plasma
Membranes--
Coupling of the
2A-adrenergic receptor
was examined in the presence and absence of exogenous Gi
protein using the agonist [3H]UK-14304. The direct
agonist binding technique is generally considered to be more sensitive
than agonist displacement studies for detecting receptor-G protein
complexes (25). When Sf9 cell membranes expressing the
recombinant
2A-adrenergic receptor were incubated in the
absence of exogenous Gi protein, a low level of specific
[3H]UK-14304 binding was detected, accounting for ~15%
of the binding that was later observed in the presence of added
Gi protein (Fig. 2). By
contrast, when Sf9 cell membranes expressing the recombinant
2A-adrenergic receptor were incubated in the presence of
exogenous Gi protein (at a molar ratio of 100:1
Gi:receptor), the level of specific
[3H]UK-14304 binding was increased by more than 5-fold,
representing coupling of the recombinant receptor to the added
Gi protein (Fig. 2). Moreover, the increased level of
[3H]UK-14304 binding was reversed by the addition of
GTP
S, reflecting uncoupling of the recombinant receptor from the
added Gi protein. Thus, reconstituted coupling was easily
distinguishable from the background coupling in this experimental
system, thereby confirming the suitability of this experimental system
for measuring the coupling of the recombinant receptor to added
Gi proteins of varying 
composition. For optimal
resolution between the reconstituted and background coupling, a 4 nM concentration of [3H]UK 14,304 was used in
subsequent experiments.

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Fig. 2.
Guanine nucleotide-sensitive
[3H]UK-14,304 binding by
2A-AR expressed in Sf9 insect
cell membranes. Triplicate binding incubations containing 2.2 µg
of membrane protein each were done in the absence or presence of 0.1 mM GTP S at the radioligand concentrations indicated.
, specific binding of [3H]UK-14,304 following
reconstitution with purified Gi heterotrimer at a ratio of
50:1, Gi to receptor; , binding in the presence of 100 µM GTP S following reconstitution with Gi;
, specific binding without added Gi; , binding in the
presence of GTP S with no added Gi. A curve was fit to
the data by non-weighted, nonlinear regression analysis using a
one-site hyperbola fit. Gi heterotrimer (9.25 pmol) was
composed of purified 6his i1 and purified
bovine brain  (3:2, mol /mol  ). Nonspecific binding
(presence of 100 µM yohimbine) was virtually identical to
low affinity binding (presence of 100 µM GTP S); thus
essentially all the radioligand binding within this concentration range
represents high affinity, receptor-G protein complexes.
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Receptor to G Protein Stoichiometry--
The requirements of the
[3H]UK-14,304 binding assay for the G protein
and

subunits were examined further. Consistent with previous studies
of the A1 adenosine receptor (9), the combined interaction
of both the G protein
and 
subunits was required in order to
detect the high affinity state of the recombinant
2A-adrenergic receptor with this binding assay. As shown
in Fig. 3A, when the
recombinant receptor was reconstituted with the
6his
i1 subunit alone (at a molar ratio of
50:1
:receptor), the level of [3H]UK-14304 binding was
low and was indistinguishable from that observed with no added
Gi heterotrimer. This result attests to the validity of the
[3H]UK-14,304 binding assay to evaluate differences in
the ability of 
dimers of varying composition to induce the high
affinity state of the recombinant receptor. Next, the recombinant
receptor was reconstituted with a constant amount of
6his
i1 subunit and increasing amounts of

dimer. As shown in Fig. 3B, raising the amount of

dimer increased the fraction of receptor in the high affinity
state as measured by the higher level of [3H]UK-14,304
binding. When the amounts of 
dimer and
6his
i1 subunit approached a 1:1 ratio, the
level of [3H]UK-14,304 binding reached a plateau,
accounting for ~60% of the total receptor population as determined
by [3H]yohimbine binding. A similar, maximal level of
coupling was observed previously for the A1 adenosine
receptor (9), suggesting that not all of the recombinant receptors are
accessible for reconstitution with added G proteins. Under these
conditions, any observed differences in the magnitude of
[3H]UK-14,304 binding can be assumed to be attributable
to selective interactions of 
dimers of varying composition with
the receptor rather than to alterations in G protein
-
subunit
interactions.

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Fig. 3.
Optimization of receptor to G protein
stoichiometry for reconstitution of high affinity, guanine
nucleotide-sensitive [3H]UK-14,304 binding.
A, binding of 4 nM [3H]UK-14,304
by 2A-AR in Sf9 insect cell membranes assayed
following reconstitution with heterotrimeric Gi,
G i subunit alone, or a mock reconstitution with no added
G protein. Triplicate receptor binding incubations contained 2 µg
each of membrane protein in the absence (filled bars) or
presence (cross-hatched bars) of 0.1 mM GTP S,
yielding mean ± S.E. values. B, ,
[3H]UK-14,304 binding by 2A-AR
reconstituted with / mixtures containing a constant amount of
purified 6his i1 incubated (2 h on ice) with
increasing amounts of purified bovine brain  to yield a final
6his i1:receptor ratio of 75:1 (mol/mol)
throughout the curve and the  :receptor ratio indicated on the
ordinate. The curve was fit to the data by non-weighted,
non-linear regression analysis using a one-site hyperbola (GraphPad
Prism). The data are representative of three similar results.
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|
Purification of G Protein
1
Dimers--
To
produce recombinant 
dimers of varying
composition, the
1 subtype was chosen since it has previously been shown
to interact with all of the
subtypes (17, 27, 28). The recombinant 
dimers were purified using a procedure originally described by
Kozasa and Gilman (11). Sf9 cells expressing the
6his
i1,
1, and one of the
following
1,
2,
3,
4,
5,
7,
10, or
11 subunits were prepared. Then,
cholate-solubilized membrane extracts from these cells were bound to
Ni-NTA agarose columns by virtue of the 6-histidine tag on the
i1 subunit; the 
dimers were eluted from the
columns by activating the bound heterotrimers with AMF; and the
6his
i1 subunit was subsequently eluted from
the columns with high imidazole. Further purification of the
recombinant 
dimers and the 6his
i1
subunit was achieved by applying their enriched fractions from the
Ni-NTA columns to Mono Q columns.
The purity of the 6his
i1 subunit was
assessed by SDS-PAGE and silver staining (29). As shown in Fig.
4A, the purified
6his
i1 preparation contained one major band
of the size expected for the
i1 subunit taking into
account the added amino-terminal tag. The purity of the recombinant

dimers was also compared by SDS-PAGE and silver staining
(Coomassie was used in the case of
1
1).
As shown in Fig. 4A, each purified 
preparation was composed of two predominant bands by protein staining as follows: a
36-kDa band representing the
1 subunit, and a 5-8-kDa
band representing one of the following
1,
2,
3,
4,
5,
7,
10, or
11 subunits. The
identity of each 
purified preparation was confirmed by
immunoblotting with antibodies specific for each
subtype.
Antibodies specific for the
1,
2,
3,
5, and
7 subunits were
used for this purpose previously (20). However, antibodies specific for
the newly described
4,
10, and
11 subunits needed to be generated against synthetic
peptides based on the unique amino acid sequences of these proteins. As
shown in Fig. 4B, the identities of the purified
1
4,
1
10,
and
1
11 preparations were confirmed by
immunoblotting with these antibodies.

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Fig. 4.
Purified recombinant G protein subunits.
A, 1st lane, 3 µg of purified
1 1 was trichloroacetic acid-precipitated
and loaded onto a 15% polyacrylamide gel in 40 µl of Laemmli sample
buffer and stained with Coomassie Blue following electrophoresis;
2nd to 9th lanes, silver-stained purified G protein
subunits. 300 ng each (determined by Amido Black staining) of purified
G protein subunits were trichloroacetic acid-precipitated and loaded
onto 15% polyacrylamide gels in 40 µl each of Laemmli sample buffer
containing 120 mM DTT. Gels were silver-stained following
the method of Fawzi et al. (30). Lanes contain the following
subunits: 2nd lane,
1 2; 3rd lane,
1 3; 4th lane,
1 4; 5th lane,
1 5; 6th lane,
1 7; 7th lane,
1 10; 8th lane,
1 11; 9th lane,
6his i1. B, following Western
transfer, 300 ng each of purified  subunits were probed with the
following -specific antibodies: 4, E59 at 1/250;
10, E57 at 1/250; and 11, E60 at 1/300.
The peak of  elution occurred at Mono Q fractions 11 and 12, corresponding to 200-240 mM NaCl, in each case.
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Comparison of 
Dimers of Varying
Composition in Terms of
Coupling to the
2A-Adrenergic
Receptor--
Gi heterotrimers of varying
composition
were tested for their relative abilities to couple with receptor as
measured by the level of high affinity [3H]UK-14,304
binding. As shown in Fig. 5, all
combinations of the 6his
i1 subunit with the
various 
dimers were capable of inducing high affinity
[3H]UK-14,304 binding, with the level of binding showing
dependence on the
composition. In all cases, the
[3H]UK-14,304 binding was completely abolished by
addition of GTP
S (data not shown). The
1
1 dimer supported only a very low level of coupling to the recombinant
2A-adrenergic receptor.
By contrast, the
1
11 dimer produced a
high level of coupling to the recombinant
2A-adrenergic
receptor. Since the
1 and
11 subtypes are
closely related, showing 76% identity (19), the observation that they promote very different levels of coupling suggests that the relatively small number of amino acid differences between the two subtypes are
important for recognition by the receptor. The
1
2,
1
3,
1
4, and
1
7
dimers also produced high levels of coupling with the recombinant
2A-adrenergic receptor. These four
subtypes are
closely related, showing 66-74% homology at the amino acid level, and
therefore, the finding that they produce essentially identical levels
of coupling is not unexpected. Finally, the
1
5 and
1
10
dimers yielded intermediate levels of coupling with the recombinant
2A-adrenergic receptor. These two subtypes are only
distantly related, showing less than 53% homology to each other or to
other
subtypes. Taken together, these results showed measurable
differences between 
dimers of varying
composition to support
coupling of the same
subunit to the recombinant
2A-adrenergic receptor. Statistical analysis revealed
the
1
1,
1
5,
and
1
10 dimers supported the lowest
levels of coupling. Previously, several groups have reported that the
1
1 dimer was less effective than other

dimers in supporting coupling to numerous receptors (8, 9, 30)
and that this problem could not be overcome by increasing its
concentration. To extend further these observations, we showed that
increasing the concentration of the
1
5
dimer did not raise the level of receptor coupling (Fig.
5B), indicating the
5 subtype has a lower
intrinsic ability to interact with the recombinant
2A-adrenergic receptor. Based on results for both the
1
1 and
1
5
dimers, a similar result could be predicted for the moderately effective
1
10 dimer.

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Fig. 5.
Comparison of reconstituted
2A-AR coupling with Gi
heterotrimers containing different 
subtypes. A, the
6his i1 subunit was incubated with each
purified  dimer for 2 h on ice. The resulting Gi
heterotrimers were reconstituted into an Sf9 cell membrane
preparation expressing the recombinant 2A-AR at a
Bmax of 2.7 pmol/mg protein. This condition
represents a ratio of : :receptor of 75:50:1. Receptor-G
protein coupling was assayed by binding of 3.5 nM
[3H]UK-14,304 ± 0.1 mM GTP S in
triplicate to obtain mean ± S.E. values. The data show total
binding minus binding in the presence of GTP S where background
coupling (obtained without added G protein) has been subtracted from
each value. The data shown are an average of three such experiments.
Asterisks indicate a significant difference ( 0.05, by
two-tailed Student's t) in agonist binding relative to
purified bovine brain  . B, the
6his i1 subunit was incubated with increasing
concentrations of the 1 5 dimer for 2 h on ice and then reconstituted into an Sf9 cell membrane
preparation expressing the recombinant 2A-AR at a
Bmax of 13.3 pmol/mg protein. The ratio of
:receptor is 75:1, and the ratio of
1 5:receptor is shown on the
ordinate. Receptor-G protein coupling was assayed by binding
of 6 nM [3H]UK-14,304 ± 0.1 mM GTP S in triplicate to obtain mean ± S.E.
values. The data show total binding minus binding in the presence of
GTP S where background coupling (obtained without added G protein)
has been subtracted from each value.
|
|
The lower activities of the
1
1,
1
5, and
1
10
dimers could be due to differences in affinities for the
subunit of
the G protein rather than for the
2A-adrenergic receptor
itself. To evaluate this possibility, the affinities of representative 
dimers for the 6his
i1 subunit were
measured by the pertussis toxin-dependent ADP-ribosylation assay. Under the reconstitution conditions used in this study, which
employed relatively high concentrations of 
dimers, there were no
real differences in the affinity of these 
dimers for the
6his
i1 subunit (Fig.
6). Thus, the observed differences
between 
dimers of varying
composition reflect their
intrinsic abilities to interact with the receptor, suggesting
structural diversity among
subtypes plays a role in
agonist-stimulated receptor-G protein complex formation.

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Fig. 6.
Islet activating protein-catalyzed
ADP-ribosylation of
6his i1 in the presence
of different  subtypes.
Using the same conditions employed for the reconstitution in Fig. 5,
500 ng of purified 6his i1 were combined with
330 ng of the various purified 1 dimers at a final
ratio of 75:50 (mol:mol) of : . Incubation of the purified
proteins in the presence of 200 ng of islet activating protein and
[32P]NAD was terminated after 20 min at 30 °C by
precipitation with 30% trichloroacetic acid followed by rapid
filtration over BA85 nitrocellulose filters. ADP-ribosylation of
6his i1 was measured by scintillation
counting to detect [32P] bound to filters.
Bars show the mean cpm ± S.E. values from triplicate
determinations with the specific 1 combination
indicated. Background [32P] bound to filters in the
absence of 6his i1 has been subtracted from
these values.
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|
Purification of G Protein
3
Dimers--
A
variety of approaches has been used to examine the ability of the 6
and 11
subtypes to form 
dimers (17, 19, 27, 28). Overall,
these approaches have provided consistent results showing that all the
known
subtypes are able to interact with the
1
subtype and, to a lesser extent, the
2 subtype. However, these approaches have yielded conflicting results regarding the abilities of the known
subtypes to interact with the
3 subtype. In this regard, the lack of a 
dimer
containing the
3 subtype to serve as a positive control
in the various assays has been a particular hindrance. To this end, the
method of Kozasa and Gilman (11) was used to obtain a 
dimer
containing the
3 subtype. Sf9 cells were infected
with recombinant viruses encoding the
6his
i1,
3, and
5 subunits either simultaneously or separately, and the
3
5 dimer was then purified by the
procedure described above. As shown in Fig.
7A, the co-expression of the
6his
i1,
3, and
5 subunits resulted in the appearance of
3 and
5 subunits in the AMF activation
fractions as detected by immunoblotting. This result was consistent
with the release of
3
5 dimer from the
6his
i1 subunit in response to AMF
activation. As shown in Fig. 7B, the identity of the
AMF-released
subunit as the
3 subunit was confirmed
by immunoblotting with a
3 subtype-specific antibody (B-34). Taken together, these results supported the conclusion that the
3 and
5 subunits interact to form a
functional 
dimer.

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Fig. 7.
Elution of
3 5
from Ni-NTA agarose column. 100-ml cultures of Sf9 insect
cells were inoculated with recombinant baculovirus encoding
6his i1, 3, and/or
5. Cholate-soluble particulate protein was loaded onto a
Ni-NTA agarose column that was washed and eluted as described under
"Experimental Procedures." Western blot was performed on aliquots
of the column fractions. A, simultaneous expression of
6his i1, 3, and
5. The upper portion with -common antibody
(SW/1, 1:5000) the lower portion with
5-specific antibody (E56, 1:250). 1st
lane, 30 µg of cholate-soluble particulate fraction;
2nd lane, 30 µg, column flow-through;
3rd lane, 30 µg, 5 mM imidazole
wash at 4 °C; 4th lane, 30 µg, 5 mM imidazole wash at room temperature (R.T.);
5th to 9th lanes, 3 µg, elution by AMF; 10th
and 11th lanes, 3 µg, elution by 150 mM imidazole. B, recognition of purified
3 5 dimer with a 3-specific
antibody (B34, 1:150). 1st lane, 300 ng of purified
1 5; 2nd lane, 300 ng of purified 2 4; 3rd
lane, 300 ng of purified 3 5;
4th lane, 300 ng of  purified from Sf9 insect
cells.
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|
An example of the purity of the
3
4,
3
5, and
3
11
subunits that can be obtained by this procedure is shown by silver
staining. As shown in Fig. 8A,
each purified
3
preparation was composed of two
predominant bands by silver staining as follows: a 37-kDa band
representing the
3 subunit and a 5-8-kDa band
representing one of the
subtypes. Confirmation that the
3 and
5 subunits interact to form a
functional 
dimer is also shown using a previously developed
tryptic digestion procedure (19, 27). This method is based on the
finding that
monomers are cleaved at numerous sites by trypsin. By
contrast, functional 
dimers are cleaved at a single site,
resulting in the appearance of a 26-kDa fragment of the
subunit
that is resistant to further digestion by trypsin. Whereas the
appearance of this stable 26-kDa fragment is a reliable marker for the
formation of 
dimers containing the
1 and
2 subtypes, it is not clear whether formation of 
dimers containing the
3 subtype yields the appearance of
a similar protected fragment. To date, such a protected fragment has
not been detected for the
3 subtype, but these results
are difficult to interpret due to the lack of availability of a
positive control at that time (19). As shown in Fig. 8B,
purified preparations of both the
1
5 and
3
5 subunits produced a 26-kDa protected
fragment when digested under identical conditions with trypsin, as
detected in each case by immunoblotting with a commercial
-antibody
(DuPont SW/1, carboxyl terminus). Since equal amounts of
1
5 and
3
5
subunits were loaded, the differences in intensities of the
1 and
3 bands presumably reflect
differences in affinities of the antibody used for immunoblotting.
Taken together, these results confirmed that the
3 and
5 subunits are able to interact to form a functional 
dimer. Moreover, these results extended the utility of the trypsin digestion method as a reliable marker of 
dimer formation to the
3 subtype.

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Fig. 8.
3 dimers
purified following expression in Sf9 insect cells.
A, silver-stained, purified 3 dimers.
1st lane, 400 ng of
3 5; 2nd lane, 400 ng of 3 4; 3rd lane,
400 ng of 3 11. B, protection
of 3 from tryptic proteolysis by association with
5.  subunits were incubated for 40 min at 30 °C
with or without 1 µg of trypsin, after which 6 µg of trypsin
inhibitor were added to each, and the samples were trichloroacetic
acid-precipitated for 15% SDS-PAGE. The products are shown by Western
blot using -common antibody (SW/1, NEN Life Science Products).
1st and 2nd lanes, 400 ng each of
1 5; 3rd and 4th
lanes, 400 ng each of
3 5.
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|
Comparison of 
Dimers of Varying
Composition in Terms of
Coupling to the
2A-Adrenergic Receptor--
As
shown in Fig. 9, all combinations of the
6his
i1 subunit with the various
3
dimers were capable of inducing high affinity [3H]UK-14,304 binding. Again, in all cases, the
[3H]UK-14,304 binding was completely abolished by
addition of GTP
S (data not shown). The
1
4 and
3
4
dimers showed similar abilities to reconstitute coupling with the
recombinant
2A-adrenergic receptor. Likewise, the
1
11 and
3
11
dimers had essentially identical activities. On the other hand, the
3
5 dimer showed a substantially higher
level of coupling with the recombinant
2A-adrenergic
receptor than the
1
5 dimer. Increasing
the concentration of the
1
5 dimer did not
raise the level of coupling (Fig. 5B), indicating the
1 subtype, when in association with the
5
subtype, has a lower intrinsic ability to interact with the recombinant
2A-adrenergic receptor. Taken together, these
differences support the conclusion that the receptor recognition of the
G protein is dependent on the particular combination of
and
subtypes.

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Fig. 9.
Comparison of
1 and
3 in reconstitution of
2A-AR coupling. Gi
heterotrimer composed with the  dimers indicated was
reconstituted into Sf9 plasma membranes expressing
2A-adrenergic receptor at a mol/mol ratio of 1:125:100
for receptor:6his i1: . Binding of 4 nM [3H]UK-14,304 was assayed in the absence
or presence of 100 µM GTP S. High affinity agonist
binding was calculated as the difference between total binding and
binding in the presence of GTP S. Background (i.e. high
affinity agonist binding without added Gi) was subtracted
from each value to show only the reconstituted coupling.
1 5 supported significantly less coupling
than 3 5 ( 0.05, Student's
t test).
|
|
 |
DISCUSSION |
The present study examined the potential of the
2A-adrenergic receptor to couple to G proteins differing
in their 
subunit composition only. The selectivity of coupling
was directly assessed by a high affinity agonist binding assay.
Importantly, this assay was found to require the addition of the 
subunits in order to detect the interaction of the
subunit with the
receptor. From previous studies, this requirement for the 
subunits appears to reflect not only a general role of the 
subunits to stabilize the
subunit (31) but also a specific role of
the 
subunits to interact directly with the receptor (6, 7). In
view of these roles and the rich diversity of 
subunit
combinations, the possibility was suggested that the nature of the

subunits might contribute to the selectivity of the receptor
interaction. Supporting such a possibility, the present study showed
clear differences in the abilities of the various 
dimers,
including those containing the
3 subtype and the newly
described
4,
10, and
11
subtypes, to promote interaction of the same
i subunit with the
2A-adrenergic receptor.
Influence of 
Composition on Receptor Coupling--
Several
lines of evidence support the validity of using Sf9 insect cell
membranes expressing the recombinant
2A-adrenergic receptor as a suitable system for examining the specificity of coupling
to purified, recombinant G proteins. First, the recombinant
2A-adrenergic receptor displayed the binding affinities
and pharmacologic properties characteristic of the native receptor.
Second, the recombinant
2A-adrenergic receptor showed a
mostly uncoupled phenotype in the absence of added G proteins and a
largely coupled phenotype in the presence of added G proteins of
defined composition and stoichiometry. Using high affinity agonist
binding as a quantitative measure of the coupled phenotype, this system
was first used to examine the influence of the
component on
receptor coupling. Gi proteins were produced from
6his
i1,
1, and varying
subtypes. Among the eight 
dimers tested, 30-fold differences
were observed in their abilities to support coupling of the
6his
i1 subunit to the
2A-adrenergic receptor, with the
1
2,
1
3,
1
4,
1
7, and
1
11 dimers displaying the most efficacy,
the
1
5 and
1
10 dimers showing intermediate
efficacies, and the
1
1 dimer exhibiting
the least efficacy. With the exception of the
11
subtype, the observed differences segregated with the structural
diversity of the
component along subclass lines. As defined
previously, the human
subunit family has been divided into three
subclasses, with each subclass showing less than 50% amino acid
homology to other subclasses (1). On this basis, subclass I contains
the
1,
c, and
11 subtypes,
which are modified by the less common C-15 farnesyl group; subclass II
includes the
2,
3,
4,
7, and
12 subtypes, which are modified by
the more common C-20 geranylgeranyl group; and subclass III contains
the
5 and
10 subtypes, which again
receive the more common C-20 geranylgeranyl group. The present study
represents the most extensive functional analysis of the
subunit
family to date.
Next, this system was used to examine the influence of the
1 versus the
3 subtype on
receptor coupling. In vitro studies have revealed little or
no functional differences due to the
subunit (16, 17). Since only
the closely related
1 and
2 subtypes were
examined, however, the present study extended this analysis to the more
divergent
3 subtype. For this purpose, the method of
Kozasa and Gilman (11) was used to produce and then purify functional

dimers containing the
3 subtype. In addition to
providing a source of material of defined composition, this approach
also revealed new information on the selectivity of
-
interaction
by confirming the ability of the
3 subtype to interact with the
4,
5, and
11
subtypes. Whereas interaction between the
3 and
4 subtypes had been predicted (2, 3), the ability of the
3 subtype to interact with the
11 subtype
was unexpected in view of the high homology between the
11 and
1 subtypes and the reported
failure of the
1 subtype to interact with the
3 subtype (28). When the various 
dimers were
tested for receptor coupling, only the
3
5
and
1
5 dimers showed substantive
differences in their abilities to support coupling of the
6his
i1 subunit to the
2A-adrenergic receptor. No such differences were
observed with the
3
4 and
1
4 dimers nor the
3
11 and
1
11
dimers. These results suggested that it is the particular combination
of
and
subtypes that ultimately determines receptor recognition. Interestingly, the
3
5 dimer
was shown previously to interact preferentially with a G
protein-coupled receptor kinase, GRK3, indicating a role of the
subtype in selective receptor desensitization (32).
Taken together, these data show that Gi proteins containing
different 
dimers produce different levels of coupling to the
2A-adrenergic receptor. This result could arise because
the composition of the 
subunits alters the formation or
stability of the G protein, the affinity of the 
dimer for the
receptor, or some combination therefrom. Our data (17) and those from
other laboratories (9, 33) suggest that formation of the G protein is
the least likely reason since the affinity of the
subunit for the
various 
dimers is similar. Instead, our data are most consistent
with the composition of the
and, particularly, the
component
affecting the affinity of the G protein for the
2A-adrenergic receptor. Studies of the
A1-adenosine receptor support a similar conclusion (33).
Sites of Interaction of
Component with Receptor--
The
observed differences in the abilities of various 
dimers to
support coupling to the
2A-adrenergic receptor reside
primarily in the
component. Although cross-linking studies have yet
to detect receptor-
contact sites (7), several studies point to the
importance of the carboxyl-terminal amino acid region and the type of
prenyl group on the
subunit in determining the interaction of the

dimer with receptor (34, 35). These latter studies may explain
the lower reconstitutive activity of the
1
1 dimer in the present study. In this
regard, the
1 subtype sequence is the most divergent of
those determined to date, and its lipid modification is a C-15 farnesyl
group rather than the C-20 geranylgeranyl group found on most other
subtypes (1). With regard to the latter, a recent study comparing

dimers with the two types of prenyl groups showed that the wild
type, geranylgeranylated
2 subunit and the mutant,
geranylgeranylated
1 subunit had similar abilities to
interact with the A1 adenosine receptor (34). By contrast,
the wild type, farnesylated
1 subunit and the mutant, farnesylated
2 subunit were much less effective. Whereas
these data indicate that type of prenyl group is critical, the primary structure of the
subunit is of equal or greater importance. Underscoring this point, a synthetic peptide derived from the carboxyl-terminal sequence of the
1 subtype was able to
stabilize the light-activated state of rhodopsin receptor to a much
greater degree when the peptide was farnesylated than not (35).
However, the effect was greatly attenuated when the amino acid sequence of the peptide changed by only two amino acid substitutions (F64T and
L67S) even though the farnesylated state was preserved. Thus, both the
primary structure and the prenylation state of the
1 subtype are likely to contribute to its poor affinity for the
2A-adrenergic receptor in the present study and its
contrastingly high affinity for the rhodopsin receptor in previous
studies (35). When compared with the
1
1
dimer, the higher reconstitutive activity of the
1
11 dimer was unexpected since the newly
described
11 subtype is farnesylated and has a
carboxyl-terminal tail nearly identical to
1 subtype.
This result suggests the importance of regions other than the
carboxyl-terminal tail of the
11 subunit in promoting
its strong interaction with the
2A-adrenergic receptor. By focusing on the few amino acid differences between the
11 and
1 subtypes and making the
appropriate mutations, it should be possible to pinpoint other regions
of
protein structure that are selectively recognized by the
2A-adrenergic receptor. Given the wide tissue
distribution of the
11 subtype (19) in contrast to the
restricted expression of the
1 subtype, it is perhaps not so surprising that a receptor other than rhodopsin would exist which prefers the farnesylated
11 subtype in tissues
other than the retina.
Finally, the high reconstitutive activities of the
1
2,
1
3,
1
4 or
1
7
dimers compared with the intermediate activities of the
1
5 and
1
10
dimers underscore the importance of primary structure in another way.
Since all of the aforementioned
subtypes are modified by the C-20
geranylgeranyl group (19), the observed differences between the two
groups must relate to the differences in protein structure. In
agreement with this, the
2,
3,
4 and
7 subtypes share a high degree of
amino acid homology (66-74%), and predictably, 
dimers
containing these
subtypes produce comparably strong levels of
coupling of the 6his
i1 subunit to the
2A-adrenergic receptor. By contrast, the
5 and
10 subtypes share only 35-50%
identity with the aforementioned group of
subunits (19), and
accordingly, 
dimers containing these
subtypes yielded
significantly lower levels of coupling in comparison with the mean
value calculated from the grouping of 
dimers consisting of the
2,
3,
4, and
7 subtypes (
= 0.025 or 0.05, respectively;
Student's t test). Taken together, these data indicate that
the primary structure, including regions other than the
carboxyl-terminal tail, of the
subunit is a most important factor
in determining the selectivity of interaction with the
2A-adrenergic receptor. In this instance, the type of prenyl group is a less critical factor.
Sites of Interaction of
Component with Receptor--
Although
the
subunits are highly conserved in their predicted amino acid
sequences, the observed difference between the
1
5 and
3
5
dimers indicates that selective receptor recognition does occur on the
basis of the
subtype. Cross-linking studies have revealed a site of
contact between the 7th or possibly 6th WD-40 repeat of the
subunit
and a peptide derived from the third intracellular loop of the
2-adrenergic receptor (7). The crystal structure of

t indicates that residue 303, which lies at the center of this segment, resides on an exposed surface of the 
dimer (36). Thus, this site has the potential to participate in the
preferential coupling of the
2A-adrenergic receptor to Gi heterotrimers containing the
3
5 dimer over those containing the
1
5 dimer. Other possibilities are
suggested: 1) regions other than the carboxyl-terminal tail of the
subunit may interact with receptor; 2) a concerted interaction between
the
and
subunits within the G protein binding pocket of the
receptor; or 3) some combination therefrom.
Influence of 
Composition on Coupling to Other G
Protein-coupled Receptors--
Various 
dimers show a different
order of potency depending on the type of receptor (8, 9, 37). It is
speculated that protein-protein interactions between the 
subunits and receptors, as well as hydrophobic interactions due to the
prenylation state of the
subunit, will be important elements in
modeling selective recognition between G protein and receptors.
Distinct, yet to be revealed, structural features within the G protein
binding regions of receptor subtypes must also be taken into account in such a model. For example, a previous study showed that the
5HT1A receptor interacts similarly with G proteins
containing
1
2,
1
3, or
1
5
dimers (8), whereas the present study revealed that the
2A-adrenergic receptor prefers G proteins containing the
1
2 or
1
3
dimer over that containing the
1
5 dimer.
Thus, the G protein binding pockets of the
2A-adrenergic
receptor and the 5HT1A may possess subtle structural
differences that result in either more or less receptor to G

contact depending on the identity of the
subunit. Active-state
receptors may possess discrete elements contacting
,
, and
subunits within the same G protein that complement one another to some
degree in order to activate the heterotrimer. The experimental approach
used here is quite amenable to manipulating both the receptor and the G protein subunit composition in order to bring together selected elements of signaling proteins.
Summary--
The data in the present study demonstrate the
specificity of
2-adrenergic receptor-G protein
interactions is affected by the 
dimer composition, with
protein-protein interactions forming the basis for the observed
differences. These in vitro results complement a growing
body of in vivo results demonstrating the 
subunit
composition is an important determinant of the specificity of signaling
pathways. Strikingly, antisense suppression of the
1
3 or
3
4
subtypes disrupts coupling between inhibition of a calcium channel and
the somatostatin or muscarinic receptors, respectively, in GH3
pituitary cells (2, 3). Similarly, ribozyme suppression of the
7 subtype attenuates coupling between stimulation of
adenylylcyclase and the
-adrenergic receptor in HEK293 cells (4).
Although these results could arise from an ordered arrangement of
signaling proteins in the cell membrane, the in vitro
results presented here implicate receptor-
"recognition" as
an additional mechanism for determining the specificity of signaling.
It is expected that a combination of in vitro and in vivo approaches will provide some of the answers needed for
construction of a mechanistic model of specificity in G
protein-mediated signaling pathways.