(Received for publication, December 11, 1995; and in revised form, February 1, 1996)
From the
The vasopressin receptor family is unique among all classes of
peptide receptors in that its individual members couple to different
subsets of G proteins. The V vasopressin receptor, for
example, is preferentially linked to G proteins of the G
class (biochemical response: stimulation of phosphatidylinositol
hydrolysis), whereas the V
vasopressin receptor is
selectively coupled to G
(biochemical response: stimulation
of adenylyl cyclase). To elucidate the structural basis underlying this
functional heterogeneity, we have systematically exchanged different
intracellular domains between the V
and V
receptors. Transient expression of the resulting hybrid receptors
in COS-7 cells showed that all mutant receptors containing V
receptor sequence in the second intracellular loop were able to
activate the phosphatidylinositol pathway with high efficiency. On the
other hand, only those hybrid receptors containing V
receptor sequence in the third intracellular loop were capable of
efficiently stimulating cAMP production. These findings suggest that
the differential G protein coupling profiles of individual members of a
structurally closely related receptor subfamily can be determined by
different single intracellular receptor domains.
An extraordinarily large number of neurotransmitters, peptide
hormones, neuromodulators, and autocrine and paracrine factors exert
their physiological actions via binding to specific plasma membrane
receptors that are coupled to distinct classes of heterotrimeric G
proteins (G protein-coupled receptors (GPCRs)). ()During the
past decade, several hundred members of this receptor superfamily have
been cloned and sequenced (Watson and Arkinstall, 1994).
Characteristically, each GPCR interacts only with specific subclasses
of the many structurally similar G proteins found within a cell (Hedin et al., 1993; Conklin and Bourne, 1993). To understand how
this selectivity is achieved at a molecular level has become the
research focus of an ever increasing number of laboratories.
Mutagenesis studies as well as experiments with short synthetic receptor peptides that can mimic or inhibit receptor-G protein interactions have shown that multiple intracellular receptor domains are involved in G protein coupling (Strosberg, 1991; Savarese and Fraser, 1992; Hedin et al., 1993). Considerable insight into the structural basis of receptor-G protein coupling selectivity has been provided by detailed mutational analysis of biogenic amine receptors such as the muscarinic acetylcholine (Wess, 1993) and adrenergic receptors (Dohlman et al., 1991; Strader et al., 1994).
In contrast, little is known about the structural
elements involved in G protein recognition by GPCRs that bind peptide
ligands. However, such receptors form one of the largest subclasses of
GPCRs, and more than sixty different peptide receptors have been cloned
to date (Watson and Arkinstall, 1994). These receptors (including, for
example, those for melanocortins, cholecystokinin, endothelins,
neurokinins, bombesin-like peptides, opioids, or somatostatin) are
known to play key roles in the regulation of a multitude of fundamental
physiological processes. Investigations into the structural basis of
the G protein coupling selectivity displayed by these receptors have
been hampered by the fact that the individual members of virtually all
peptide receptor subfamilies couple to similar G proteins. All
cholecystokinin, endothelin, neurokinin, and bombesin receptors, for
example, are preferentially coupled to G proteins of the G family, whereas the various opioid and somatostatin receptors are
all selectively linked to G proteins of the G
class
(Watson and Arkinstall, 1994). This pattern has precluded the use of
hybrid peptide receptors (in which distinct domains are exchanged
between functionally different members of a receptor subfamily) to
study the structural basis underlying the selectivity of protein
recognition displayed by these receptors.
In contrast to all other
peptide receptor subfamilies, the group of vasopressin receptors is
exceptional in that its members clearly differ in their G protein
coupling profiles. The vasopressin receptor family is formed by three
distinct subtypes, V, V
, and V
,
which share a high degree (40-50%) of sequence identity
(Birnbaumer et al., 1992; Lolait et al., 1992; Morel et al., 1992; Sugimoto et al., 1994; de Keyzer et
al., 1994). However, the V
and V
receptors are selectively coupled to G proteins of the G
family (Laszlo et al., 1991), which mediate the
activation of distinct isoforms of phospholipase C
, resulting in
the breakdown of phosphoinositide lipids (PI hydrolysis). The V
receptor, on the other hand, preferentially activates the G
protein G
(Laszlo et al., 1991), resulting in the
activation of adenylyl cyclase(s).
The individual vasopressin
receptors mediate numerous important physiological effects including
hepatic glycogenolysis, contraction of vascular smooth muscle and
mesangial cells, aggregation of platelets, and antidiuresis in the
kidney (Laszlo et al., 1991). Moreover, recent studies have
shown that mutations in the V receptor gene are responsible
for the X-linked form of nephrogenic diabetes insipidus (for recent
reviews see Birnbaumer (1995) and Spiegel(1996)).
To study the
structural elements responsible for the functional diversity found
within the vasopressin receptor family, we have created a series of
V/V
hybrid receptors in which distinct
intracellular domains were exchanged between the two wild type
receptors (Fig. 1). Functional characterization of the resulting
hybrid receptors in transfected COS-7 cells led to the novel
observation that different single receptor segments determine the
differential G protein binding profiles of two structurally closely
related peptide receptors.
Figure 1:
Structure,
ligand binding properties, and functional profile of wild type and
mutant V/V
vasopressin receptors.
[
H]AVP saturation binding studies were carried
out as described under ``Experimental Procedures.'' K
and B
values are
given as means ± S.E. of three independent experiments, each
performed in duplicate. The functional properties of the various
receptors (for experimental data, see Table 1and Fig. 2and Fig. 3) are summarized underneath the receptor
structures (PI, stimulation of PI hydrolysis; AC,
stimulation of adenylyl cyclase). The symbols are defined as the
percentage of maximum PI and cAMP responses induced by the wild type
V
and V
receptor, respectively:
++++, 90-100%; +++,
80-90%; +, 10-30%; -, no significant response.
The following sequences were exchanged between the rat V
(Morel et al., 1992) and human V
(Birnbaumer et al., 1992) receptor (amino acid numbers in parentheses): V2i1, V
(1-82)
V
(1-94); V2i2, V
(138-160)
V
(150-171); V2i3, V
(225-279)
V
(237-305); V2i4, V
(331-371)
V
(359-395); V1i1, V
(77-94)
V
(63-82); V1i2, V
(152-172)
V
(140-161); V1i3, V
(237-303)
V
(225-277); V1i4, V
(349-395)
V
(321-371).
Figure 2:
AVP-induced cAMP accumulation mediated by
wild type V and hybrid V
/V
vasopressin receptors. Transfected COS-7 cells transiently expressing
the different receptors were incubated in 6-well plates for 1 h at 37
°C with the indicated AVP concentrations, and the resulting
increases in intracellular cAMP levels were determined as described
under ``Experimental Procedures.'' The data are presented as
fold increase in cAMP above basal levels in the absence of AVP. Basal
cAMP levels for the wild type V
receptor amounted to 870
± 390 cpm/well. The basal cAMP levels observed with the
different mutant receptors were not significantly different from this
value. Each curve is representative of three independent experiments,
each carried out in duplicate.
Figure 3:
AVP-induced stimulation of PI hydrolysis
mediated by wild type V and hybrid V
/V
vasopressin receptors. Transfected COS-7 cells transiently
expressing the various receptors were incubated in 6-well plates for 1
h at 37 °C with the indicated AVP concentrations, and the resulting
increases in intracellular IP
levels were determined as
described (Berridge et al., 1983; Blin et al., 1995).
The data are presented as fold increase in IP
above basal
levels in the absence of AVP. Basal IP
levels for the wild
type V
receptor amounted to 1500 ± 310 cpm/well.
The basal IP
levels observed with the various mutant
receptors were not significantly different from this value. Each curve
is representative of three independent experiments, each carried out in
duplicate.
To explore the structural basis
underlying this selectivity, a series of hybrid V/V
receptors (Fig. 1) were created in which distinct
intracellular domains were systematically exchanged between the two
wild type receptors (note, however, that V2i1 contains V
receptor sequence not only in the first intracellular loop (i1)
but also in the extracellular N-terminal domain and the first
transmembrane segment). Saturation binding studies showed that all
mutant receptors, when transiently expressed in COS-7 cells, retained
the ability to bind the agonist radioligand
[
H]AVP with high affinity (Fig. 1).
Moreover, all hybrid receptors were expressed at levels similar to
those found with the two wild type receptors (B
= 480-690 fmol/mg; Fig. 1).
Whereas the
wild type V receptor bound [
H]AVP
with 2-3-fold higher affinity than the wild type V
receptor (p < 0.05), this affinity pattern was
reversed when the i3 loop was exchanged between the two wild type
receptors (Fig. 1). In agreement with previous studies using
hybrid m2/m3 muscarinic (Wess et al., 1990) and hybrid
D
/D
dopamine receptors (Robinson et
al., 1994), this finding may indicate that the i3 loop can exert
indirect conformational effects on the configuration of the AVP binding
site predicted to be formed by amino acids located on the extracellular
receptor surface (Chini et al., 1995).
Consistent with these
results, substitution of the i1, i2, or i4 domain of the V receptor into the V
receptor resulted in mutant
receptors that, similar to the wild type V
receptor,
lacked the ability to mediate stimulation of adenylyl cyclase ( Table 1and Fig. 2). However, a mutant V
receptor in which the i3 domain was replaced with the homologous
V
receptor sequence (V1i3) gained the ability to stimulate
cAMP production with high efficacy (9.5 ± 1.4-fold increase in
cAMP above basal) and high AVP potency (EC
= 0.88
± 0.12 nM) ( Table 1and Fig. 2).
In agreement with these results,
substitution of the i1, i3, or i4 domain of the V receptor
into the V
receptor yielded mutant receptors that were
unable to efficiently stimulate PI hydrolysis ( Table 1and Fig. 3). Remarkably, however, replacement of the i2 loop in the
V
receptor with the homologous V
receptor
sequence yielded a hybrid receptor (V2i2) that gained the ability to
stimulate inositol phosphate production with high efficacy (7.4
± 0.6-fold increase in IP
above basal) and high AVP
potency (EC
= 1.82 ± 0.21 nM), in a
fashion very similar to that of the wild type V
receptor ( Table 1and Fig. 3).
To exclude the possibility that the ability of
V2i2 and V1i3 to couple to both stimulation of PI hydrolysis and
adenylyl cyclase was due to a complete loss of G protein coupling
selectivity (coupling promiscuity; Wong and Ross(1994)), we examined
the ability of these two mutant receptors to mediate coupling to
G, a G protein (family) recognized by neither of the two
wild type receptors. It is known that wild type or mutant GPCRs that
can couple to both G
and G
can stimulate
adenylyl cyclase activity with markedly increased efficacy after
inactivation of G
by PTX treatment (Liggett et
al., 1991; McClue et al., 1994). This is illustrated in Fig. 4for a mutant m2 muscarinic receptor (m2
i3) in which
the i3 loop was replaced with the corresponding
-adrenergic receptor sequence (a structurally
homologous m2 muscarinic/
-adrenergic mutant receptor
can couple to G
, G
, G
, and
G
; Wong and Ross(1994)). PTX pretreatment (500 ng/ml; 24 h)
of cells expressing this mutant receptor resulted in a marked increase
in cAMP production at all agonist (carbachol) concentrations tested (Fig. 4). In contrast, PTX pretreatment had only little effect
on the magnitude of the AVP-induced cAMP responses mediated by the wild
type V
as well as the V2i2 and V1i3 mutant receptors (Fig. 4).
Figure 4:
Effect of PTX on receptor-mediated
stimulation of adenylyl cyclase. COS-7 cells transiently expressing the
indicated receptors were studied for their ability to mediate
AVP-induced increases in intracellular cAMP levels, either in the
absence or in the presence of PTX (500 ng/ml). Assays were carried out
as described under ``Experimental Procedures.'' The
structures of V2i2 and V1i3 are given in Fig. 1. m2i3
represents a human m2 muscarinic receptor (Bonner et al.,
1987) in which the i3 loop was replaced with the corresponding human
-adrenergic receptor sequence (Chung et al.,
1987). Basal cAMP levels for the wild type V
receptor
amounted to 984 ± 243 cpm/well and remained virtually unaffected
by PTX pretreatment. The basal cAMP levels observed with the different
mutant receptors were not significantly different from this value. The
data are given as means ± S.E. and are representative of three
independent experiments, each carried out in
duplicate.
In another set of experiments, we examined whether
the ability of the V2i2 mutant receptor to productively couple to PI
hydrolysis was specifically dependent on the presence of V receptor sequence in the i2 loop of this hybrid construct or was
rather due to the ``loss'' of V
-i2-loop sequence
that could at least theoretically play a role in preventing access to
G
proteins. To distinguish between these two
possibilities, a mutant V
receptor was constructed in which
the i2 loop was replaced with the corresponding sequence of the ss4
somatostatin receptor (Fig. 5A), which does not couple
to G
but to G
proteins (O'Carroll et al., 1992). Similar to V2i2, the resulting mutant receptor
(V2i2ss; B
= 624 ± 45 fmol/mg)
retained the ability to stimulate adenylyl cyclase with high efficacy (Fig. 6). However, in contrast to V2i2, the V2i2ss mutant
receptor did not gain the ability to efficiently couple to stimulation
of PI hydrolysis (Fig. 6). Moreover, PTX pretreatment (500
ng/ml; 24 h) of V2i2ss-expressing cells had no significant effect on
the magnitude of AVP-induced increases in cAMP levels (data not shown),
indicating that the V2i2ss mutant receptor and the wild type V2
receptor share similar functional properties.
Figure 5:
Comparison of the i2 and i3 loop sequences
of members of the vasopressin/oxytocin (OXY) receptor family. A, comparison of i2 loop sequences. The underlined sequences of the rat V and human V
receptor were replaced with the corresponding V
and
V
sequences, respectively (see also Fig. 1). The boxed sequence of the rat ss4 somatostatin receptor
(O'Carroll et al., 1992) was substituted into the human
V
receptor, yielding hybrid receptor V2i2ss (see Fig. 6). *, positions at which all vasopressin/oxytocin
receptors have identical residues. #, positions at which the
G
-coupled vasopressin/oxytocin receptors (OXY,
V
, and V
) have identical residues that differ
from those present in the V
receptor(s). Gaps were
introduced to allow for maximum sequence identity. Sequences were taken
from: Kimura et al., 1992 (human OXY); Rozen et al.,
1995 (rat OXY); Gorbulev et al., 1993 (pig OXY); Sugimoto et al., 1994 (human V
); Lolait et al.,
1995 (rat V
); Thibonnier et al., 1994 (human
V
); Morel et al., 1992 (rat V
);
Birnbaumer et al., 1992 (human V
); Lolait et
al., 1992 (rat V
); Gorbulev et al., 1993 (pig
V
). B, comparison of i3 loop sequences. For
explanations, see description of A.
Figure 6:
Functional profile of wild type and mutant
V vasopressin receptors. COS-7 cells transiently expressing
the indicated receptors were studied for their ability to mediate AVP
(1 µM)-induced increases in intracellular cAMP and
IP
levels. Functional assays were carried out as described
under ``Experimental Procedures.'' V2i2 and V2i2ss represent
mutant V
receptors in which the i2 loop was replaced with
V
or somatostatin receptor sequence (rat ss4 subtype;
O'Carroll et al.(1992)), respectively (see Fig. 5A for replaced sequences). Basal cAMP and
IP
levels for the wild type V
receptor amounted
to 944 ± 162 and 1865 ± 296 cpm/well, respectively. The
corresponding levels observed with the two mutant receptors were not
significantly different from these values. The data are given as means
± S.E. and are representative of three independent experiments,
each carried out in duplicate.
Figure 7:
Functional interaction of hybrid
V/V
vasopressin receptors with G
.
COS-7 cells were cotransfected with G
(Amatruda et al., 1991) and the indicated wild type or mutant
vasopressin receptors (Control, transfection with receptor DNA
alone). AVP (1 µM)-induced increases in intracellular
IP
levels were determined as described under
``Experimental Procedures.'' The structures of hybrid
receptors V1i2 and V2i3 are shown in Fig. 1. Basal IP
levels for the wild type V
receptor in the absence
or the presence of G
amounted to 1638 ± 214
and 2034 ± 355 cpm/well, respectively. Similar values were found
with the wild type V
and the two mutant receptors. The data
are given as means ± S.E. and are representative of three
independent experiments, each carried out in
duplicate.
The vasopressin receptor family represents an ideal model
system to study the molecular basis of G protein recognition by peptide
receptors, because its individual members (V,
V
, and V
) clearly differ in their G protein
coupling properties. In this study, we have created and functionally
analyzed a series of V
/V
hybrid receptors in
which distinct intracellular domains (i1-i4; Fig. 1) were
systematically exchanged between the two wild type receptors. cAMP
assays showed that all mutant receptors that contained V
receptor sequence in the i3 loop were able to stimulate adenylyl
cyclase activity with high efficacy and AVP potency, whereas all mutant
receptors in which the i3 loop was derived from the V
receptor had little or no effect on intracellular cAMP levels (Fig. 1). These data strongly suggest that the i3 loop of the
V
receptor plays a key role in proper recognition and
activation of G
.
On the other hand, all hybrid
constructs in which the i2 loop consisted of V receptor
sequence were able to activate the PI cascade in a fashion very similar
to the wild type V
receptor, whereas all mutant receptors
that contained V
sequence in this receptor region displayed
only residual PI activity, similar to the wild type V
receptor (Fig. 1), indicating that the i2 loop of the
V
receptor is critically involved in selective activation
of G
.
Consistent with this pattern, substitution of
the i2 loop of the V receptor into the wild type V
receptor resulted in a mutant receptor (V2i2) that gained the
ability to efficiently couple to G
but still retained
the ability to productively couple to G
. Analogously,
replacement of the i3 loop in the V
receptor with the
homologous V2 receptor sequence yielded a hybrid construct (V1i3) that
gained efficient coupling to G
but was still able to
activate G
in a fashion similar to the wild type
V
receptor. The ability of V2i2 and V1i3 to couple to both
G
and G
is fully consistent with the notion
that different single receptor domains determine the differential G
protein coupling profiles of the V
and V
vasopressin receptors.
Interestingly, Wong and Ross(1994)
recently described chimeric m2 muscarinic/-adrenergic
receptors, which are completely nonselective among the known mammalian
G proteins. These mutant receptors could activate G
and
G
as well as G
, which is not a target of
either of the two parent receptors. Moreover, Wong and Ross(1994) found
that a mutant m1 muscarinic receptor containing
-adrenergic receptor sequence in the i3 loop does not
only activate G
and G
but also
G
, which is not a target of either the m1 muscarinic or the
-adrenergic receptor. Like this mutant receptor, the
bifunctional receptors described in the present manuscript (V2i2 and
V1i3) were composed of sequences derived from G
- and
G
-coupled receptors. Therefore, to rule out the possibility
that the ability of V2i2 and V1i3 to couple to both G
and G
was caused by a general loss of G protein
coupling selectivity, we studied whether these two mutant receptors
also gained the ability to couple to G proteins of the G
class, which are activated by neither of the two wild type
receptors. Because receptor-mediated activation of G
counteracts the stimulation of adenylyl cyclase mediated by
activated G
, selective inactivation of G
by PTX
is known to lead to an increase in the magnitude of the cAMP responses
induced by (mutant) receptors that couple to both G
and
G
(Liggett et al., 1991; McClue et al.,
1994). We found that PTX pretreatment had little effect on the
magnitude of the cAMP responses mediated by V2i2, V1i3, or the wild
type V
receptor (Fig. 4). In contrast, PTX
pretreatment of cells expressing a mutant m2 receptor that contained
-adrenergic sequence in the i3 loop (a modification
predicted to lead to a complete loss in G protein coupling selectivity;
Wong and Ross(1994)) resulted in a pronounced increase in
agonist-induced adenylyl cyclase activity. Thus, the functional
properties of the V2i2 and V1i3 mutant receptors clearly differ from
those of the generally ``promiscuous'' hybrid receptors
described by Wong and Ross(1994), indicating that the ability of V2i2
and V1i3 to couple to both G
and G
is not
due to a general loss of G protein coupling selectivity.
It might
also be argued that the i2 loop of the V receptor plays a
specific role in preventing access to G
proteins.
Analogously, the i3 loop of the V
receptor may be involved
in preventing interactions with G
. To exclude such a
mechanism as a possible cause of receptor-G protein coupling
selectivity, an additional hybrid receptor (V2i2ss) was created in
which the i2 loop of the V
receptor was replaced with the
corresponding segment of the G
-coupled ss4 somatostatin
receptor (O'Carroll et al., 1992). The i2 loop of the
ss4 somatostatin receptor shares considerably more sequence homology
with the corresponding region of the V
receptor
(35-40% sequence identity) than with that of the V
receptor (20-25%; Fig. 5A). In contrast to
the mutant V
receptor containing V
receptor
sequence in the i2 loop (V2i2), the V2i2ss hybrid receptor did not gain
the ability to efficiently couple to G
, indicating that
the bifunctionality of V2i2 is not due to the ability of the i2 loop of
the V
receptor to prevent access to G
proteins or to a loss of specific interactions between the i2 and
i3 loop (in the wild type V
receptor) that constrain G
protein coupling selectivity. Taken together, these data suggest that
the i2 loop of the V
receptor is in fact directly involved
in G
recognition and activation.
Consistent with the
proposed roles of the i2 loop of the V receptor and the i3
loop of the V
receptor in selective recognition of
G
and G
, respectively, two mutant receptors,
V2i3 and V1i2, were identified that showed only residual or no
functional activity at all (Fig. 1). However, we could
demonstrate that both mutant receptors retained the ability to
productively couple to G
(upon coexpression with
G
), a G protein known to be activated by most GPCRs
(Offermanns and Simon, 1995). This observation strongly suggests that
the inability of the V2i3 and V1i2 mutant receptors to interact with
G
and G
is not caused by a generalized
misfolding of the intracellular receptor surface.
Taken together, these data provide compelling evidence that different single receptor domains are responsible for the functional diversity found within the vasopressin receptor family. The possibility therefore exists that the G protein coupling selectivity of other classes of peptide receptors is also determined by a clearly delineated intracellular receptor region. In agreement with this view, it has been demonstrated that the G protein coupling properties of a series of splice variants of the pituitary adenylyl cyclase-activating polypeptide receptor critically depend on the sequence present at the C terminus of the i3 loop (Spengler et al., 1993).
It should be of interest to
investigate the functional effects of substituting the i2 loop of the
V receptor or the i3 loop of the V2 receptor into other
GPCRs (nonvasopressin receptors). Such experiments could provide
information as to whether these specific loop sequences are sufficient
for proper recognition and activation of G
and
G
, respectively. Alternatively, this question could be
addressed by randomizing intracellular vasopressin receptor sequences
while leaving the i2 loop of the V
receptor or the i3 loop
of the V
receptor intact.
A sequence comparison (Fig. 5) shows that the i2 and i3 loops of the V and V
vasopressin receptors and the oxytocin
receptor (which is structurally closely related to the vasopressin
receptors and, like the V
and V
receptors, is
selectively coupled to G
; Kimura et al.(1992))
are quite similar to each other but substantially differ from the
corresponding V
receptor sequences. Each of the two loops
contains a number of residues that are conserved only within the two
functional receptor subclasses. It is therefore likely that these amino
acids play key roles in determining the distinct G protein coupling
profiles of the different vasopressin/oxytocin receptors.
In contrast to the findings reported here for different members of a peptide receptor family, multiple intracellular domains are known to be involved in determining the G protein coupling properties of receptors activated by biogenic amine ligands such as the adrenergic or muscarinic acetylcholine receptors (Wong et al., 1990; Liggett et al., 1991; Wong and Ross, 1994; Blin et al., 1995). Such regions have been shown to include the i2 loop, the N- and C-terminal segments of the i3 loop, and the membrane-proximal portion of the C-terminal tail (i4). It could be demonstrated that these regions act in a cooperative fashion to select and activate the proper set of G proteins (Wong et al., 1990; Liggett et al., 1991; Wong and Ross, 1994; Blin et al., 1995).
Interestingly, several peptide receptors (including, for example,
the receptors for calcitonin, glucagon, vasoactive intestinal
polypeptide, or secretin) have recently been identified that, similar
to two of the hybrid receptors examined in this study (V2i2 and V1i3),
can couple to both G and G
(Chabre et
al., 1992; Abou-Samra et al., 1992). This property is
also shared by the receptors that are activated by the glycoprotein
hormones follicle-stimulating hormone, luteinizing hormone, and
thyrotropin (Kosugi et al., 1993a; Allgeier et al.,
1994). Loss-of-function mutagenesis studies showed, for example, that
mutational modification of the N- and C-terminal segments of the i3
loop of the thyrotropin receptor virtually abolished coupling to
G
but had little effect on efficient activation of
G
(Kosugi et al., 1993a, 1993b). In agreement with
the results of the present study, these data suggest that the region(s)
in the thyrotropin receptor critical for activation of G
differ(s) from that (those) required for productive coupling to
G
.
Despite the existence of distinct vasopressin
receptor sequences dictating specificity of G protein recognition, it
is likely that most (if not all) intracellular receptor regions are
generally required for the proper formation of the receptor-G protein
complex. This notion is based on a large number of biochemical and
molecular genetic studies with other GPCRs (Strosberg, 1991; Dohlman et al., 1991; Savarese and Fraser, 1992; Strader et
al., 1994) demonstrating that there are multiple receptor-G
protein contact sites involving at least three domains on the G
subunits (Rens-Domiano and Hamm, 1995). Because all GPCRs and all G
proteins are predicted to share a similar three-dimensional structure,
the molecular architecture of the receptor-G protein interface may be
generally conserved.
In conclusion, this study introduces the novel concept that the differential G protein coupling profiles of individual members of a peptide receptor subfamily are determined by different single intracellular receptor domains. All our experimental data are consistent with the notion that these domains are directly involved in G protein binding and/or activation. The identification of the site(s) on the G protein(s) involved in these interactions should eventually lead to the delineation of three-dimensional models of the receptor-G protein complex and provide novel insights into the molecular basis of peptide receptor-mediated G protein activation.