(Received for publication, December 21, 1994; and in revised form, May 15, 1995)
From the
The gonadotropin-releasing hormone (GnRH) receptor belongs to
the superfamily of heptahelical G protein coupled receptors, most of
which have a highly conserved DRYXXV/IXXPL sequence
in the second intracellular (2i) loop that has been implicated in G
protein coupling. The predicted 2i loop of the GnRH receptor contains
serine rather than tyrosine in the DRY sequence but retains the
conserved hydrophobic Leu residue, which is required for G protein
coupling and internalization of muscarinic receptors. The present study
examined the effects of mutating the unique Ser to the
conserved Tyr, and the conserved Leu
to Ala or Asp, on
agonist binding, internalization, and signal transduction. The S140Y
mutant showed a 100% increase in agonist binding affinity, and its
internalization was increased by 60% above that of the wild-type
receptor. The binding characteristics of the Leu
mutants
were indistinguishable from those of the wild-type receptor, but their
internalization was reduced by about 50%. The L147A and L147D mutants
also showed significant impairment of GnRH-stimulated inositol
phosphate production. These findings demonstrate that substitution of
Ser
by Tyr does not affect G protein coupling but
significantly increases receptor affinity and internalization rate. In
contrast, replacement of a conserved aliphatic residue
(Leu
) impairs both G protein coupling and agonist-induced
receptor internalization.
The hypothalamic decapeptide, gonadotropin-releasing hormone
(GnRH), ()acts via its specific receptors in the anterior
pituitary gland to regulate the synthesis and secretion of luteinizing
hormone (LH) and follicle-stimulating hormone, and thus plays a pivotal
role in reproduction(1, 2) . The cDNAs for the GnRH
receptors of several species, including mouse(3, 4) ,
rat(5, 6, 7) , sheep (8, 9) , cow(10) , and
human(11, 12) , have recently been cloned. The deduced
amino acid sequences of the receptor share >85% amino acid identity
among species. The coding region of the GnRH receptor contains seven
putative transmembrane domains (TM I-VII) as well as many of the
conserved residues and sequences typical of other members of the G
protein-coupled receptor (GPCR) superfamily. However, the GnRH receptor
exhibits several unique features that include (a) the presence
of a long and highly basic first intracellular loop; (b) the
replacement of Tyr by Ser in the conserved GPCR ``signature''
motif DRY located at the junction of TM III and the second
intracellular loop; and (c) the absence of a cytoplasmic
COOH-terminal tail. Another interesting feature of the receptor is that
the highly conserved Asp in TM II and Asn in TM VII are reciprocally
exchanged(13) . Upon agonist binding, the GnRH receptor
activates phosphoinositide hydrolysis to diacylglycerol and inositol
1,4,5-trisphosphate, thereby mobilizing Ca
and
activating protein kinase C to initiate a variety of cellular
responses. Receptor activation is followed by extensive internalization
and down-regulation of the receptor via endocytosis and processing (14) . The molecular mechanism(s) of GnRH receptor activation
have yet to be clarified.
Mutagenesis and chimeric studies have
suggested that the intracellular regions of the G protein-coupled
receptors, in particular the second and third intracellular loops and
sometimes the cytoplasmic tail, interact with G proteins and mediate
signal transduction(15, 16, 17) . Most of
these structural and functional studies have been performed on
-adrenergic and muscarinic receptors, and data on these properties
of the peptide hormone receptors are more
limited(16, 17, 18) . In addition, recent
reports have suggested that the domains/residues involved in G protein
activation and receptor internalization processes are multi-site in
nature and that some of these regions may either be similar or
overlapping. In the muscarinic receptor, a conserved leucine residue in
the second intracellular loop has been implicated in both signal
transduction and receptor sequestration(19, 20) . This
hydrophobic amino acid is also conserved (as Leu
) in the
GnRH receptor and is present at the equivalent position in the 2i loop
(see Fig. 1). In studies on the structure-function relationships
of the GnRH receptor, we examined the roles of the conserved
Leu
residue and the unique Ser
residue,
which is Tyr or Trp in most other GPCRs, in agonist-induced signal
transduction and receptor internalization. To this end, these residues
were replaced with other amino acids by site-directed mutagenesis, and
the expressed receptors were analyzed for ligand binding, GnRH-induced
inositol phosphate formation and agonist-induced internalization. The
results show that replacement of Leu
with either Asp or
Ala impairs both G protein coupling and receptor internalization.
However, replacement of Ser
by Tyr did not affect the
signaling response but increased ligand binding and receptor
internalization.
Figure 1:
Amino acid sequence alignment of the
second intracellular loop of several G protein coupled receptors.
Above, the putative structure of the GnRH receptor, with cylinders
representing transmembrane regions I to VII. Below, the amino acid
sequence of the second intracellular (2i) loop of the GnRH receptor is
aligned with selected members of the GPCR family, including the human
muscarinic (m1-R)(57) , human
-adrenergic (58) , human serotonin
1d(59) , odorant F3(60) , mouse thyrotropin-releasing
hormone (TRH)(61) , human rhodopsin (62) , rat
cannabinoid(63) , bovine angiotensin II(64) , rat
luteinizing hormone(65) , and rat thyroid stimulating hormone (TSH) (66) receptors. The numbers above the
sequence indicate the amino acid residues of the GnRH receptor. The
precise boundaries of the 2i loop may extend by one or more residues
(for details see Refs. 16 and 17). The G protein signature motif and
the conserved hydrophobic amino acid are boxed. It should be
noted that the consensus sequence DRY is present in most of the GPCRs,
except in GnRH-R where Tyr is replaced by Ser, and in LH/CG and TSH
receptors, where Tyr is replaced by Trp. In the remainder of the
consensus sequence, DRYXXV/IXXPL, the conserved Leu
or another hydrophobic amino acid flanked by an amino-terminal Pro is
present in all receptors. i and e indicate
intracellular and extracellular loops,
respectively.
For assays of GnRH-induced receptor
internalization, cells were allowed to bind the radioligand for
appropriate times, followed by two rapid washes with ice-cold
phosphate-buffered saline. The extracellular receptor-associated ligand
was then removed by washing once with 1 ml of acid solution (50 mM acetic acid and 150 mM NaCl (pH 2.8)) for 12 min. The
acid wash was collected to determine the surface-bound radioactivity,
and the internalized radioactivity was quantitated after solubilizing
the cells in NaOH/SDS solution. The internalized radioactivity is
expressed as a percent of the total binding at that time interval. For
displacement of I-labeled GnRH agonist binding with
native GnRH and GnRH agonist or antagonist analogs, cells were
incubated with radioligand in the absence or presence of increasing
doses of displacing ligands for 1 h at 37 °C. IC
values for the three ligands were estimated using the FLEXIFIT
program(24) . All time points were performed in triplicate, in
at least three separate experiments. Binding parameters were determined
by the LIGAND program(25) .
Figure 2:
Time and temperature dependence of I-GnRH-Ag binding and internalization by GnRH receptors
expressed in COS-7 cells. COS-7 cells expressing wild-type GnRH
receptors were incubated with
I-GnRH-Ag for the indicated
times at room temperature (24 °C) (panelA) or at
37 °C (panelB). The specific acid-releasable
(surface binding) (
) and acid-resistant (internalized) (
)
radioactivities were measured as described under ``Experimental
Procedures.'' The total binding (
) was calculated as the sum
of these two values. The data shown are representative of three
separate experiments, and each plotted value is the mean of triplicate
assays. In panelC, internalization data are
expressed as percent of total binding for each time point and are means
± S.E. from three independent experiments, each performed in
triplicate.
The signal transduction efficiency of
the wild-type and mutant GnRH receptors was determined by assaying
GnRH-induced inositol phosphate production in receptor-transfected
COS-7 cells. The inositol phosphate responses of cells expressing
wild-type receptors to a maximally effective concentration of GnRH are
shown in Fig. 3. The metabolic products of inositol
1,4,5-trisphosphate (Ins(1,3,4)P, InsP
, and
InsP) were increased severalfold during the 15-min period of
stimulation (see panelA for unstimulated cells and panel B for stimulated cells). Under the experimental
conditions used here, i.e. in the presence of 10 mM
LiCl, InsP
was the major accumulated product and showed a
19 ± 5-fold increase after 15 min of stimulation, and InsP
showed a 7 ± 2-fold increase over the basal level (n = 3). The basal levels of inositol monophosphates were
higher and showed relatively smaller (2.4-fold) increases than
InsP
and InsP
during agonist stimulation.
Figure 3:
GnRH-induced changes in inositol phosphate
levels in COS-7 cells expressing wild-type GnRH receptors. COS-7 cells
transiently expressing the wild-type GnRH receptors were labeled with
[H]inositol for 24 h, then preincubated in the
presence of 10 mM LiCl for 30 min, and subsequently stimulated
with 50 nM GnRH in the presence of 10 mM LiCl for 15
min as described under ``Experimental Procedures.'' The
figure shows representative HPLC elution profiles of
[
H]inositol phosphates extracted from non-treated
cells (panelA) and cells stimulated with GnRH (panelB). Data are from a single experiment
performed in duplicate. Similar results were obtained in two separate
experiments.
The ability of guanine
nucleotides to cause a significant decrease in agonist binding affinity
is a characteristic feature of most of the G protein-coupled receptors.
GnRH agonist binding to wild-type and S140Y mutant receptors was
analyzed in the presence of the GTP analog GTPS. As expected,
agonist binding to the wild-type GnRH receptors was inhibited by
GTP
S in a dose-dependent manner, with half-maximal displacement
occurring in a concentration range of 1-10 nM and
maximum reduction of about 55% at 100-1000 nM (see Fig. 4). Scatchard analysis showed that the reduction in binding
was a consequence of decreased receptor affinity rather than a change
in receptor number. GnRH-Ag binding to S140Y receptors was also reduced
in the presence of GTP
S, and the inhibitory effect was essentially
the same as for the wild-type receptor (Fig. 4). This finding
suggests that the higher binding affinity of this mutant, reflected by
its increased binding of the agonist ligand, is independent of its
interaction with G proteins.
Figure 4:
Effects of GTPS on
I-GnRH-Ag binding to wild-type and S140
Y
mutant GnRH receptors. Binding to membranes prepared from transfected
COS-7 cells was measured in the absence or presence of the varying
concentrations of GTP
S as described under ``Experimental
Procedures.'' Results are expressed as percent of the binding
determined in the absence of GTP
S and are shown as means ±
S.E. of values obtained from three separate experiments, each performed
in duplicate.
, wild-type;
, S140Y
mutant.
The ability of GnRH receptor ligands to
compete for the binding of I-GnRH-Ag to COS-7 cells
expressing the wild-type and mutant receptors was also determined.
Radioligand binding to the wild-type receptor was competitively
displaced in a dose-dependent manner by the unlabeled agonist
(IC
= 1.5 nM) as well as by native GnRH
(IC
= 23 nM) and an antagonist (IC
= 4.0 nM). Essentially similar patterns of
inhibition and IC
values were obtained for the mutant
receptors (not shown).
Figure 5:
Effect of site-directed mutations in the
2i loop on internalization of GnRH receptors. Wild-type or S140Y,
L147A, and L147D mutant GnRH receptors were transiently expressed in
COS-7 cells, and the internalization kinetics of I-
GnRH-Ag by wild-type or mutant GnRH receptors were measured at 37
°C as described under ``Experimental Procedures.'' PanelA shows the time course of internalization of
the radioligand by wild-type and mutant receptors. Values are expressed
as percent of total binding for each time point and are means ±
S.E. from three independent experiments, each performed in triplicate.
In panelB, data on internalization at 60 min (from panelA), expressed as percent of the wild-type, are
shown as bar graphs. In this study, 26% of the radioligand
bound to the wild-type receptors was internalized after 60
min.
Figure 6:
Effect
of site-directed mutations in the 2i loop of GnRH receptor on
GnRH-induced inositol phosphate production. COS-7 cells transiently
expressing wild-type or S140Y, L147A, and L147D mutant GnRH receptors
were labeled with [H]inositol for 24 h, then
preincubated in the presence of 10 mM LiCl for 30 min followed
by 15 min of treatment with 50 nM GnRH in the presence of 10
mM LiCl as described under ``Experimental
Procedures.'' Inositol phosphates were extracted and separated by
HPLC as described under ``Experimental Procedures.'' Shown is
the
H radioactivity of InsP
(panelA) and InsP
(panelB)
fractions in counts/min after stimulation with (+) or
without(-) GnRH of wild-type and mutant GnRH receptors. The data
shown are representative of three separate experiments, and each
plotted value is the mean of duplicate assays. PanelC shows the combined InsP
and InsP
responses
expressed as percentages of the wild-type receptor responses, after
subtracting the respective basal levels present in unstimulated
cells.
The functional roles of the unique Ser and the
conserved Leu
residues in the second intracellular loop
of the GnRH receptor were analyzed in mutant receptors in terms of
their involvement in agonist-induced receptor internalization and
signal transduction. Our findings indicate that both residues influence
internalization of the GnRH receptor, but do so in different ways.
Thus, the S140Y mutation increased ligand-induced internalization of
the receptor by 60%, while substitution of Leu by Ala or Asp reduced
receptor internalization by almost 50% (Fig. 5). Signal
generation efficiency, measured by the stimulation of inositol
phosphate production by GnRH, was also impaired in the Leu
mutant receptors but was unaffected by mutation of the
Ser
residue. These results demonstrate that the
Leu
residue is required for efficient signal transduction
by the GnRH receptor, but the presence or absence of the conserved Tyr
residue in the DRY sequence has little effect on this process. The
expression and affinity of the Leu
mutant receptors for
GnRH or its agonist were largely unchanged from those of the wild-type
receptor, indicating that these substitutions did not alter the
integrity of the receptor and that impairment of signal generation was
not caused by reduction of binding affinity. Thus, it appears that
Leu
is critical for G protein coupling of the GnRH
receptor and subsequent phospholipase C activation. However, the
Ser
to Tyr mutation reduced the K
by
2-fold, a change that was reflected by the increase in its radioligand
binding (Table 1). The ability of a single residue mutation in
intracellular loops remote from the binding pockets to increase ligand
binding affinity has also been observed in studies on the
-adrenergic receptor(28) .
In the present
study, the effects of Leu mutations on agonist-induced
internalization and G protein coupling of a peptide hormone receptor
were found to resemble those reported for the muscarinic acetylcholine
receptor(19, 20) . A comparison of the amino acid
sequence in the second intracellular loop of members of the GPCR
superfamily indicates that all receptors have a conserved Leu or
another hydrophobic residue in the DRYXXV/IXXPL sequence (29) (see also Fig. 1). Given the highly
conserved nature of the hydrophobic residue at this position, even
among distantly related members of the G protein-coupled receptor
superfamily, it is not unexpected that this region plays an important
role in universal receptor functions such as G protein activation and
ligand-induced internalization. The effects of this mutation could
result from either the introduction of a conformational change that
reduces its internalization/sequestration and G protein interaction, or
the prevention of an agonist-induced conformational change due to loss
of contact sites required for these responses to activation of the
receptor.
Although significant reductions in signal generation
efficiency were observed for the Leu mutants, the fact
that inositol phosphate production was not completely abolished
indicates that other residues or regions of the intracellular loops are
involved in G protein activation. This was also the case for
ligand-induced receptor internalization, which was significantly
reduced but not abolished. It has become clear from studies employing
mutagenesis(15, 17) , receptor-domain based synthetic
peptides(30, 31) , and receptor
antibodies(32) , that multiple regions within the transmembrane
domains and intracellular loops participate in the activation of G
proteins. Previous mutagenesis studies on
-adrenergic,
muscarinic, and rhodopsin receptors have shown that the NH
-
and COOH-terminal regions of the 3i loop, and often the amino-terminal
region of the cytoplasmic tail, are important for coupling to G
proteins(33, 34, 35) . In the TSH receptor,
the 1i loop and the carboxyl-terminal regions of the 2i and 3i loops
are involved in signal transduction(36) , suggesting that the
presence and location of regions involved in G protein coupling may
vary among individual receptors. Such regions in the primary structure
of GPCRs are usually non-contiguous and thus are multi-site. In the
absence of a cytoplasmic tail, specific regions of the intracellular
loops of the GnRH receptor must be responsible for its interaction with
G proteins. Further mutagenesis studies combined with the application
of physical methods should aid in determining the regions/residues that
are directly involved, and those which act indirectly by stabilizing a
specific receptor conformation. The present results using mutagenesis
clearly demonstrate the importance of the conserved Leu in signal
transduction by the GnRH receptor.
In previous studies, the highly
conserved ``DRY'' sequence in the amino-terminal region of
the 2i loop has been shown to be involved in signal transduction.
Single residue mutations of the highly conserved Asp in this sequence
of - and
-adrenergic, and muscarinic m
receptors, caused reduction or loss of G protein coupling with
retention of high-affinity ligand binding (37, 38, 39, 40) . The corresponding
Glu residue in rhodopsin has likewise been implicated in the
interaction with its G protein, transducin
(G
)(41) . In the angiotensin II receptor,
replacement of the DRY sequence by GGA indicated that these residues
are important for G protein coupling(42) . However, mutation of
Ser
to Tyr (converting the unique DRS sequence in the
GnRH receptor to the conserved DRY sequence) indicates that this
residue is not critical for efficient signal transduction. These
results are consistent with a preliminary report (43) that
replacement of Ser by Ala at position 140 of the GnRH receptor did not
affect inositol phosphate production. Our studies also demonstrated
that receptors bearing a point mutation of Ser
to Tyr
were more rapidly internalized than the native receptors, in the
absence of a change in second messenger production. Interestingly, the
internalization kinetics of the Ser
to Ala mutant
receptor were similar to those of the wild-type receptor, with
sequestration of approximately 27% of the bound ligand over a period of
1 h (data not shown). These findings suggest that the polar group of
the Ser residue is not an important factor in controlling GnRH receptor
internalization. They also demonstrate the ability of an aromatic amino
acid such as Tyr, located within a motif that is highly conserved even
among distantly related GPCRs, to promote receptor internalization.
However, the possibility that the unique Ser
of the GnRH
receptor may be involved in receptor regulation or desensitization
awaits future studies.
The impaired ability of the L147D and L147A mutants to mediate inositol phosphate production and to undergo agonist-induced internalization might appear to support the idea that internalization of the hormone-receptor complex is causally related to receptor activation and signal transduction. However, recent studies on angiotensin II (44) and insulin-like growth factor-I (45) receptors, involving mutational studies in the 3i loop and cytoplasmic region, respectively, have demonstrated that these two events can be dissociated.
Agonist-induced internalization of
transferrin and low density lipoprotein receptors is believed to
involve endocytosis via clathrin-coated pits, but the molecular
determinants and mechanism of GPCR internalization have not been
defined. It is likely that the situation for each receptor is
different. For example, regions in the COOH-terminal tail with multiple
Ser and Thr residues that are potential phosphorylation sites are
critical for the internalization of -adrenergic (46) and gastrin-releasing peptide (47) receptors as
well as the angiotensin II receptor(48) . However, although
deletion of the cytoplasmic tail decreased the internalization rate of
the TSH receptor (49) it increased that of the LH/CG receptor (50) . Interestingly, although the GnRH receptor lacks a
cytoplasmic tail it still internalizes to an appreciable extent, albeit
more slowly than some of the other GPCRs. Thus, for angiotensin II and
GRP receptors, 50-60% (44) and 80% (47) of the
bound ligand was internalized by 1 h, compared to 26% of the
radioligand bound to the GnRH receptor at this time ( Fig. 2and Fig. 5). Furthermore, the GnRH receptor does not possess
multiple Ser and Thr residues in any of its intracellular loops,
suggesting that other regional signal motifs are involved in this
process. In this regard it is noteworthy that sequences resembling the
conserved NPXXY motif that is present in the seventh
transmembrane domains of most GPCRs tend to form tight
turns and
have been implicated as recognition signals for high efficiency
ligand-induced receptor internalization(51) . For example, Tyr
in the NPXXY motif was shown to be important for
internalization of the
-adrenergic
receptor(52) . However, this sequence was not found to be
involved in the internalization of the receptors for gastrin-releasing
peptide (53) or angiotensin II(54) . The present data
suggest that Leu
is required for normal internalization
of the GnRH receptor. However, it appears that additional receptor
subdomains are required to regulate the endocytosis mechanism. The GnRH
receptor deviates from the NPXXY sequence in that it has a
DPXXY sequence in the equivalent position. The role of this
sequence in receptor internalization remains to be evaluated.
Our
observations on the effects of the Ser to Tyr mutation on
receptor internalization are relevant to the reported importance of
aromatic amino acids in maintaining efficient endocytosis. McGraw et al.(55) showed that substitution of a Tyr for a
Ser within the cytoplasmic domain of the transferrin receptor can
reconstitute internalization activity in an internalization-defective
mutant. Also, it was shown that a single amino acid change in the
cytoplasmic domain of the influenza virus hemagglutinin, substituting
Tyr for Cys at position 543, caused its more rapid internalization than
the wild-type hemagglutinin, while replacement with Ser or Phe had no
such effect(56) . It is possible that Tyr may be a part of a
recognition feature comprised of two to four residues, as is the case
for the motifs recognized by the oligosaccharyl transferase responsible
for glycosylation of asparagines, or by individual protein kinases
including cAMP-dependent protein kinase, Ser/Thr kinase, and Tyr
kinase. In addition to such motifs, there may be
topological/conformational requirements conferred by interactions with
cytoplasmic loops and/or other proteins.
In conclusion, we have
analyzed the functional significance of two residues, a unique
Ser and a conserved Leu
, in the second
intracellular loop of the GnRH receptor. Leu
appears to
play an important role in receptor-G protein coupling and in
agonist-induced internalization of the receptor. In contrast,
Ser
is not required for G protein coupling and
stimulation of inositol phosphate production, but influences the
binding affinity and internalization of the receptor.