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Oregon Regional Primate Research Center (A.U.-A., D.S., V.A., J.V., S.B., J.A.J., P.M.C.), Beaverton, Oregon 97006; and the Department of Physiology and Pharmacology, Oregon Health Sciences University (P.M.C.), Portland, Oregon 97201
Address all correspondence and requests for reprints to: Dr. P. Michael Conn, Oregon Regional Primate Research Center, Beaverton, Oregon 97006.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1B-adrenergic
(Gq/11-coupled) receptors resulted in 25–55% inhibition
of maximal GnRH-evoked IP turnover. Paradoxically, loop 3i from the
M1Ach-muscarinic receptor also maximally inhibited GnRH
agonist-stimulated cAMP accumulation and PRL release by 40% (both
effects mediated through activation of the Gs protein).
Transfection of loop 3i from the D1A -dopamine receptor
(coupled to the Gs protein) produced a selective
attenuation (40%) in Gs-mediated cellular responses. In
contrast, receptor/G protein coupling appeared unaffected by expression
of loop 3i domains derived from two receptors coupled to
Gi/o proteins (M2Ach-muscarinic and
2A-adrenergic receptors). These data indicate that the
third intracellular loop of the rat GnRH-R is involved in receptor
Gq/11 protein coupling and/or selectivity, and in the
GGH31' cell line, this loop is also involved in signal
transduction mediated through the Gs protein pathway. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been shown that the intracellular domains of the rhodopsin/ß-adrenergic-like family of GPCRs, particularly the regions closest to the plasma membrane in intracellular loops 2 (2i) and 3 (3i) as well as some specific regions located in the membrane-proximal portion of the COOH-terminus, are important sites for G protein coupling and specificity determination (1, 9, 11, 12, 13, 14, 15, 16). Studies on the mouse and the human GnRH-R indicated that loop 2i is a critical element in determining the Gq/11-mediated transduction mechanism of this receptor (17, 18, 19, 20). However, given the functional similarity among the members of this GPCR subfamily, it is also likely that multiple intracellular domains may be required for optimal signal transduction (1, 3, 12, 13).
In the present study, we analyzed the role of GnRH-R loop 3i in GnRH-R coupling to G proteins. For this purpose we used the rat GnRH-R expressed in GH3 cells, a pituitary-derived lactotrope cell line that does not ordinarily express this moiety (21). These cells (GGH3) express a GnRH-R similar in ligand binding affinity and specificity to the receptor found in the gonadotrope (8). In response to GnRH and its agonists, GGH3 cells produce inositol phosphates (IP) via Gq/11 as well as cAMP and PRL via Gs-mediated signal transduction pathways in a dose-dependent manner (22, 23). To study the role of GnRH-R loop 3i in G protein coupling, GGH31' cells were transiently transfected with a plasmid DNA containing a complementary DNA (cDNA) construct encoding the full 3i of the rat GnRH-R as well as with expression plasmids containing cDNA constructs encoding loop 3i of other receptors that normally couple Gs, Gi/o, or Gq/11 proteins. The effects of coexpression of these loops with the wild-type GnRH-R on IP production, cAMP accumulation, and PRL release were examined.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). This
minigene construct was spliced into the EcoRV site of
pcDNA3.1(+) (Invitrogen, San Diego, CA), and the orientation of the
insert was determined by DNA sequencing using an Applied Biosystems DNA
Sequencer (Branchburg, NJ). The 5'-end of the minigene contained the
ribosomal binding consensus sequence GCCGCCACCATG (25). A methionine
codon was added upstream of the receptor-specific sequence as a
translation initiation site followed by a Gly codon (GGA). At the end
of the sequence, a TAA stop codon was added. The GPCR loop 3i
expression plasmids pRK2A3i-adrenergic receptor
(2A3i) and pRKM23i-muscarinic receptor
(M2Ach3i; both coupled to the Gi/o protein),
pRKM13i-muscarinic receptor (M1Ach3i) and
pRK1B3i-adrenergic receptor (1B3i;
Gq/11 coupled), and pRKD1A3i-dopamine receptor
(D1A3i; Gs coupled) were provided by Dr.
R. J. Lefkowitz (Duke University, Durham, NC). All of these
minigene sequences were confirmed by dideoxynucleotide sequencing of
double-stranded DNA, as previously reported (26, 27). An expression
plasmid containing the ß-galactosidase-coding sequence (ßGal) was
provided by Dr. Tae H. Ji (University of Wyoming, Laramie, WY).
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2A3i, or ßGal minigenes) mixed
with 2 µl Lipofectamine (Life Technologies) in 0.25 ml OPTI-MEM were
added to each well in triplicate or quadruplicate. After a 5-h
incubation, 0.25 ml DMEM containing 20% FCS was added to each well.
Twenty-four hours after the start of transfection, medium was removed,
and the cells were washed twice with DMEM-0.1% BSA (Irvine Scientific,
Santa Ana, CA) and prepared for measurement of GnRH-stimulated IP
production, cAMP accumulation, or PRL release.
In a separate study, cells were grown to confluence in
75-cm2 T-flasks (Costar) and then transiently transfected
with 11 µg DNA/flask from the M1Ach3i-,
1B3i-, or D1A3i-containing expression
plasmids by the Lipofectamine procedure described above. In these
experiments, empty pRK5 vector was added to the control flasks to keep
the total mass of DNA added per flask constant within each experiment.
After transfection, cells were split into 24- or 48-well plates and
assayed 24 h later for GnRH-stimulated IP production, cAMP
accumulation, and PRL release. Transfection efficiency by these
procedures was determined to be 35–45% by galactose histochemical
staining of control cells transfected with the ßGal cDNA (28).
Measurement of IP production
For quantitation of IP production, cells were incubated
initially in 0.5 ml inositol-free DMEM containing 4 µCi/ml
[3H]inositol for 18 h at 37 C. After the preloading
period, cells were washed twice with DMEM (inositol free) containing 5
mM LiCl and incubated for 2 h at 37 C in the absence
or presence of increasing concentrations of the GnRH agonist buserelin
(GnRHa) (29) dissolved in 0.5 ml DMEM (inositol free)-LiCl.
At the end of the incubation period, medium was removed, and 0.1
M formic acid (1 ml) was added to each well. Cells were
then frozen and stored (at -20 C) until the IP assay was performed. IP
accumulation was measured by Dowex anion exchange chromatography and
liquid scintillation spectroscopy, as previously described (30).
Quantitation of PRL release and cAMP accumulation
PRL release and cAMP accumulation were measured after a 24-h
incubation period at 37 C in the absence or presence of increasing
doses of buserelin dissolved in DMEM (0.5 ml) containing 0.1% BSA, 20
µg/ml gentamicin, and 0.2 mM methylisobutylxanthine
(Sigma Chemical Co., St. Louis, MO). PRL release was measured by RIA as
previously described (21), using materials obtained from the Hormone
Distribution Program of the National Pituitary Agency, NIDDK (Bethesda,
MD). cAMP accumulation (intra- and extracellular) was measured in
acetylated samples by RIA as previously described (31). cAMP antiserum
C-1B (prepared in our laboratory) (21) was used at a titer of 1:5000;
this antiserum showed less than 0.1% cross-reactivity with cGMP,
2',3'-cAMP, 5'-cAMP, 3'-cAMP, ADP, GDP, ATP, CTP, and
methylisobutyl- xanthine.
Western blots
GGH31' cells were grown in 75-cm2
T-flasks (Costar) and transfected as described above with 20 µg cDNA
encoding the 2-adrenergic receptor loop 3i or with cDNA
encoding the ßGal gene sequence as a control. Forty-eight hours after
transfection, cells were lysed by freeze-thawing, and the cell lysates
were solubilized with sample buffer. Proteins were resolved by 12%
SDS-PAGE and then transferred to nitrocellulose paper (Hoefer
Scientific Instruments, San Francisco, CA) as described previously
(32). Polyclonal antiserum made against this loop 3i (33) (provided by
Dr. Hitoshi Kurose, University of Tokyo, Tokyo, Japan) was used at a
1:500 titer. Color was developed on Western blots using
4-chloro-1-napthol (horseradish peroxidase) color development reagent
(Bio-Rad Laboratories, Richmond, CA). Standards were color-stained
proteins (Rainbow markers, Amersham, Arlington Heights, IL) with
molecular masses of 200K (myosin), 92.5K (phosphorylase), 69K (BSA),
46K (ovalbumin), 30K (carbonic anhydrase), 21.5K (trypsin inhibitor),
and 14.3K (lysozyme).
Statistical analysis
Differences between the maximally GnRHa-stimulated
responses (IP and cAMP accumulation as well as PRL release) in
GGH31' cells transfected with the different loops 3i and
the cells expressing the cDNA for ß-galactosidase were calculated
employing the Student’s unpaired t test (when n = 2)
or one-way ANOVA followed by t tests (for n > 2).
Maximal responses (Rmax) were calculated from the
dose-response curves following the method of De Lean et al.
(34). P < 0.05 was considered statistically
significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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shows a topographical map of the GnRH-R, indicating the
sequence of the third intracellular loop used to construct the
minigene. In all experiments, exposure of GGH31' cells to
increasing doses of GnRHa resulted in a significant
stimulation of IPs and cAMP as well as in PRL release. Transfection of
GGH31' cells with the cDNA for the 3i of the rat GnRH-R
inhibited maximal GnRHa-stimulated IP turnover by 20%
(Fig. 2). This inhibition increased to
33% when the cells were exposed to higher amounts of loop 3i cDNA (1.8
µg/well; not shown). Likewise, cellular expression of this loop
yielded a 30% inhibition of cAMP accumulation and PRL secretion
stimulated by GnRHa (Figs. 3 and 4). This attenuation in cellular
responses to GnRHa was statistically significant
(P < 0.05) compared with the responses exhibited by
GnRHa-stimulated GGH31' cells transfected with
the plasmid bearing the cDNA of ßGal (Table 1).
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1B-adrenergic receptors
(Gq/11 coupled) efficiently antagonized
GnRHa-stimulated IP production by 55% and 25%,
respectively, compared with that by GGH31' cells
transfected with the empty pRK5 vector (Fig. 5A and Table 1). Paradoxically, loop 3i
from the M1Ach-muscarinic receptor also inhibited
GnRHa-stimulated cAMP accumulation and PRL release by 40%
(Fig. 5, B and C). D1A3i (derived from a
Gs-coupled receptor) transfected in GGH31'
cells exhibited a selective attenuation (
40%) of
Gs-mediated cellular responses (Fig. 5, B and C). In
contrast to the effects elicited by all of these heterologous loops,
the Gq/11- and Gs-mediated cellular responses
to GnRHa stimulation of cAMP accumulation and PRL release
appeared unaffected by expression of the heterologous 3i domains
derived from receptors coupled to Gi/o proteins
(M2Ach-muscarinic and 2A-adrenergic
receptors; Fig. 6).
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2A3i was confirmed by Western blot analysis. As Fig. 7 shows, a single band was seen at the
apparent molecular mass of 21.5K. Due to the lack of availability of
antisera against other loops 3i, we were unable to confirm the
expression of other loops 3i. However, the facts that the same or a
similar expression vector was employed for all studies and that all
vectors were driven by the cytomegalovirus promoter suggest that the
remaining loop 3i cDNAs transfected may have been expressed with a
similar efficiency as 2A3i.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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q/11 protein subclass (38). The requirement for several
intracellular loops for optimal signal transduction is also present in
other GPCRs not belonging to this particular family. For example, it
has recently been shown that loop 2i of the metabotropic glutamate
receptor 1 (coupled to the Gq protein) plays a critical
role in the G protein coupling selectivity of this receptor, although
optimal coupling and activation of the G protein involve cooperation
among all of its intracellular domains (39). In the mouse GnRH-R, substitution of the membrane-proximal residue Ser140 (corresponding to Tyr in several GPCRs exhibiting the highly conserved DRY tripeptide at the NH2-terminus of loop 2i) with Tyr had no effect on coupling to the Gq/11 protein, whereas replacement of Arg139 with Gln significantly impaired both its expression level and the GnRH-stimulated IP response (17, 20). Meanwhile, replacement of the conserved Leu147 [a hydrophobic residue located in the middle of loop 2i and implicated in signal transduction of the Hm1 and Hm3 muscarinic receptors (15)] with either Asp or Ala profoundly impaired receptor Gq/11 protein coupling (17). On the other hand, mutation of Arg145 to Pro in loop 2i of the human counterpart (creating a Pro-Pro motif that disrupts the secondary structure of the loop) resulted in defective coupling to signal transduction, thus suggesting that a specific conformational structure of this loop domain appears to be necessary for efficient G protein coupling (19). More recently, it was shown that coexpression of the wild type and a truncated form of the human GnRH-R lacking the entire loop 3i resulted in a profoundly suppressed signaling capability of the wild-type receptor, presumably due to a direct and specific physical interaction between the intracellular domains of the wild-type receptor and the splice variant (40). Although this series of studies suggested a role for loop 2i in receptor coupling to G proteins, recent studies employing computational simulations and protein database searches with the wild-type and Pro-Pro mutant receptor loop segment sequences have identified some of the conformations that may be preferred by this receptor for G protein coupling (19). This analysis has suggested that an association of loop 2i with other loop domains appears to be required for optimal coupling of this receptor to G proteins. In fact, a more recent study has shown that an Ala residue at position 261 in loop 3i of the human GnRH-R is an important structural determinant for binding of the receptor to and/or activation of the Gq/11 protein (41).
The results of the present study indicate that cellular expression of loop 3i of the rat GnRH-R was able to partially, yet significantly, antagonize receptor coupling to G proteins in GGH31' cells. The finding that, under optimized transfection efficiency for this particular cell line, the free loop 3i was able to inhibit the Gq/11- and Gs-mediated pathways by only 20% and 30%, respectively, may be due to several factors: 1) the normally occurring, time-dependent increase in receptor density (21), thereby permitting the formation of more hormone-receptor complexes able to counteract the effect of the free loop 3i; 2) the concomitant (and perhaps predominant) participation of loop 2i in G protein coupling (17, 20); and 3) the likelihood that only a fraction (35–45%) of the GnRHa-responding cells were expressing loop 3i. The recognition that loop 2i of mouse and human GnRH plays an important role in signal transduction (17, 19, 20) and that cellular expression of loop 3i of the rat GnRH-R disrupts receptor/G protein interaction (present study) suggests cooperativity among several intracellular domains in controlling the efficacy of coupling to G proteins.
The precise mechanism(s) subserving the GnRH-R loop 3i-induced
attenuation of GPCR signaling is unclear. Although the GnRH-R loop 3i
might have interacted directly with the G protein by competing with the
homologous loop of the intact receptor for G protein binding, as
indicated by the greater inhibition of the cellular response when
higher amounts of GnRH-R loop 3i minigene were transfected, the
possibility also exists that the interaction of loop 3i with the
receptor itself disrupted receptor conformation, thus impairing
receptor/G protein coupling. The finding that transfection of
GGH31' cells with the heterologous M2Ach3i and
2A3i did not affect the Gq/11- and
Gs-mediated signal transduction pathways coupled with the
observation of a significant antagonism of receptor signaling elicited
by the D1A3i and 1B3i domains strongly
suggest that the observed inhibition was mediated through a mechanism
involving a G protein-specific disruption of receptor/G protein
coupling. In this setting, the more profound attenuation of receptor
signaling provoked by the heterologous loops (D1A3i and
1B3i) compared with that elicited by the homologous
GnRH-R loop 3i may be due to a higher affinity and/or specificity of
the former loops for the same G protein (Gs or
Gq/11), to the simultaneous participation of the
nonantagonized GnRH-R loop 2i in receptor-mediated activation of G
proteins, or to the fact that the GnRH-R-unlinked 3i may be able to
couple to more than one G protein class. The possibility also exists
that the particular expression vector containing the cDNA sequence of
ßGal may have altered the cellular transcriptional and/or
translational machinery, thus attenuating the production of protein
molecules involved in the cellular response to ligand-induced
activation of G protein-mediated pathways. On the other hand, the
significant antagonism produced by loop 3i derived from a
Gq/11-coupled receptor (i.e. the
M1Ach-R) on both GnRHa-activated signal
transduction pathways (cAMP and IP) suggests that the observed
attenuation of G protein-coupled receptor signaling by this loop was
nonspecific. However, in a previous study (27), cotransfection of
HEK-293 cells with the 2A-adrenergic receptor
(Gi coupled) and the M1Ach3i resulted in a
significant inhibition of maximal agonist-stimulated
Gq/11-mediated IP accumulation. Therefore, the possibility
that the presence of additional residues, at the NH2-
and/or COOH-terminal ends of the loop (42) [or even in other
intracellular domains of its homologous receptor (43)] may be
necessary to confer this particular loop with a greater specificity for
its cognate G protein cannot be completely excluded.
The results presented here extend those of a previous study employing
dispersed rat pituitary cell cultures as an experimental model (44). In
this study, expression of a heterologous loop 3i from the
1B-adrenergic, but not the 2A-adrenergic,
receptor and the M2-muscarinic and dopamine D1A
receptors resulted in a 10–12% inhibition of maximal GnRH-evoked IP
turnover. The data indicate that the third intracellular loop of the
rat GnRH-R is involved in receptor Gq/11 protein coupling
and/or selectivity, and that in the GGH31' cell line, this
loop is also involved in signal transduction mediated through the
Gs protein pathway.
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Footnotes |
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2 Present address: Department of Reproductive Biology, Instituto
Nacional de la Nutrición SZ, Vasco de Quiroga 15, Mexico City
14000 D.F., Mexico.
Received October 29, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-adrenergic receptors based upon
characteristics of primary structure. J Biol Chem 267:8342–83461-adrenergic
receptor determine the selectivity of coupling to phosphatidylinositol
hydrolysis. J Biol Chem 267:1633–16392-adrenergic receptor
subtype-specific antibodies. Mol Pharmacol 43:444–450[Abstract]
q/11. Cell Signal 10:101–105[CrossRef][Medline]
by the gonadotropin-releasing
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