Salt bridge integrates GPCR activation with protein trafficking.

Abstract

G protein-coupled receptors (GPCRs) play central roles in almost all physiological functions; mutations in GPCRs are responsible for more than 30 disorders. There is a great deal of information about GPCR structure but little information that directly relates structure to protein trafficking or to activation. The gonadotropin releasing hormone receptor, because of its small size among GPCRs, is amenable to preparation of mutants and was used in this study to establish the relation among a salt bridge, protein trafficking, and receptor activation. This bridge, between residues E(90) [located in transmembrane segment (TM) 2] and K(121) (TM3), is associated with correct trafficking to the plasma membrane. Agonists, but not antagonists, interact with residue K(121), and destabilize the TM2-TM3 association of the receptor in the plasma membrane. The hGnRHR mutant E(90)K has a broken salt bridge, which also destabilizes the TM2-TM3 association and is typically retained in the endoplasmic reticulum. We show that this mutant, if rescued to the plasma membrane by either of two different means, has constitutive activity and shows modified ligand specificity, revealing a role for the salt bridge in receptor activation, ligand specificity, trafficking, and structure. The data indicate that destabilizing the TM2-TM3 relation for receptor activation, while requiring an intact salt bridge for correct trafficking, provides a mechanism that protects the cell from plasma membrane expression of constitutive activity.

Full text

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Salt bridge integrates GPCR activation with
protein trafficking
Jo Ann Janovickaand P. Michael Conna,b,1
aDivisions of Reproductive Sciences and Neuroscience, Oregon National Primate Research Center, andbDepartments of Physiology and Pharmacology, Cell
and Developmental Biology and Obstetrics and Gynecology, Oregon Health and Science University, Beaverton, OR 97006
Edited* by Susan E. Leeman, Boston University School of Medicine, Boston, MA, and approved January 26, 2010 (received for review December 9, 2009)
G protein–coupled receptors (GPCRs) play central roles in almost all
physiological functions; mutations in GPCRs are responsible for
more than 30 disorders. There is a great deal of information about
GPCR structure but little information that directly relates structure
to protein trafficking or to activation. The gonadotropin releasing
hormone receptor, because of its small size among GPCRs, is ame-
nable to preparation of mutants and was used in this study to
establish the relation among a salt bridge, protein trafficking, and
receptor activation. This bridge, between residues E90[located in
transmembrane segment (TM) 2] and K121(TM3), is associated with
correct trafficking to the plasma membrane. Agonists, but not
antagonists, interact with residue K121, and destabilize the TM2–
TM3 association of the receptor in the plasma membrane. The
hGnRHR mutant E90K has a broken salt bridge, which also destabil-
izes the TM2–TM3 association and is typically retained in the endo-
plasmic reticulum. We show that this mutant, if rescued to the
plasma membrane by either of two different means, has constitu-
tive activity and shows modified ligand specificity, revealing a role
for the salt bridge in receptor activation, ligand specificity, traffick-
ing, and structure. The data indicate that destabilizing the TM2–
TM3 relation for receptor activation, while requiring an intact salt
bridge for correct trafficking, provides a mechanism that protects
the cell from plasma membrane expression of constitutive activity.
constitutive activity|hormone action|receptor|G-protein coupled
receptor|receptor trafficking
H
stitutive activity (CA) (1). This observation is surprising, because
many G protein coupled receptors (GPCRs) have mutants (2) or
WT receptors (3) with CA. A highly conserved (4) structural fea-
ture of the GnRHR is a salt bridge between transmembrane seg-
ment (TM) 2 and TM3 (residues E90and K121in the human
sequence) (5, 6). Gonadotropin releasing hormone (GnRH) ago-
nists, but not peptide antagonists, bind receptor residues D98and
K121(5,6)andaltertherelationbetweenTM2andTM3;thissame
relation is important for trafficking of the human GnRHR
(hGnRHR) to the plasma membrane (4, 7). We considered that
perturbingthisrelationisalsoadeterminantofreceptoractivation.
Another way to perturb this relation, other than ligand binding,
istoalterthechargeofoneofitsconstituents; mutantE90Kwould
result in charge repulsion between K90and K121. This mutant,
which occurs in some cases of human hypogonadotropic hypo-
gonadism, is typically recognized by the quality control system
(QCS) as a misfolded protein (8), retained in the endoplasmic
reticulum (ER) (9) and does not traffic to the plasma membrane.
E90K can be rescued by pharmacoperones, drugs that diffuse
into cells and provide a folding template. This template enables
(otherwise) misfolded mutants to fold or refold correctly (10),
pass a quality control system (constituted of chemically hetero-
geneous chaperone proteins of the ER that either promote
correct folding or retain misfolded proteins) (11), traffic to the
plasma membrane, bind agonist, and produce a signal. Pharma-
coperones of the hGnRHR rescue mutant E90K by creating a sur-
rogate bridge between D98and K121that also stabilizes the relation
undreds of gonadotropin releasing hormone receptor
(GnRHR) mutants have been reported but none have con-
betweenTM2andTM3(4).Wetookadvantageofpharmacoperone
rescue of mutants and a genetic modification that enhances traf-
fickingtotheplasmamembranetoexaminetherelationamongthis
bridge, receptor trafficking, and activation.
Results
Mutations from patients with hypogonadotropic hypogonadism
are widely distributed over the 328 residues of the hGnRHR
structure. Most are misfolded and misrouted molecules (9) that
are rescuable with pharmacoperones, such as In3 (12–16), and
rerouted to the plasma membrane (10). In Fig. 1 Upper, the
dashed lines show the range of basal activity (no agonist) of cells
transfected with each of the mutants, but which have not been
rescued.These dashedlinesshowthatactivity doesnotexceed the
vector-only control. When rescued by pharmacoperone In3, only
a single mutant, E90K, shows constitutive activity, producing
inositol phosphate (IP) in the absence of agonist. Fig. 1 Lower
shows pharmacoperone-rescued mutants subsequently incubated
with the stable GnRHR agonist, 10−7M Buserelin. This obser-
vation confirms that rescue occurs for most mutants and that they
are able to couple to IP, but most exhibit no constitutive activity.
S168R and S217R cannot be rescued for thermodynamic reasons
(17), and A129D is minimally rescuable for unknown reasons.
Fig. 2 Upper (radioligand binding) and Lower (IP production)
show cells expressing WT hGnRHR, E90K, or sequences from
which K191was deleted, then either rescued by pharmacoperone
In3 or unrescued. Like pharmacoperone rescue, deletion of res-
idue K191is known to rescue misrouted GnRHR mutants,
includingE90K(8).Likepharmacoperonerescue,deletionofK191
also reveals constitutive activation by E90K. When E90K-desK191
was subjected to pharmacoperone rescue, the constitutive activity
(IP) was increased more dramatically than for E90K-desK191
alone, because routing to the plasma membrane is increased by
both deletion of the K191and pharmacoperone. Radioligand
binding confirms that pharmacoperones and deletion of residue
K191increase the number of mutant receptors at the plasma
membrane (Fig. 2 Upper). Because In3 was identified in a screen
relying on rat GnRH (which does not contain K191), it is difficult
to wash out of cells expressing hWT-desK191; accordingly, the
specific binding after In3 rescue may not quantitatively reflect the
number of receptors at the PM. The increased fold stimulation
(and responsiveness) of E90K-desK191to agonist, compared with
constitutive activity shown by the mutant, suggests that the K90-
K121repulsion precludes attaining the optimal activation struc-
ture, but that this is corrected by the presence of the agonist
(Fig. 2 Lower).
Because of the close relation of the agonist binding site and the
salt bridge, we compared the ligand specificity of the CA mutant
to that of the WT receptor. Fig. 3 shows the effects of GnRH
Author contributions: J.A.J. and P.M.C. designed research; performed research; analyzed
data; and wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
1To whom correspondence should be addressed. E-mail: connm@ohsu.edu.
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peptide analogs (groups A, B, and D) and selected irrelevant
compounds (group C) on signaling by the WT hGnRHR (Fig. 3
Upper)andbytheconstitutivelyactivemutant,E90K-desK191(Fig.
3 Lower). High affinity antagonists of the WT (Fig. 3 Upper, group
D) inhibit responsiveness to the agonist, Buserelin; they are also
inverse agonists of the mutant (that is, they inhibit constitutive
activity).Irrelevantpeptidesthathavenoagonisticorantagonistic
activity on the WT also have no activity on the rescued mutant
(Fig. 3 Lower, group C). Replacement of the His2in the natural
ligand by Gly2, Asp2, or Glu2results in little or no agonism with
the WT receptor (i.e., in medium without Buserelin) (Fig. 3
Upper, group A; ref. 18) but strong agonism with the mutant (Fig.
3 Lower, group A). Deletion of His2or its replacement by D-
amino acid residues, along with D-amino acids at position 6,
actions that stabilize the peptide against degradation, produce
weak antagonists or no activity on the WT, but weak agonists of
the mutant (group B). This observation suggests that the mutant
possesses an altered ligand binding site compared with WT, has
reduced specificity required for receptor activation by ligands and
that both agonistic and antagonistic peptides, which bind near the
activesiteofthemutant,canproducetheconfigurationassociated
with activated receptor.
To determine whether the same chemical interactions of phar-
macoperones with hE90K were involved in both mutant rescue and
development of CA, we assessed (i) whether these agents had a
similar efficacy order for the development of CA and for mutant
rescue and (ii) whether pharmacoperones from different chemical
classes acted as inverse agonists (inhibiting constitutive activity). In
theprotocolsdescribedthusfar,oncethemutantisrescued,In3was
removed so that the unoccupied receptor could be assessed for
constitutive activity. Fig. 4 Top shows, using this approach, that the
fivedifferentpharmacoperonestested(fromfourdifferentchemical
classes)rescuemutantE90Kandleadtosomedegreeofconstitutive
activity, although this is quite modest for A177775. The concen-
trations used were selected as the optimum for rescue from prior
studies(4,12).InFig.4Middle,allcellswererescuedbyIn3,thenIn3
was washed out and each of the five pharmacoperones was added
to assess constitutive activation; this observation shows that the
pharmacoperonesalsobehaveasinverseagonists,agentsthatinhibit
constitutive activity (compare with dashed line, showing IP pro-
duction by mutant E90K that was not rescued).
Becausethepharmacoperonesusedinthisstudyallformabridge
between receptor residues D98and K121(4, 11, 19), other inter-
actions notwithstanding, they stabilize the TM2–TM3 relation, an
event that allows correct trafficking but precludes constitutive
activity, because of occupancy. When the pharmacoperones are
washed out and the metabolically stable GnRH agonist, Buserelin,
is present after rescue of E90K by each of the pharmacoperones,
effectorcouplingisobserved.ItisinterestingtonotethatIn3ismore
effective than TAK-013 in producing constitutive activity (Fig. 4
Top), while it is less effective in rescuing Buserelin-stimulated
activity(Fig.4Bottom).Thisobservationsuggeststhatthebindingor
steric interactions required for production of constitutive activity
and for mutant rescue may be subtly different between different
classes of pharmacoperones.
Discussion
The concept of constitutive activity for GPCRs was introduced
in 1989 (20), and it has been theorized that virtually all
members of this class have mutants that couple to effectors in
the absence of agonist (2). Inhibitors of constitutive activity,
inverse agonists, are drug candidates for lowering unstimulated
receptor activity. It was curious, in light of the clinical impor-
tance of the GnRHR and the many hundreds of mutants
reported for this GPCR, that no mutant with CA was identified
among either naturally occurring or designed mutants. The
present work suggests that this constitutive activity has likely
been missed because the cell recognizes a constitutively active
Fig. 1.
occurringmutationsofthehGnRHRafterpharmacoperonerescue.COS-7cells
were transfectedwith 100ngof WTormutantcDNA as described inMaterials
and Methods. Mutants were incubated in media alone (shown as two dashed
lines showing upper and lower level of IP production) or rescued with phar-
macoperone (In3) as described in Materials and Methods; In3 was then
washed out and IP production was measured in response to media alone (no
agonist added) (Upper) or rescued with pharmacoperone (In3), then In3 was
washed out and agonist added. In all figures, SEMs are shown for least three
independent experiments performed in replicates of 4–6 (Lower).
Assessment of constitutive activity in IP production of 14 naturally
Fig. 2.
were transfected with 25 ng of WT or mutant (each with or without K191)
cDNA and rescued with or without pharmacoperone (In3), as described in
Materials and Methods for binding studies. The In3 was washed out and
specific binding was determined by using 2 × 106cpm/mL [125I-Buserelin] for
90 min at room temperature (Upper). The tracer was removed, cells were
washed twice, and radioactivity was measured. *, P < 0.05 for the compar-
ison of each mutant that is rescued or not. (Lower) Cells were transfected
with 100 ng of WT or mutant (hE90K or hE90K-desK191) cDNA and rescued
with or without pharmacoperone (In3), as described in Materials and
Methods, and total IP production was measured in response to medium
alone or the addition of agonist (10−7M Buserelin). The values for hE90K and
hE90K-desK191are 156 ± 19 and 440 ± 50 (DMSO) and 292 ± 33 and 2,246 ±
300 (In3), respectively.
Effect on constitutive activity of deleting Lys191on E90K mutant. Cells
Janovick and ConnPNAS
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PHARMACOLOGY

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mutant as misfolded and prevents it from being expressed at
the plasma membrane.
We performed these studies having noted that hGnRHR
residue D98forms both an interhelical interaction with K121and
binds His2in the naturally occurring agonist (5). It is likely that
agonist binding interferes with the TM2 and TM3 interaction,
both by breaking the interhelical interaction and because of the
physical interpositioning of the peptide. In addition, noncon-
servative substitutions at K121interfere with GnRH agonist but
not antagonist binding (6), suggesting that antagonists, although
competing for the binding site, are not candidates for altering the
TM2–TM3 relation. For these reasons, we considered that pos-
itively charged residues at K90(mutant E90K) and K121would
activate the receptor by a mechanism similar to that which occurs
during agonist binding and leading to receptor activation. We
knew that this mutant was recognized as incorrectly folded by the
QCS and retained in the ER, so we examined two methods to
rescue it.
Pharmacoperones of the hGnRHR rescue misfolded mutant
E90K(whichisunabletocreatethesaltbridgebetweenresiduesE90
and K121) by creating a surrogate bridge between D98and K121(4)
that stabilizes the relation between TM2 and TM3. After rescue,
the mutants were stabilized by hydrophobic interactions with the
seven TMs, and pharmacoperones were removed to allow agonist
occupancy without competition.
Mutant E90K was rescued in cells expressing it with pharma-
coperone, and both constitutive activity and radioligand binding
were assessed after removal of the drug. In other studies, mutant
E90K was rescued by deleting amino acid K191, which promotes
the formation of a C14–C200bridge, a modification that decreases
the requirement for the E90–K121salt bridge (8, 17). It was not
possible to quantitate receptors with immunological approaches
because there is no known antibody for the hGnRHR that can be
used for this purpose and tagging sequences, such as GFP and
HA-antigen, alter the trafficking of this receptor (21).
All pharmacoperones examined were inverse agonists, block-
ing constitutive activity. Pharmacoperone structures associated
with the highest amount of rescue were not those associated with
production of the highest amount of constitutive activity. This
observation suggests that the receptor structure associated with
proper routing to the plasma membrane is distinct from the
structure associated with receptor activation.
The present studies show that the cell protects itself against
constitutive activity by requiring a bridge between TM2 and TM3
for correct routing to the plasma membrane. Because WT re-
ceptors are frequently only partially expressed at the plasma
membrane (11, 19), these findings may explain why inverse
agonists (in this case, pharmacoperones), which stabilize recep-
tors in the configuration that is acceptable to the QCS, have been
Fig. 4.
pharmacoperones on rescue. All cells were transfected with 100 ng of cDNA
of hE90K mutant, and rescue was attempted with each indicated pharma-
coperone. The cells were then incubated with media alone to assess con-
stitutive activity (Top). a, P < 0.05 compared with DMSO only; b, P < 0.05
compared with Q103; c, P < 0.05 compared with In3. (Middle) Rescued with
In3 and then incubated with various pharmacoperones to assess their actions
as inverse agonists. (Bottom) Stimulated with 10−7M of the GnRH agonist,
Buserelin to assess the ability to couple. All values are significant compared
with DMSO only.
Comparison of constitutive activity of hE90K with four classes of
Fig. 3.
hE90K-desK191mutant IP production. (Upper) Cells were transfected with 100
ng of human WT GnRHR cDNA and left unrescued. Various GnRH peptides or
irrelevant compounds were added in medium alone (1 μg/mL) or with 10−9M
Buserelin to show agonistic or antagonistic activity on total IP production.
For comparison, a maximal concentration (10−9M) of the GnRH agonist,
Buserelin, produces a response of 6,517 ± 93 cpm. (Lower) Cells were
transfected with 100 ng of hE90K-desK191cDNA, then rescued with phar-
macoperone (In3). The In3 was then washed out. After 18 h of preloading
the cells with
analogs or irrelevant compounds to assess agonistic or antagonistic activity
on total IP production. Group A (Asp2, Glu2, Gly2); Group B (DPhe2DAla6,
DGlu1DPhe2DTrp3DLys6, DesHis2-GnRH, DLeu2DLys6, DPhe2DPhe6); Group C
(sCalcitonin, Vapreotide, hGalanin, GHRP6, TRH); Group D (In3, Acyline,
Azaline B, FE486, Nal-Arg, Nal-Glu, Antide). For comparison, a maximal
concentration (10−7M) of the GnRH agonist, Buserelin, produces a response
of 11,144 ± 1,098 cpm.
Assessment of GnRH peptides and irrelevant compounds on WT and
3H-inositol, the cells were incubated with 1 μg/mL GnRH
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shown to produce greater receptor up-regulation in many sys-
tems than do neutral antagonists (22). This effect may occur by
increasing the percentage of protein that passes the QCS. Ago-
nist activation of the receptor is also associated with disruption
of this bridge, making it attractive to consider that the cell may
also use this structure as a signal for recognition of agonist
occupancy before agonist-activated receptor down-regulation.
Materials and Methods
pcDNA3.1 (Invitrogen), GnRH analog, D-tert-butyl-Ser6-des-Gly10-Pro9-ethyl-
amide-GnRH (Buserelin, Hoechst-Roussel Pharmaceuticals), myo-[2-3H(N)]-
inositol (PerkinElmer; NET-114A), DMEM, OPTI-MEM, lipofectamine, PBS
(GIBCO, Invitrogen), competent cells (Promega), and Endofree maxi-prep kits
(Qiagen), were obtained as indicated. Mutant receptors: WT and mutant
GnRHR cDNAs for transfection were prepared as reported (13); the purity
and identity of plasmid DNAs were verified by dye terminator cycle
sequencing (Applied Biosystems). GnRH analogs were obtained as indicated:
DPhe2-DAla6-GnRH; DLeu2-DAla6GnRH; Des-His2-GnRH; DPhe2, DPhe6-GnRH
(Wyeth-Ayerst Laboratories); “Nal-Arg,” [Ac-DNal1-DCpa2-DPal3, Arg5-D-
Arg6-D-Ala10]- GnRH; “Nal-Glu,” [Ac-DNal1, DCpa2, D3Pal3, Arg5, DGlu6,
DAla10]-GnRH; acyline, [Ac-d-2Nal1, D4Cpa1,2-D3Pal3, Ser4Aph(Ac), d-4Aph
(Ac)6-Leu7-ILys8-Pro9- D-Ala10-NH2]-GnRH; azaline B, [Ac-DNal1-DCpa2-DPal3-
Aph5(atz)-DAph6(atz)-ILys8-DAla10]-GnRH; Asp2-GnRH, Glu2-GnRH, Gly2-
GnRH (Jean Rivier, Salk Institute, La Jolla, CA); “FE486” [Ac-D2Nal1-D4Cpa2-
D3Pal3-Ser4- 4Aph(l-hydroorotyl)3-D4Aph
DAla10]-GnRH (Ferring Research Institute); Antide, [Ac-(DNal) -(DpClPhe)-
(DPal)-Ser-Lys(nicotinoyl)-[D-Lys(nicotinoyl)]-Leu-Lys(isopropyl)-Pro-[DAla]-
NH2 (Serono Laboratories) and DpGlu1-DPhe2-DTrp3-DLys6-GnRH (John
Stewart, University of Colorado, Denver). Irrelevant molecules were obtained
as follows: GHRP6 (His1-DTrp2-Ala3-Trp4-DPhe5-Lys6-NH2), human galanin,
salmon calcitonin, and TSH-releasing hormone (Phoenix Pharmaceuticals);
vapreotide was obtained from Debiopharm. Pharmacoperones In3, Q89,
Q103 (Merck), A177775 (Abbott Laboratories), and TAK-013 (Takeda Phar-
maceuticals) were obtained as indicated (4, 11). The drugs are, respectively,
from the following chemical classes: indoles, quinolones (both Q89 and
Q103), erythromycin macrolides and (N–1-(2,6-difluorobenzyl)-2,4-dioxo-3-
phenyl-1,2,3,4-tetrahydrothieno[2,3-d]pyrimidin-6-yl]phenyl}-N’-methox-
yurea). Chemical structures (11, 19) and the mechanism of action on the
hGnRHR (4) have been reported.
(carbamoyl)6-Leu7-ILys8-Pro9-
Transient Transfection. Cos-7 cells were cultured in growth medium (DMEM,
10% FCS, 20 μg/mL gentamicin) at 37 °C in a 5% CO2humidified atmosphere.
For transfection of WT or mutant receptors into cells, 5 × 104cells were
plated in 0.25 mL growth medium in 48-well Costar cell culture plates.
Twenty-four hours after plating, the cells were washed with 0.5 mL of OPTI-
MEM then transfected with WT or mutant receptor DNA with pcDNA3.1
(empty vector) to keep the total DNA constant (100 ng per well). Lipofect-
amine was used according to the manufacturer’s instructions. Five hours
after transfection, 0.125 mL DMEM with 20% FCS and 20 μg/mL gentamicin
was added. Twenty-three hours after transfection, the medium was replaced
with 0.25 mL of fresh growth medium. Where indicated, pharmacoperones
(indicated concentration) in 1% DMSO (“vehicle”) were added for 4 h in
respective media to the cells and then removed 18 h before agonist treat-
ment (16). In this study, we used Trypan blue exclusion to show cell viability
after drug exposure.
IP Assays. Twenty-seven hours after transfection, cells were washed twice
with 0.50 mL DMEM/0.1% BSA/20 μg/mL gentamicin then “pre-loaded” for
18 h with 0.25 mL of 4 μCi/mL myo-[2-3H(N)]-inositol in inositol-free DMEM,
then washed twice with 0.30 mL DMEM (inositol free) containing 5 mM LiCl
and treated for 2 h with 0.25 mL of a saturating concentration of Buserelin
(10−7M) in the same medium. When constitutive activity was assessed,
Buserelin was omitted from the assessment period. Total IP was then
determined (23). This assay has been validated as a sensitive measure of PME
for functional receptors when expressed at low amounts of DNA (<100 ng
per well) and stimulated by excess agonist (12, 14, 16, 24–30).
Binding Assays. Cellswere culturedandplatedingrowth mediumasdescribed
above, except 105cells in 0.5 mL of growth medium were added to 24-well
Costar cell culture plates (cell transfection and medium volumes were dou-
bled accordingly). Twenty-three hours after transfection, the medium was
replaced with 0.5 mL of fresh growth medium with or without pharmaco-
perone (1 μg/mL of In3). Twenty-seven hours after transfection, cells were
washed twice with 0.5 mL of DMEM containing 0.1% BSA and 20 μg/mL
gentamicin, then 0.5 mL of DMEM was added. After 18 h, cells were washed
twice with 0.5 mL of DMEM/0.1% BSA/10 mM hepes, then 2 × 106cpm/mL of
[125I]-Buserelin, prepared in our laboratory (specific activity, 700–800 μCi/μg),
was added to the cells in 0.5 mL of the same medium and allowed to
incubate at room temperature for 90 min, consonant with maximum bind-
ing (31). New receptor synthesis during this period is negligible at room
temperature. After 90 min, the media was removed and radioactivity was
measured (22). To determine nonspecific binding, the same concentrations
of radioligand were added to similarly transfected cells in the presence of
10 μg/mL unlabeled GnRH.
Statistics. Data (n ≥ 3) were analyzed with one-way ANOVA and then Holm-
Sidak test and paired with Student’s t test (SigmaStat 3.1, Jandel Scientific
Software). SEMs are shown.
ACKNOWLEDGMENTS. We thank Jo Ann Binkerd for formatting the manu-
script and Drs. J. Fielding Hejtmancik, Jon Hennebold, and Richard Stouffer
for commenting on a draft. This work was supported by National Institutes
of Health Grants HD-19899, RR-00163, and HD-18185.
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