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Division of Molecular Medicine (M.C.G.), Department of Medicine, Weill Medical College and Graduate School of Medical Sciences of Cornell University, New York, New York 10021; and Department of Physiology and Biophysics (R.O.), Mount Sinai School of Medicine, New York, New York 10029
Address all correspondence and requests for reprints to: Dr. Marvin C. Gershengorn, Weill Medical College of Cornell University, 1300 York Avenue, Room A328, New York, New York 10021-4896. E-mail: mcgersh{at}med.cornell.edu
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 Top Abstract IntroductionInitial Models of Ligand-GPCR...Use of Models to...ConclusionsReferences |
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Introduction |
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TopAbstractIntroductionInitial Models of Ligand-GPCR...Use of Models to...ConclusionsReferences |
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GPCRs may be grouped into three subfamilies that exhibit little
sequence homology but appear to share the same overall topology. In
this review, we will focus on receptors in the largest subfamily, which
comprises more than 90% of GPCRs, that includes rhodopsin (Rh) and
receptors for small neurotransmitters, such as opioids and
catecholamines, peptides such as bradykinin, GnRH, and TRH, and
glycoprotein hormones. The two-dimensional topology of a typical member
of this subfamily, a receptor for TRH, is illustrated in Fig. 1
1.
GPCRs contain an extracellular amino terminus, three extracellular
loops (ECLs), seven transmembrane-spanning helices (TMHs), three
intracellular loops (ICLs) and an intracellular carboxyl terminus. The
specific domains within which different regulatory molecules bind to
GPCRs, and the changes in a GPCR that constitute conversion of the
receptor from an inactive to an active state are under intense
investigation. In general, evidence has been presented that small
ligands bind primarily in the core of the TMHs, intermediate size
peptides to the ECLs and TMHs, and larger peptides and proteins to the
amino terminus, ECLs and, most likely, the TMHs of their specific
receptors. Upon binding an agonist, it is thought that changes are
induced in the conformation of the receptor, in particular, changes
involving the relative positions of the seven TMHs. The movements of
the TMHs are thought to cause changes in the ICLs leading to increased
coupling to a heterotrimeric G protein(s) and G protein activation.
These hypotheses, however, have only begun to be tested by direct
experimentation.
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Some investigators used bacteriorhodopsin (Br), for which a crystal structure at 1.55 Å resolution is now available (2), as a template for constructing the TMHs of GPCRs (3). Br, however, is not a GPCR even though it is a seven transmembrane-spanning protein. Other investigators used a GPCR template constructed by Baldwin (4). This TMH template was based on a comprehensive analysis of hundreds of GPCR sequences and was shown to be consistent with many mutational analyses. This template agrees well with the recently reported crystal structure of Rh that was resolved to 2.8 Å (1). It is now clear that position and tilt of TMHs in Rh, and by inference in all GPCRs, are different from those in Br. Nevertheless, models based on both Rh and Br have proven useful in generating hypotheses that have been tested experimentally.
In the majority of reports that describe computer simulations of GPCRs, the purpose of model construction was to describe the binding pocket for the ligand within the receptor. Models of ligand-receptor complexes were constructed incorporating experimental evidence of the specific receptor amino acid residues with which the ligands were found to interact. These models are being used, for example, in efforts to discover GPCR agonist or antagonist drugs. Another important use of GPCR models is to generate hypotheses regarding the binding and signaling functions of these receptors for experimental testing. That is, to direct experimentation to define the structure-function relationships of GPCRs. We think the most effective use of models, however, is as part of an iterative, bidirectional approach in which models are used to generate hypotheses that are tested by experimentation and the experimental findings are, in turn, used to revise and refine the model. The success of this approach is due to the synergistic interaction between theory and experiment. We will emphasize this last approach in this review.
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Initial Models of Ligand-GPCR Complexes |
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TopAbstractIntroductionInitial Models of Ligand-GPCR...Use of Models to...ConclusionsReferences |
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). The TMH
bundle of the receptor was based on either the Rh or Br template. In
every case, the ligand was docked within the receptor based on
experimental results that identified receptor amino acid residues which
directly interact with the ligand. That is, in no case was a stable
model of a ligand/receptor complex constructed using a computer
simulation independent of experimental evidence delineating the binding
pocket. (This is true for ligand/GPCR complexes of peptide and protein
ligands also.) A typical model of a GPCR for a small ligand, TRH
(pyroGlu-His-ProNH2), is illustrated in Fig. 2 and a close-up of the TRH/receptor
complex is shown in Fig. 3. The details
of our construction have been described elsewhere (5). TRH
is predicted to interact with residues in TMH3, 6 and 7 within the
extracellular part of the TMH bundle (see below).
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). A typical example of a model of a
peptide ligand/receptor complex is that of cholecystokinin-8, an
octapeptide that is the most biologically active form of this peptide,
with its receptor (6, 7). The interactions of
cholecystokinin-8 with its receptor are predicted to involve residues
in the amino terminus, ECL2, ECL3, and the extracellular aspect of
TMH6. The nonapeptide arginine-vasopressin is predicted to bind to its
cognate receptor by interacting with residues in ECL1 and TMH2, 3, 4,
6, and 7 (8, 9) and the decapeptide GnRH is predicted to
bind to residues in all TMHs and ECL2 and 3 in its cognate
receptor (10, 11). A model for the binding of the
nonapeptide bradykinin predicts that bradykinin binds to residues
in TMH5, 6, and 7 near the TMH/ECL boundary, whereas a peptide
antagonist binds to residues in TMH5, 6, and 7 that are deeper within
the TMH bundle (12). Different sites for binding peptide
agonists, peptide antagonists and nonpeptide antagonists have been
reported in a number of GPCRs.
Large polypeptide/protein ligands
As noted above, larger peptides and proteins bind to the amino
terminus, ECLs and, most likely, the TMHs of their specific receptors.
Chemokines are 70 to 100 amino acid polypeptides that bind primarily to
the amino termini and ECL1 and 2 of their cognate receptors (13, 14). Models of receptors for glycoprotein hormones do not
include the amino termini (15, 16), but it has been
predicted that these ligands bind primarily to the amino termini and
ECLs of their receptors (17).
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Use of Models to Generate Hypotheses of GPCR Structure and Function |
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TopAbstractIntroductionInitial Models of Ligand-GPCR...Use of Models to...ConclusionsReferences |
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) were
used to guide the initial construction. In our initial model of the
TRH/TRH receptor complex, we docked TRH into the TMH bundle of TRH
receptor and, using a novel simulation approach, optimized the complex
by extensive sampling of the configurational and conformational degrees
of freedom (18). This model predicted that, in addition to
the experimentally determined interaction of pyroGlu with Tyr-106 and
Asn-110 in TMH3, Tyr-282 in TMH6, and Arg-306 in TMH7 could interact
with the His and ProNH2 of TRH, respectively.
These predictions were tested using synthetic analogs of TRH and
mutational analysis of TRH receptor, and the experimental observations
were shown to be consistent with the predictions (19, 20, 21).
The final model shows that the pyroGlu moiety of TRH forms H-bonds with
Tyr-106 and Asn-110 in TMH3, His interacts hydrophobically with Tyr-282
in TMH6, and the ProNH2 forms H-bonds with
Arg-306 in TMH7. Thus, these studies allowed us to describe the
molecular interactions of TRH with the binding pocket within the TMH
bundle of TRH receptor. Ter Laak, Timmerman, Leurs, and colleagues (22, 23) constructed a model of an agonist/histamine H1 receptor complex based on a Br TMH template using experimental evidence for the interactions between histamine agonists and specific receptor residues. The model predicted an Asp in TMH3, a Trp in TMH4, a Lys in TMH5 and two Phe residues in TMH6 with which ligands would interact. Experiments involving functional studies with mutated receptors and different antagonists provided evidence that the predictions were correct. These studies allowed delineation of agonist and antagonist binding pockets in histamine H1 receptor.
Befort and colleagues (24) constructed a model of the TMH
bundle of the 
-opioid receptor based on a Br template and
sequence alignment with other GPCRs. The model predicted that a series
of aromatic residues in TMHs project into the bundle. These residues
were mutated and binding of opioid analogs was measured. Evidence was
obtained that a Tyr in TMH3, a Trp in TMH4, a Phe in TMH5, a Trp in
TMH6, and a Tyr in TMH7 provide interactions with the ligands. These
findings further demonstrated the importance of aromatic residues in
binding small ligands.
Almaula and co-workers (25) constructed a model of the serotonin type 2A receptor that predicted that the ligand would interact with a Ser one helical turn below the Asp in TMH3 that binds the cationic primary amino group of serotonin. This prediction was shown to be consistent with data from experiments using Ser to Ala and to Cys mutations and serotonin analogs.
Sequential binding of small ligands to GPCRs
Based on our model of the unoccupied TRH receptor (26, 27) (Fig. 4), we proposed that TRH
binds to TRH receptor in at least three discrete steps. TRH interacts
with: 1) Tyr-181 on the surface of the ECLs; 2) with Asn-289 and
Ser-290 at the ECL/TMH boundary; and 3) with TMH residues (see above).
Specifically, it was predicted that the pyroGlu of TRH interacts with
Tyr-181 then with Asn-289/Ser-290 and then with Tyr-106/Asn-110. These
predictions were tested experimentally. Using a mutation of Tyr-181 to
Phe, we showed that TRH interacted with Tyr-181 via the pyroGlu moiety
because a mutant receptor in which Phe was substituted for Tyr-181
(Y181F) was not able to discriminate between TRH analogs changed in the
pyroGlu position but was able to distinguish between TRH and analogs
substituted in the His or ProNH2 positions.
Similar findings were obtained with Y106F and N110A mutant TRH
receptors, and with N289A and S290A mutant receptors. Most importantly,
N289A exhibited a decreased binding affinity for TRH that was accounted
for by a decreased rate of association rather than N110A that exhibited
a similar decrease in affinity but in which the decreased affinity was
caused by an increased rate of dissociation
(27). Thus, we concluded that TRH is attracted to TRH
receptor via interactions with Tyr-181, is conducted down a funnel
formed by the ECLs through which it enters the TMH binding pocket in
which it exerts its effect to change the conformation of TRH
receptor that comprises the conversion of the receptor from an inactive
to active state (see below).
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Thus, it appears that small ligands enter their binding pockets within the TMH bundle after attraction and sequential interactions with ECL residues.
Peptide ligand binding pockets
Several groups of investigators have used initial models of
ligand/GPCR complexes to make predictions regarding other interactions
between receptor residues and ligands to define the binding pockets of
receptors for peptides more completely. The binding pockets for
nonpeptide ligands in these same receptors were analyzed also. In a
series of reports (6, 7, 29), Fourmy and colleagues
described experiments in which they used a series of cholecystokinin
(CCK) analogs and cholecystokinin-A receptor (CCK-AR) mutants coupled
with computer simulations to develop a model of CCK binding and CCK-AR
activation. An initial model of CCK-AR was constructed based on the TMH
template of Br, and Rh and ß2-adrenergic receptor for sequence
alignment. Based on experimental findings that two residues in the
amino terminus of CCK-AR bind the amino terminus of CCK-9 and docking
the remainder of the CCK-9 peptide into the TMH bundle of CCK-AR, a
model of the CCK/CCK-AR complex was constructed. (The 3D structure of
CCK-9 was based on biophysical measurements.) The model was optimized
by energy minimization and molecular dynamics simulation protocols.
This model predicted that a Met residue in ECL2 of CCK-AR interacted
with the sulfated Tyr in position-3 of CCK-9. Using site-specific
mutagenesis of this Met and CCK-9 analogs, these authors tested this
hypothesis and presented evidence that supported an interaction between
this Met and Tyr(SO3H)-3 as being important for
ligand binding and receptor activation. In a subsequent study,
predictions of the model that the carboxylate of Asp-8 and the carboxyl
terminal amide of CCK-9 interacted with Asn and Arg residues at the
boundary of TMH6 and ECL3 were tested, and data supporting these
interactions were obtained. From these findings, the authors were able
to identify a hydrophilic group of receptor residues that interact with
CCK-9 and hypothesized that electrostatic interactions were important
in converting CCK-AR from an inactive to an active state. Gouldson and
co-workers (30) constructed a model of CCK-AR and
presented evidence based on mutational analyses that a nonpeptide
agonist and antagonist bind via interactions with different residues
within the TMH bundle. The antagonist was predicted to bind near the
extracellular surface of the TMH bundle via a limited number of
contacts whereas the nonpeptide agonist, like CCK-8, bound deeper
within the bundle and made many more contacts with receptor
residues.
Jarnagin and colleagues (12) constructed a model of the TMH bundle of the B2 bradykinin (BK) receptor based on Br and mutagenesis experiments of several GPCRs. The conformation of BK in solution resolved by nuclear magnetic resonance spectroscopy was docked into the receptor model. Based on predictions of the model, a number of residues in the TMH5, 6, and 7 were mutated and data consistent with interactions between these residues and BK were provided.
Feighner and colleagues (31) constructed an energy-minimized model for the hexapeptide GH-releasing peptide (GHRP-6) receptor. The model predicted interactions of the ligand with residues in TMH2, 3, 5, and 6 and in ECL1 that were tested by site-specific mutations. The data obtained were consistent with the predictions.
Macdonald and colleagues (32) constructed a melanin-concentrating hormone (MCH)/MCH receptor complex by docking MCH in a conformation obtained by molecular dynamics simulation into a receptor model based on a Br template. The model predicted an interaction between an Asp in TMH3 and Arg-11 in the 19-amino acid, cyclic peptide. The prediction was tested experimentally by mutation of the receptor and synthesis of position-11 MCH analogs. The findings were consistent with a role for the Asp in TMH3 in binding MCH and in receptor activation.
Mouillac, Barberis, Hibert, and co-workers (8, 9, 33, 34) have used models of the vasopressin V1a receptor to study binding of agonists and antagonists. Their model predicted that a primary binding pocket for the cyclic, nonapeptide vasopressin was within the TMH bundle (see above), and this was confirmed by mutational analysis of the receptor. They next studied the binding of linear peptide and nonpeptide antagonists. They found that aromatic residues in TMH6 of the receptor were needed for binding antagonists even though they are not involved in binding agonists. These findings provide new insights that could be used in rational design of antagonists vs. agonists.
Role of highly conserved Asn in TMH1, Asp in TMH2 and Asn in TMH7
in GPCR structure
Sealfon, Weinstein, and colleagues (35, 36) used
models of the GnRH receptor to predict structural features that were
important for receptor function. They noticed that the highly conserved
pattern of Asn in TMH1, Asp in TMH2 and Asn in TMH7 was changed in GnRH
receptor to Asn in TMH1, Asn in TMH2 and Asp in TMH7. A model of the
TMH bundle of GnRH receptor was constructed and energy minimized. The
model predicted that these three residues interact to stabilize the
helical bundle. The findings from experiments using site-specific
mutations of these residues and functional analyses of the mutant
receptors were consistent with this prediction.
Our model of the unoccupied TRH receptor (see above) predicted that Asn-43, Asp-71and Asn-316 interacted via H-bonds to hold TMH1, 2 and 7 together (37). This prediction was tested by site-specific mutagenesis, and the data obtained were consistent with this hypothesis.
In the 3D crystal structure of Rh (1), the conserved Asn in TMH1 and the Asp in TMH2 interact with the peptide backbone carbonyl one helical turn above the conserved Asn in TMH7, and Asp in TMH2 and Asn in TMH7 interact via a water molecule also. Thus, models provided the first insights into how residues in TMH1, 2, and 7 through intramolecular interactions constrained the conformation of TMH bundle of GPCRs.
Role of highly conserved Asn-Pro/Asp-Pro motif in the structure of
TMH7
Weinstein and colleagues (38) used results from
analysis of a structure database search and dynamic simulations to
construct a model of TMH7 of a GPCR. Their model predicted that TMH7 is
not an ideal 
-helix but contains a kink caused by the
Asn-Pro/Asp-Pro motif. Using the serotonin receptor type 2a as a
prototype, they suggested that a model incorporating this kinked helix
explained previously generated experimental data. However, the Rh
structure shows that the Asn-Pro-Xaa-Xaa-Tyr sequence has a regular
helical structure, and a kink occurs at a point further along the
helix. Other parts of TMH7, in particular the area around the Lys that
covalently attaches to retinal and the sequence near the Ala residue
whose carbonyl interacts with Asn in TMH1, are distorted.
Role of highly conserved Asp/Glu-Arg-Tyr sequence in GPCR
activation
The amino acid sequence Asp/Glu-Arg-Tyr (D/E-R-Y) at the
intracellular end of TMH3 has been shown to be important in receptor
activation in several GPCRs. It was initially shown that the charged
pair of Glu-Arg was needed for rhodopsin activation because double
mutants of these residues failed to activate transducin
(39). Scheer, Fanelli, and colleagues (40, 41) constructed models of native
1B-adrenergic receptors and of receptors with
mutations of the Asp and Arg residues of the D-R-Y sequence
that exhibited constitutive signaling activities. Functional
experiments with these receptors showed that mutation of Arg to several
other amino acids caused inhibition of stimulated signaling and to Lys
increased basal signaling, whereas mutation of Asp to Ala caused a
marked increase in basal activity. Their models predicted that the Arg
residue was located in a hydrophilic pocket formed by the highly
conserved Asn in TMH1, Asp in TMH2 and Asn and Tyr in TMH7 (see above)
in the receptors that were not basally active and that this Arg was not
in this pocket in basally active 1B-adrenergic
receptor mutants. Based on their experimental observations and model
predictions, and the finding that activation of Rh is associated with
proton uptake (42), these investigators proposed the
following hypothesis for activation of the
1B-adrenergic receptor. The inactive receptor
is restrained by interactions between Arg of D-R-Y and the
hydrophilic residues of the pocket formed between TMH1, 2 and 7.
Activation is secondary to protonation of the Asp in D-R-Y
that causes the Arg of the D-R-Y sequence to move out of
the TMH bundle and changes the orientation of residues in ICL2 and 3
that allow for increased affinity of coupling to G protein-coupled.
Fanelli and colleagues (43) analyzed the Asp-Arg-Cys,
rather than Asp-Arg-Tyr in most GPCRs, sequence at the end of TMH3 in
the oxytocin receptor by mutation and modeling. Mutation of the Arg to
Ala caused this receptor to become constitutively active. Nevertheless,
they predicted that the same movement of Arg from a hydrophilic pocket
formed by TMH1, 2, and 7 in the oxytocin receptor creates a crevice
formed by ICL2 and 3 and the intracellular extension of TMH5 to which a
G protein may couple. This hypothesis is similar to the one they
proposed for the 1B-adrenergic receptor (see
above).
A different hypothesis regarding a protonation-dependent mechanism of activation involving Arg at the intracellular aspect of TMH3 has been proposed for the GnRH receptor. From their models, Ballesteros and colleagues (36) predicted that the Arg of the Asp-Arg-Ser sequence at the end of TMH3 interacts with the adjacent Asp in the inactive state of the GnRH receptor. Activation involves release of the Arg from interacting with Asp by Asp protonation and promotes movement of the Arg into a hydrophilic pocket in the TMH bundle. An Ile one helical turn above the Arg in TMH3 sterically directs the Arg into the TMH hydrophilic pocket. They provided experimental support for this hypothesis by finding that mutation of Arg markedly decreased agonist-stimulated activation whereas receptors with mutations of Asp to Asn or Ile to Ala were less well stimulated than GnRH receptor.
Thus, these models predict that movement of the sidechain of the Arg of the highly conserved E/D-R-Y sequence is involved in receptor activation. In particular, they support a role for the Arg as a switch to convert a GPCR from an inactive to an active state in response to agonist-stimulated receptor protonation. A recent report, however, has provided evidence that the conserved Asp in the D-R-Y sequence is not necessary for protonation-dependent activation of the ß2-adrenergic receptor even though protonation may be involved (44).
Role of transmembrane helix movement in GPCR activation
Experiments with mutant rhodopsins in which double site-specific
substitutions by Cys labeled for electron spin resonance spectroscopy
demonstrated that movement of TMHs relative to one another were
involved in receptor activation (45). In the case of Rh,
TMH3 and 6 were shown to exhibit relative motion upon light activation.
Movement of TMHs of Rh upon conversion of 11-cis-retinal to
all trans-retinal is predicted from the crystal structure
also (1). Gether and co-workers (46)
performed experiments using a conformationally sensitive Cys-reactive
reagent and found that positions in TMH3 and 6 changed their
environments upon binding ß2-adrenergic
agonists. Based on their model of the
ß2-adrenergic receptor, they concluded that
these movements were similar to those proposed in Rh.
Based on our analyses of unoccupied and TRH-occupied TRH receptor models, we (47) predicted relative movement of TMH5 and 6 upon activation of TRH receptor. To provide evidence in support of this hypothesis, we mutated residues in TMH5 and 6 that our model predicted were involved in interhelical aromatic interactions that constrain the receptor in the inactive conformation. Mutation of these residues to amino acids that could not form aromatic interactions should allow these helices to move apart and thereby activate TRH receptor. As predicted, mutation of Phe-199 in TMH5 and of Trp-279 in TMH6 to Ala produced receptors that were constitutively active.
Lin and colleagues (15) constructed a model of the LH/CG receptor and used it along with an analysis of naturally occurring constitutively activating mutations to predict TMH movements that constitute LH/CG receptor activation. They concluded that hydrophobic interactions among residues in the intracellular halves of TMH5 and 6, and polar interactions between residues in TMH6 and 7 constrain LH/CG receptor in an inactive conformation. They suggested that activation involves agonist-induced disruption of these interactions that allows TMH6 movement. Findings inconsistent with some of these predictions, however, have been reported (16). An analysis of a number of constitutively active LH/CG receptor mutants supports the idea that activation is caused by release of intrahelical interactions (48).
Fanelli and colleagues (43) predicted movements of TMH3, 4, 5, and 6 as components of activation of the oxytocin receptor secondary to movement of the Arg sidechain of the E/D-R-Y sequence (see above). Zhang and Weinstein (49) predicted that TMH5 and 6 underwent the greatest motion when agonists bound to serotonin type 2 receptors.
Biologically active conformation of an agonist
Prevailing theory of GPCR activation holds that the receptor can
assume many conformations within a spectrum from inactive to fully
active and that different agonists will lead to different subsets of
receptor conformations. In general, especially for small ligands,
conformational changes of the agonist are not considered. However, to
understand receptor activation, the conformation of the active
pharmacophore is critical. We (50) found that the
conformation of TRH in our model of the TRH/TRH receptor complex was
different from the predominant conformations of TRH in solution. To
gain insight into the biologically active TRH conformation, Dr. Kevin
Moeller (Washington University, St. Louis, MO) synthesized
conformationally restricted analogs of TRH. We characterized two
diastereomers of an analog restricted by addition of a methylene bridge
between the second and third positions of a TRH analog,
pyroGlu-cyclohexylAla-ProNH2. We found that one
diastereomer bound with higher affinity and exhibited higher intrinsic
activity than pyroGlu-cyclohexylAla-ProNH2,
whereas the other diastereomer bound with lower affinity and was less
active than pyroGlu-cyclohexylAla-ProNH2.
Computer simulations of these analogs predicted that the higher
affinity, more active diastereomer assumed a conformation that was
similar to TRH in our model of the TRH/TRH receptor complex whereas the
lower affinity, less active diastereomer was similar in structure to
TRH in solution. Thus, our model of the TRH/TRH receptor complex
predicted a biologically active conformation of TRH. These types of
predictions may allow for the design of better agonists of GPCRs.
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Conclusions |
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TopAbstractIntroductionInitial Models of Ligand-GPCR...Use of Models to...ConclusionsReferences |
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Acknowledgments |
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We apologize to those investigators whose relevant contributions we failed to include in this review.
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Footnotes |
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Received August 28, 2000.
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References |
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TopAbstractIntroductionInitial Models of Ligand-GPCR...Use of Models to...ConclusionsReferences |
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-opioid receptor
in ligand recognition. J Biol Chem 271:10161–101681B-adrenergic receptor: role of highly
conserved polar amino acids in receptor activation. EMBO J 15:3566–3578[Medline]
1b-adrenergic
receptor: effects on receptor isomerization and activation. Mol
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 B. Karges, W. Karges, M. Mine, L. Ludwig, R. Kuhne, E. Milgrom, and N. de Roux Mutation Ala171Thr Stabilizes the Gonadotropin-Releasing Hormone Receptor in Its Inactive Conformation, Causing Familial Hypogonadotropic Hypogonadism J. Clin. Endocrinol. Metab., April 1, 2003; 88(4): 1873 - 1879. [Abstract] [Full Text] [PDF]
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X.-Q. Ding, D. I. Pinon, K. E. Furse, T. P. Lybrand, and L. J. Miller Refinement of the Conformation of a Critical Region of Charge-Charge Interaction between Cholecystokinin and Its Receptor Mol. Pharmacol., May 1, 2002; 61(5): 1041 - 1052. [Abstract] [Full Text] [PDF]
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 W. Wang, D. J. Seward, L. Li, J. L. Boyer, and N. Ballatori Expression cloning of two genes that together mediate organic solute and steroid transport in the liver of a marine vertebrate PNAS, July 19, 2001; (2001) 161099898. [Abstract] [Full Text] [PDF]
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M. H. Perrin, W. H. Fischer, K. S. Kunitake, A. G. Craig, S. C. Koerber, L. A. Cervini, J. E. Rivier, J. C. Groppe, J. Greenwald, S. M. Nielsen, et al. Expression, Purification, and Characterization of a Soluble Form of the First Extracellular Domain of the Human Type 1 Corticotropin Releasing Factor Receptor J. Biol. Chem., August 17, 2001; 276(34): 31528 - 31534. [Abstract] [Full Text] [PDF]
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W. Wang, D. J. Seward, L. Li, J. L. Boyer, and N. Ballatori Expression cloning of two genes that together mediate organic solute and steroid transport in the liver of a marine vertebrate PNAS, July 31, 2001; 98(16): 9431 - 9436. [Abstract] [Full Text] [PDF]
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