(Received for publication, June 28, 1996, and in revised form, October 2, 1996)
From the Department of Obstetrics and Gynecology, Robert Wood Johnson (Rutgers) Medical School, Piscataway, New Jersey 08854
Bovine lutropin (bLH) and human chorionic
gonadotropin (hCG) are heterodimeric glycoprotein hormones required for
reproduction. Both bind rat LH receptors (rLHRs), but hCG binds human
LH receptors (hLHRs) 1000-10,000 fold better than bLH. We tested the
premise that this difference in affinity could be used to identify
lutropin receptor contacts. Heterodimers containing hCG/bLH - or
-subunit chimeras that bound hLHR like hCG (or bLH) were expected to
have hCG (or bLH) residues at the receptor contact sites. Analogs
containing one subunit derived from hCG bound hLHR much more like hCG
than bLH, indicating that each bLH subunit contains all the residues sufficient for high affinity hLHR binding. Indeed, the presence of
bovine
-subunit residues increased the activities of some hCG
analogs. The low hLHR activity of bLH was due primarily to an
interaction between its
-subunit and
-subunit residue
Leu95. Leu95 does not appear to contact the
hLHR since it did not influence the hLHR activity of heterodimers
containing human
-subunit. These observations show that interactions
within and between the subunits can significantly influence the
activities of lutropins, thereby confounding efforts to identify ligand
residues that contact these receptors.
The gonadotropins human lutropin (hLH),1 human chorionic gonadotropin (hCG), and human follitropin (hFSH) are essential for reproduction and have been used for many years to enhance human fertility. Development of clinically useful agonist and antagonist analogs would be facilitated by knowledge of how these ligands interacted with their receptors. Two radically different models of gonadotropin-receptor interaction have been proposed (1, 2) based on the crystal structures of hCG (3, 4) and ribonuclease inhibitor (5, 6), a protein containing a leucine-rich repeat motif thought to be similar to those in the glycoprotein hormone receptors. These models could be readily distinguished if the portions of the hormone that contacted their receptors were known.
Like other glycoprotein hormones, the gonadotropins are heterodimers
that contain a conserved -subunit and a hormone-specific
-subunit
(7). Each subunit is divided into three large loops by a cysteine knot
(3, 4), and the heterodimer is stabilized by a portion of the
-subunit termed the "seat belt" (3) that is wrapped around
-subunit loop 2. Based on the activities of chemically and
enzymatically modified hormones (summarized by Pierce and Parsons (7)),
synthetic hormone fragments (8-12), and analogs prepared by
site-directed mutagenesis (13-19), residues throughout both hormone
subunits have been suggested to participate in essential high affinity
hormone receptor contacts. Surprisingly, some hCG residues proposed to
contact LH receptors are in regions recognized by monoclonal antibodies
that bind to hCG-receptor complexes (1, 20). Others thought to be
essential for receptor contacts can be replaced without disrupting
receptor binding. For example, replacing hCG seat belt residues
101-109 with their hFSH counterparts led to analogs that bound FSH
receptors with high affinity (21, 22), even though they contained hCG
-subunit residues at positions thought to interact with the FSH
receptor. These findings raise signficant questions about the
identities of residues that have been proposed to be responsible for
high affinity receptor contacts.
Studies described in this report were initiated with the goal of identifying residues in hCG that are essential for high affinity contacts with the human LH receptor. The heterodimeric nature of the glycoprotein hormones confounds efforts to identify residues needed for essential high affinity receptor contacts, and with few exceptions (23), neither free subunit has significant biological activity (7). Thus, any modification of the hormone that distorts the interaction between its subunits has the potential to disrupt hormone activity, a problem that has been recognized for many years (7) but largely ignored. In the experiments reported here, we sought to minimize this problem by limiting our studies to lutropins such as hCG and bLH that bind the rat lutropin receptor well, an indication that their overall conformations are very similar. We anticipated that the dramatic differences in the affinities of these hormones for the human lutropin receptor (24, 25) would be caused by a few key residues in the primary receptor contact site that could be identified by comparing the abilities of hCG/bLH chimeras to bind the human and rat receptors. Chimeras that had hCG residues in the contact site were expected to bind the hLHR like hCG; those that had bLH residues in this site were expected to bind hLHR like bLH. As we show here, the low affinity of bLH for hLHR appears to involve an interaction between its subunits that either distorts the region of the high affinity contact and/or creates a steric interaction between the hormone and the receptor. This implies that subtle changes in hormone conformation can exert a much more dramatic influence on ligand binding than commonly perceived.
Purified hCG was obtained from Drs. Robert Canfield and Steven
Birken (Columbia University, NY). Purified bLH was obtained from Dr.
John Pierce (University of California at Los Angeles, CA). Antibodies
were obtained from Dr. Canfield, Drs. Glenn Armstrong and Robert
Wolfert (Hybritech Inc., CA), and Dr. Janet Roser (University of
California at Davis, CA) as noted previously (1, 20). The hLHR cDNA
was obtained from Dr. Aaron Hsueh (Stanford University, CA) in a vector
that was used without modification to make stable CHO cell lines that
express the human receptor. These lines were prepared by
co-transfecting CHO cells with the hLHR vector and pSV2-Neo, a vector
encoding aminoglycoside phosphotransferase downstream of the SV40 early
promoter (26), selecting stable transformants in the presence of 500 µg G418/ml, and identifying receptor-expressing cell lines based on
their abilities to bind 125I-labeled hCG. A similar
strategy was used to select CHO cell lines that express the rat LH
receptor. The rLHR cDNA (27) was inserted downstream of the
metallothionein promoter in a vector (pLEN) kindly supplied by Dr.
Peter Kushner (University of California at San Francisco, CA). This was
accomplished by cloning the XhoI-BamHI fragment
of pSVL-hCG (21) into the unique XhoI-BamHI
sites of pLEN to create a vector (pLEN-hCG
) that has a unique
XbaI site immediately downstream of the XhoI
site. The XbaI-BamHI fragment containing the
coding region of hCG
was excised and replaced with an
XbaI-BamHI fragment containing the entire rLH
receptor cDNA prepared as described earlier (27).
Figs. 1 and 2 illustrate the amino acid sequences of the hormones and
analogs used in this study relative to the overall structures of the
- and
-subunits. Characterization of the immunological properties
of each
-subunit analog has been described (1). All but two of the
-subunit analogs have also been described (20, 28). CLC89-96, an
hLH/hCG
-subunit chimera having hCG
-subunit residues 89-92
replaced by their hLH counterparts, was made by swapping parts of the
cDNA that encode hCG and CLC77-96 (28) at PvuII
restriction sites common to both. These are located in the codons for
Ala86-Ser87-Cys88 and in the pSVL
vector (Pharmacia Biotech Inc.). Specifically, the small fragment
prepared by PvuII digestion of pSVL-hCG
(21) was ligated
to the large fragment created by PvuII digestion of pSVL-hCLC
77-96 (28), and a clone having the proper orientation of
the insert was selected by endonuclease restriction mapping. The vector
encoding hCG
-subunit in which Arg95 was converted to
Leu, pSVL-hCG
R95L, was made by simultaneously ligating three
fragments including the large XhoI-PpuMI fragment of the vector pKBM-hCG
(21) that contained the vector and codons
for hCG
-subunit residues 103-145, an
XhoI-BssHII fragment that contained the codons
for the hCG
-subunit signal sequence and amino acids 1-92, and a
synthetic oligonucleotide cassette that had BssHII and
PpuMI overhangs and encoded residues 93-102. This restored
all hCG
-subunit codons except that for Arg95, which was
changed to Leu during synthesis of the DNA cassette. The coding
sequence was confirmed by standard dideoxy methods and the
XhoI-BamHI fragment was cloned into pSVL for
expression in COS-7 cells.
Glycoprotein hormone heterodimers were prepared by transient
co-transfection of COS-7 cells with vectors encoding - and
-subunits as described (21). Hormone and analog heterodimers
released into the medium were quantified using a sandwich immunoassay
similar to one we reported (29) except that different antibodies and standards were used, depending on the hormone or analog being measured.
To quantify heterodimers consisting of human/bovine
-subunit
chimeras and hCG
-subunits, we captured the analogs with an
anti-
-subunit antibody (B112) having high affinity for an hCG
-subunit epitope that included Asn77 (28). We detected
the complex using a radiolabeled antibody (B109) that recognized a
-subunit epitope specific to the heterodimer (28). Highly purified
urinary hCG was used as the standard. This dimer-specific assay has
been shown to be unaffected by the presence of bovine
-subunit
residues (1). Heterodimers containing hCG
-subunit and bLH/hCG
-subunit chimeras were assayed using an
-subunit antibody (A113)
as a capture agent and a labeled antibody (B410) that has high affinity
for nearly all mammalian LH
-subunits, including those from hCG and
bLH (30). Again, we used highly purified urinary hCG as a standard. We
also used this same assay to quantify heterodimers containing bovine
hCG/bLH
-subunit chimeras. However, because A113 has lower affinity
for the
-subunit from bLH than that from hCG (1), we used highly purified bLH as the standard in these latter assays. Procedures for
preparing radiolabeled hCG and antibodies using IODO-GEN (Pierce) to
specific activities of approximately 50 µCi/µg have been described (31). Methods for measuring the abilities of hCG, bLH, and analogs to
inhibit binding of 125I-labeled hCG to CHO cells expressing
lutropin receptors have also been cited (22). Briefly, we mixed the
hormones and analogs with 125I-labeled hCG (100,000 cpm,
approximately 1.5 ng) and then added cells expressing the receptors.
Following 1 h at 37 °C, we added 2 ml of 0.9% NaCl solution
containing 2 mg bovine serum albumin, sedimented the cells at 1000 × g (10 min), aspirated the supernatant, and measured the
radioactivity in the pellet using a
counter.
Consistent with earlier reports (32), we found that the
heterodimer consisting of the bovine -subunit and hCG
-subunit bound rLHR well (Table I). This analog bound hLHR about
half as well as hCG (Table I), but more than 1000-fold better than bLH,
indicating that differences between the human and bovine
-subunits
per se were not responsible for the very low activity of bLH
in hLHR assays. To localize residues for the small loss in binding to
the hLHR, we compared the activities of heterodimers containing hCG
-subunit and human/bovine
-subunit chimeras (Figs. 1
and 2). Many of these were more active
than those containing either the human or bovine
-subunits (Table
I). While some of the most active analogs (e.g.
bH1-6/41-81) contained bovine residues in loop 1 and human residues
in all or parts of loop 3, the presence of bovine residues in loop 1 was not required for increased activity. Other analogs (e.g.
bH11-26/64-68, bH11-26/73-75, and bH11-26/81) were more active
than hCG even though they contained human residues in loop 1 and bovine
residues in parts of loop 3 (Table I). This suggested that the
increased activities of some chimeras was caused by interactions
between residues in the regions of the
-subunit that differ most in
the human and bovine proteins (i.e. the N terminus, loop 1, and loop 3) rather than by interactions between bovine-specific
-subunit residues and either of the receptors.
|
Heterodimers containing the
human -subunit and bLH/hCG
-subunit chimeras bound hLHR at least
100-fold better than bLH, suggesting that residues in the bLH
-subunit per se accounted for only a fraction of the low
affinity of bLH for the hLHR. The design of the chimeras permitted an
assessment of differences in
-subunit loop 1, loops 2-3, and the
seat belt (Table II). The presence of bLH residues in
loops 2-3 had a greater influence than those in loop 1 or the seat
belt (Fig. 3, top, Table II, column labeled "human
-subunit") as seen by comparing the relative activity of
an analog containing bLH
-subunit residues only in loop 1 (i.e. CbL36-145) with that of an analog containing bLH
-subunit residues in loops 1-3 (i.e. CbL88-145). The
influence of loops 2-3 can also be seen by comparing the activity of
an analog containing bLH seat belt residues (i.e. CbL1-87)
with that of an analog containing the bLH seat belt and
-subunit
residues derived from loops 2-3 (i.e. CbL1-35).
Heterodimers containing human
-subunit and bLH
-subunit residues
only in loop 1 (i.e. CbL36-145) or the seat belt
(i.e. CbL1-87) were about as active as hCG.
|
The Low Potency of bLH in the hLHR Binding Assay Is Due to a Combined Influence of the Bovine
To test the idea that the inactivity of
bLH was due to the combined effects of bovine residues in both
subunits, we assayed heterodimers that contained bovine residues in
parts of both the - and
-subunits. We anticipated that the least
active analogs would contain the bovine
-subunit and portions of the
-subunit in which loops 2-3 originated from bLH. Although analogs
containing the bovine
-subunit and these bLH
-subunit residues
were less active than those containing the human
-subunit (Table
II), they were more active than bLH in hLHR assays. For example, the
hLHR activities of heterodimers containing the entire bovine
-subunit and bLH
-subunit residues solely in loop 1 (CbL36-145)
or loops 1-3 (CbL88-145) were reduced 50-80% relative to those that
contained the human
-subunit (Fig. 3, top and
bottom, and Table II), yet they remained at least 100-fold
greater than bLH.
The least active chimeras contained the bovine -subunit and the bLH
seat belt. This can be seen by comparing the activities of heterodimers
containing the human or bovine
-subunits and CbL1-87, a
-subunit
chimera containing hCG loops 1-3 and the bLH seat belt (Figs. 3
and 4, Table II). The heterodimer containing the human
-subunit had nearly the same potency as hCG; that containing the
bovine
-subunit was inactive at the highest concentration available
for testing.
The relative influences of -subunit loops 1-3 and the seat belt can
be seen by comparing the activities of heterodimers containing the
CbL1-87 and CbL88-145
-subunits. That containing the bovine
-subunit, bLH
-subunit loops 1-3, and the hCG seat belt
(i.e. CbL88-145) was at least 100-fold more active than
that containing the bovine
-subunit, hCG
-subunit loops 1-3, and
the bLH seat belt (i.e. CbL1-87). These observations showed
that the effect of the interactions between bLH-specific residues in
the
-subunit and seat belt was considerably greater than those in
the
-subunit and
-subunit loops 1-3.
Previous studies showed that seat belt residues influence
the receptor binding specificity of hCG (21). Those between
Cys93 and Cys100 had a greater influence on
binding to LH receptors than those between Cys100 and
Cys110 (22). Residues in the N-terminal half of the bLH
seat belt appeared to exert a greater influence on hLHR binding than
those in the C-terminal half. This can be seen by comparing the hLHR binding activities of heterodimers containing the bovine -subunit and
-subunit chimeras CbL88-145 or CbL103-145, analogs that differ only by the presence of hCG and bLH residues in the N-terminal halves
of the seat belt, respectively (Table II). That containing hCG residues
in the N-terminal half of the seat belt (i.e. CbL88-145) was much more active than that containing bLH residues in this region
(i.e. CbL103-145).
The amino acid sequences of the entire N-terminal halves of the seat
belts of hCG, hLH, and bLH are
C90ALCRRSTTDC100,
C90GPCRRSTSDC100, and
C90GPCRLSSTDC100, respectively. Residues
Gly91, Pro92, Leu95, and
Ser97 differ in this region of the hCG and bLH
-subunits. Although residues Gly91 and Pro92
are found in the seat belt of hLH, a hormone that binds hLHR well, we
considered it possible that an interaction between the bovine
-subunit and Gly91/Pro92 could lead to a
reduction in hLHR binding. To test this, we compared the activities of
heterodimers containing either the human or bovine
-subunit and an
hCG/hLH chimeric
-subunit (CLC89-92) containing Gly91
and Pro92. In rLHR assays, the bovine
-subunit/CLC89-92
-subunit heterodimer was twice as active as that containing the
human
-subunit (Fig. 5), an increase similar to what
was observed when the bovine
-subunit was expressed with the
-subunit of hCG (Table II). In hLHR assays, the bovine
-subunit/CLC89-92
-subunit heterodimer was similar in activity
to that containing the bovine
-subunit and the hCG
-subunit. Both
had approximately half the activity of hCG (Table II). Thus, the
differences at
-subunit residues 91 and 92 were not responsible for
the very low ability of bLH to bind to the hLHR.
Finally, we studied the role of Leu95, the residue that was
the least conserved in this region of the hCG and bLH seat belts. Heterodimers containing the human -subunit and an hCG
-subunit analog in which Arg95 was converted to Leu (i.e.
hCG
R95L) bound hLHR like hCG (Fig. 6), suggesting
that neither Arg nor Leu at this position participated in essential
hLHR contacts needed for high affinity ligand binding. In contrast, the
heterodimer containing the bovine
-subunit and the hCG
R95L
-subunit had much less ability to bind hLHR since concentrations of
more than 10
7 M were needed to observe 50%
inhibition of binding. While this heterodimer was more active than bLH,
it was clear that the presence of a single amino acid change, namely
Leu for Arg95, accounted for much of the influence of the
bLH seat belt.
Several mutagenesis strategies are available for identifying ligand residues that may be involved in receptor contacts. For example, to identify interactions between lutropins and their receptors, one might begin with hCG and change conserved residues to search for those that disrupt its ability to interact with receptors. Several of these have been found (13-16, 34). We assumed that it would be more efficient to distinguish receptor contacts using a homolog scanning approach in which we characterized the hLHR binding activities of chimeras containing parts of lutropins that had both high and low affinities for the hLHR. In principle, this would enable us to identify clusters of amino acids that would contain residues responsible for the inactivity of bLH. Subsequent mutagenesis would need focus only on these smaller regions. This strategy has been shown to be highly efficient (35).
When these studies were initiated, we expected residues, which differed
in hCG and bLH in one or both subunits, would participate in high
affinity hLHR contacts and be recognized by contributions they made to
the overall affinity of the hormone for the receptor. Given that bLH is
virtually inactive in hLHR assays, we were surprised to find that
heterodimers containing one hCG subunit were highly active. Those
containing the hCG -subunit and a chimeric
-subunit were at least
half as active as hCG; some were even more active than hCG in both rLHR
and hLHR assays. The activities of heterodimers containing the hCG
-subunit and bLH-specific residues in
-subunit loops 2-3 were
reduced only 5-10 fold. Even when present throughout loops 1-3 of
both subunits, bLH-specific residues reduced binding only 10-20 fold.
Taken together, this suggests that residues that differ in hCG and bLH
are probably not essential for receptor binding. In marked contrast to
the effects of residues in
-subunit loops 1-3, the combined
influence of a single residue (Leu95) in the bLH seat belt
and the bLH
-subunit accounted for most of the inactivity of bLH in
hLHR assays. Thus, while neither the entire bLH seat belt nor
Leu95 had a significant influence on the hLHR binding
activities of heterodimers containing the human
-subunit, the
activities of heterodimers containing these residues and the bLH
-subunit were reduced 1000-fold, nearly to the level of bLH. This
extreme synergism between the bLH
-subunit and the bLH seat belt
suggested that differences in the abilities of hCG and bLH to bind hLHR
are due primarily to an influence of the seat belt on subunit
interaction.
These observations have important implications for efforts to identify
residues responsible for high affinity glycoprotein-hormone receptor
interactions. Because there is no crystal structure of any glycoprotein
hormone-receptor complex, most efforts to decipher portions of the
hormone that might interact with the receptor are based on monitoring
changes in receptor binding or function following chemical or enzymatic
modification (7), site-directed mutagenesis (13-19), chemical
cross-linking (36-38), or competition with hormone fragments (8-12).
Due to the unusually high proportion of residues in the subunit
interface (39), many mutations distant from the receptor contact sites
may influence subunit interaction and thereby alter receptor binding
and/or signal transduction. This would account for the observations
noted earlier that some parts of the hormones previously suggested to
make important receptor contacts appear to be exposed in the
hormone-receptor complex or can be deleted without disrupting hormone
activity. The influence of small changes in gonadotropin conformation
on receptor binding is readily explained by the model in which the
hormone binds to the concave surface of a horseshoe-shaped receptor
extracellular domain and contacts it at two distinct sites (1). A small
change in the conformation of a heterodimer that interferes with either of these contacts would be expected to reduce binding in a fashion similar to that observed between the interaction between the bovine -subunit and the bLH seat belt.
We thank the following individuals for reagents used in these studies: Drs. Robert Canfield and Steven Birken for purified urinary hCG and antibodies B105, B109, and B201; Dr. John Pierce for purified bLH; Drs. Glenn Armstrong and Robert Wolfert for antibodies A113 and B112; Dr. Janet Roser for the universal anti-lutropin antibody; and Dr. Aaron Hsueh for the hLHR cDNA.
While this manuscript was in review,
Szkudlinski et al. (40) reported that the presence of bovine
-subunit residues in loop 1 increased the potency of hCG similar to
what we report in Table I. However, they reached a different
conclusion, namely that these residues contact the receptor as
predicted by the model of Jiang et al. (2).