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Characterization of Lactogen Receptor-binding Site 1 of
Human Prolactin*
(Received for publication, January 16, 1996, and in revised form, March 18, 1996)
Sandrina Kinet‡, Vincent Goffin§, Ve´ ronique Mainfroid, and Joseph A. Martial¶
From the Laboratory of Molecular Biology and Genetic Engineering, Alle´e du 6 Aouˆ t, University of Lie`ge,
B-4000 Sart-Tilman, Belgium
Prolactin (PRL) binds to two molecules of PRL recep-
tor (PRLR) through two regions referred to as binding
sites 1 and 2. Although binding site 1 has been generally
assigned to the pocket delimited by helix 1, helix 4, and
the second half of loop 1, the residues involved in recep-
tor binding have not yet all been precisely identified. In
an earlier alanine-scanning mutational study, we iden-
tified three major binding determinants in loop 1 of
human PRL (hPRL) (Goffin, V., Norman, M. & Martial, J.
A. (1992) Mol. Endocrinol. 6, 1381–1392). Here we focus
on the two other regions that form binding site 1,
namely helices 1 and 4. Putative binding residues, se-
lected on the basis of a three-dimensional model of
hPRL constructed in this laboratory, were mutated to
alanine, and recombinant hPRL mutants produced in
Escherichia coli were tested for their ability to bind to
the PRLR and to stimulate Nb2 cell proliferation. We
thus identified nine single mutations (three in helix 1
and six in helix 4) whose effect was to reduce both bind-
ing and mitogenic activity by more than half as com-
pared with wild-type hPRL, indicating the functional
involvement of the corresponding residues. Adding
these to the three binding determinants identified in
loop 1, we now propose a complete picture of PRLR-
binding site 1 of hPRL. As we earlier hypothesized, the
binding site 1 determinants of hPRL differ from those of
human growth hormone, a hPRL homolog.
Prolactin (PRL)
1
and growth hormone (GH) are homologous
hormones primarily secreted by the pituitary gland in all ver-
tebrates (for reviews, see Refs. 1 and 2). PRL is involved in a
wide variety of biological functions, mainly related to reproduc-
tion, lactation, osmoregulation, and immunomodulation (re-
viewed in Ref. 3), while GH is involved primarily in growth and
morphogenesis (4). The multiple bioactivities of these hor-
mones are mediated by homologous membrane receptors, the
prolactin or lactogen receptor (PRLR) and the growth hormone
or somatogen receptor (GHR) (for reviews, see Refs. 5 and 6).
Both receptors belong to class I of the newly described cytokine
receptor superfamily (7–9). These receptors are all activated by
clustering of two or more membrane subunits (for reviews, see
Refs. 10 –13). On the one hand, receptor activation can result
from the hetero-oligomerization of different subunits, such as a
ligand-specific binding subunit (the
a
-chain) and a common
signal transducer subunit (the
b
-chain). On the other hand,
activation of some receptors can also result from the ho-
modimerization of two identical binding components. This has
been reported for the receptors of erythropoietin (14, 15), gran-
ulocyte colony-stimulating factor (16), GH (17, 18), and PRL
(19 –22).
The mechanism of activation of the human (h) GHR by hGH
has been extensively studied, and a sequential dimerization
model was proposed by Wells and co-workers in 1992 (17).
According to their model of activation, the hormone first binds
to its receptor through a set of amino acids forming the so-
called binding site 1. The complex composed of one molecule of
receptor and one molecule of hormone (H1zR1) remains inactive
until it associates with a second (and identical) receptor mole-
cule to yield an active H1zR2 complex. Receptor clustering leads
to interactions between both receptors as well as between the
hormone and the second receptor molecule (18). Binding of the
two receptor molecules is thus sequential, and the set of amino
acids of hGH interacting with the second hGHR molecule is
called binding site 2. Interestingly, a sequential two-site model
has also been hypothesized for interleukin-4, another four-
helix bundle cytokine (25).
Mutational (23, 24) and crystallographic (18) studies of hGH
have led to the identification of the amino acids belonging to
both binding sites. Binding site 1 of hGH involves residues of
helices 1 and 4 and of the second half of the long loop (loop 1)
joining helices 1 and 2. On the other hand, binding site 2 is
formed by residues belonging to the facing sides of helices 1 and
3 and a few residues in the small N-terminal loop. Due to the
numerous structural and functional similarities between the
PRL-PRLR and GH-GHR systems, we hypothesized earlier
that the sequential receptor dimerization model described for
the hGHR might also apply to activation of the PRLR (21, 26,
27). In agreement with this assumption, we showed that steric
hindrance introduced in the helix 1/helix 4/loop 1 pocket (bind-
ing site 1) (28) or in the helix 1-helix 3 interface (binding site 2)
of hPRL (21) is detrimental to activity. While these studies
clearly indicate the general location of both binding sites on
hPRL, not all residues involved in receptor binding have been
identified. With respect to binding site 1, segment 58 –74 (loop
1) has been characterized through systematic alanine-scanning
mutagenesis (26). To the best of our knowledge, however, no
systematic mutational study has yet focused on helices 1 and 4,
so these helical segments, strongly suspected of containing
several residues critical for tight receptor binding, remain es-
sentially uncharacterized. To date, Arg-177 is the only amino
* This work was supported in part by Grants PAI P3-044 and PAI
P3-042 from the Services Fe´de´raux des Affaires Scientifiques, Tech-
niques, et Culturelles. The costs of publication of this article were
defrayed in part by the payment of page charges. This article must
therefore be hereby marked “advertisement” in accordance with 18
U.S.C. Section 1734 solely to indicate this fact.
‡ Recipient of a fellowship from the Fonds pour la Formation a`la
Recherche dans l’Industrie et dans l’Agriculture.
§ Recipient of a fellowship from the European Communities Biotech-
nology Program. Present address: INSERM Unit 344, Molecular Endo-
crinology, 156, rue de Vaugirard, 75730 Paris Cedex 15, France.
¶To whom correspondence should be addressed. Tel.: 32-41-66-33-71;
Fax: 32-41-66-29-68.
1
The abbreviations used are: PRL, prolactin (prefixes “h” and “b”
indicate human and bovine, respectively); PRLR, PRL receptor;
hPRLbp, human PRL-binding protein; GH, growth hormone; GHR, GH
receptor; hGHbp, human GH-binding protein.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 271, No. 24, Issue of June 14, pp. 14353–14360, 1996
© 1996 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
14353
This is an open access article under the CC BY license.
acid within these helical segments to have been unambiguously
identified as very important for the mitogenic activity of bovine
PRL (bPRL) toward Nb2 cells (29).
Growth hormones, and presumably PRL, are composed of
four
a
-helices and adopt the “four-helix bundle” fold (18, 27, 30,
31). In these proteins, the hydrophobic faces of amphiphilic
helices form the hydrophobic core (18, 27, 30). Therefore, mu-
tational analysis within
a
-helices must be conducted with cau-
tion since any mutation affecting the hydrophobic core can
alter the global folding pattern, as reported for some bPRL
mutants (32). To date, no crystallographic structure has been
reported for PRL; this has prevented any structure-based mu-
tational study. Therefore, we have recently developed a three-
dimensional model of hPRL (27), constructed on the basis of the
crystallographic coordinates of porcine GH, the first elucidated
structure for a protein of the PRL/GH family (30). On the basis
of these data, we selected 7 residues in helix 1 and 10 residues
in helix 4 whose side chain orientations were compatible with
an involvement in the pocket of binding site 1 (see Fig. 1).
Although meeting this criterion, Arg-177 was not considered
again in the present study since its importance has been dem-
onstrated by others (29). The 16 remaining amino acids were
individually mutated to alanine, and the effect of each muta-
tion was examined by measuring the binding and mitogenic
activity of the hormone mutants. In agreement with our hy-
pothesis, both helical regions contain several residues that are
required for the hormone’s biological potency. Linking the pres-
ent study with our previous analysis of loop 1 (26) and with
structural data now available for hPRL (27), we can provide a
complete picture of receptor-binding site 1 of hPRL.
EXPERIMENTAL PROCEDURES
Materials
Restriction enzymes and DNA ligase were purchased from Boeh-
ringer Mannheim (Mannheim, Germany), Amersham International
(Buckinghamshire, United Kingdom), Life Technologies, Inc., and Eu-
rogentec (Seraing, Belgium). IODO-GEN was purchased from Sigma,
and carrier-free Na
125
I was obtained from Amersham International.
Ampholytes (pH range of 5–7) and pI protein markers were from Phar-
macia (Uppsala). Oligonucleotides were from Eurogentec. Culture me-
dia and sera were purchased from Life Technologies, Inc.
Methods
Oligonucleotide-directed Mutagenesis
All mutated hPRL cDNAs (33) were constructed as previously re-
ported (21, 26) by the oligonucleotide-directed mutagenesis method of
Sayers et al. (34). The vector used was single-stranded M13. We used
the oligonucleotide-directed mutagenesis system of Boehringer Mann-
heim and strictly followed the manufacturer’s instructions. Clones con-
taining the expected mutation were identified by DNA sequencing; the
mutated cDNAs were digested with HindIII and NdeI (helix 1 mutants)
or with HindIII and BglII (helix 4 mutants); and isolated fragments
(661 and 222 base pairs, respectively) were reinserted into the pT7L
expression vector (35). The sequences of the mutated oligonucleotides
are as follows (59339-noncoding strands; mutated codons are under-
lined). For helix 1: R16A, 59-AAA CAG GTC TGC AAG GGT CAC-39;
V23A, 59-GGA CAG GAC GGC GGC GCG GTC-39; S26A, 59-GAT G-
TA GTG GGC CAG GAC GAC-39; H30A, 59-GGA GAG GTT AGC GAT-
GTA GTG-39; S34A, 59-GAA CAT TTC TGC GGA GAG GTT-39; F37A,
59-GAA TTC GCT GGC CAT TTC TGA-39; and S38A, 59-ATC GAA T-
TC GGC GAA CAT TTC-39. For helix 4: Y169A, 59-GAG CAG GTT AG-
C ATA AGC AGA-39; H173A, 59-GCG TAG GCA GGC GAG CAG GTT-
39; R176A, 59-TGA ATC CCT GGC TAG GCA GTG-39; H180A, 59-
GTC GAT TTT AGC TGA ATC CCT-39; K181A, 59-ATT GTC GAT-
TGC ATG TGA ATC-39; K181E, 59-ATT GTC GAT TTC ATG TGA-
ATC-39; N184A, 59-CTT GAG ATA AGC GTC GAT TTT-39; Y185A, 59-
GAG CTT GAG AGC ATT GTC GAT-39; L188A, 59-GCA CTT GAG-
GGC CTT GAG ATA-39; and R192A, 59-GTG GAT GAT TGC GCA-
CTT CAG-39.
Production and Purification of Proteins
Recombinant native hPRL and hPRL mutants were overproduced in
500-ml cultures of Escherichia coli BL21(DE3) cells and purified as
described previously (35). Purity was assessed by SDS-polyacrylamide
gel electrophoresis according to Laemmli (37).
Quantification of Proteins
Proteins were quantified physically by weighing the lyophilized pow-
der on a precision balance (Electrobalance, Cahn 26) and chemically by
the Bradford method (36). The disparity between weight and chemical
measurements never exceeded 20%.
Isoelectrofocusing
The isoelectric point of the hPRL mutants was estimated by isoelec-
trofocusing (pH range of 5–7) as described previously (21).
Structural Analyses
Circular Dichroism—Lyophilized proteins were resuspended in 50
mMNH
4
HCO
3
, pH 8, at a concentration ranging from 300 to 500
m
g/ml.
Spectra were recorded with a CD6 dichrograph (Instruments SA-JO-
BIN YVON, Longjumeau, France) linked to a personal computer for
data recording and analysis (dichrograph software, Instruments SA-
JOBIN YVON). For each protein, four spectra recorded between 195
and 260 nm were averaged. Measurements were performed in a 0.1-cm
path length quartz cell. The helicity was calculated at 222 nm according
to Chen et al. (38).
Apparent Molecular Mass—The apparent molecular masses of all the
hPRL mutants were measured by high pressure liquid-gel filtration
chromatography. 100-
m
l samples (500
m
g/ml) were loaded on a Superose
12 molecular sieve (Pharmacia) equilibrated in 20 mMTris-HCl, pH 8,
100 mMNaCl. Elution was carried out in the same buffer at a constant
flow rate of 0.5 ml/min, and protein elution was monitored at 280 nm.
The column was calibrated with several molecular mass markers: dex-
tran blue (void volume), bovine serum albumin dimers (136 kDa), bo-
vine serum albumin (68 kDa), ovalbumin (45 kDa), carbonic anhydrase
(30 kDa), and myoglobin (17.5 kDa).
Nb2 Cell Culture and in Vitro Bioassay
The bioactivity of the hPRL mutants was estimated by their ability to
stimulate growth of lactogen-dependent Nb2 lymphoma cells (39). The
procedure used (40) has been previously detailed (21, 26). Briefly, cells
were cultured in Fisher’s medium containing 10% horse serum and 10%
fetal calf serum. 24 h before the bioassay, the cells were synchronized in
culture medium containing only 1% fetal calf serum. Bioassays were
performed in fetal calf serum-free Fisher’s medium (starvation medi-
um). Various amounts of hPRL samples diluted in starvation medium
(from 25 to 100
m
l) were added to 2.5 ml of cells (1–2 310
5
cells/ml)
plated in 6-well Falcon plates. Nb2 cells were counted with a Coulter
counter (Coulter Electronics Ltd., Harpenden, Hertfordshire, United
Kingdom) after 3 days. Two to four experiments were performed in
duplicate for each mutant. The ED
50
value (amount of hormone needed
to achieve half-maximal cell growth) was calculated, and the relative
bioactivity of each mutant with respect to native hPRL was estimated
as the ratio of the native versus mutant ED
50
values.
Binding Experiments
Binding of hPRL mutants to the lactogen receptor was performed as
reported earlier (21, 26, 28). Briefly, homogenates from 3 310
6
Nb2
cells were incubated for 16 h at 25 °C with 30,000 –50,000 cpm
125
I-
hPRL in the presence of increasing amounts of unlabeled native hPRL
or hPRL analog (final reaction volume of 0.5 ml). The assay was termi-
nated by addition of 0.5 ml of ice-cold buffer (0.025 MTris-HCl, 0.01 M
MgCl
2
, pH 7.5) followed by centrifugation (5 min, 11,000 3g). The
supernatants were removed carefully, and the radioactivity of the pel-
lets was analyzed in a
g
-counter (Hybritech 002011B).
Each mutant was tested at least three times in duplicate. Specific
binding was calculated as the difference between radioactivity bound in
the absence (B
0
, maximal binding) and in the presence (nonspecific
binding) of 2
m
g of unlabeled native hPRL. In the different experiments,
nonspecific binding never exceeded 20% of maximal binding. Data are
presented as percentages of specific binding. Competition curves were
analyzed with the LIGAND PC program (41). The relative binding
affinity of each mutant was estimated as the ratio of the native versus
mutant IC
50
values.
Prolactin-binding Site 114354
RESULTS
Structure-based Design of the Mutational Study
Residues to be mutated were selected on the basis of a
three-dimensional model of hPRL constructed in our laboratory
(27), which is to date the only atomic structure available for
any PRL. Selection was based on two criteria. First, residues
had to be located on the exposed (hydrophilic) faces of helices 1
and 4. Second, only residues whose side chain orientations
were compatible with an involvement in the predicted binding
site 1 were selected. We thus did not consider residues such as
the helix 1 residues pointing toward the helix 1-helix 3 inter-
face, which forms binding site 2 (21). From this structure-based
prediction of binding determinants, a set of 17 residues was
selected: for helix 1, Arg-16, Val-23, Ser-26, His-30, Ser-34,
Phe-37, and Ser-38; and for helix 4, Tyr-169, His-173, Arg-176,
Arg-177, His-180, Lys-181, Asn-184, Tyr-185, Leu-188, and
Arg-192.
To assess the functional involvement of each residue, we
used the alanine-scanning approach since this strategy ap-
pears to be the most appropriate for identifying binding resi-
dues in both hGH and hPRL (24, 26, 42). Arg-177, previously
reported to be extremely important for bPRL mitogenic activity
(29), was not retested. To evaluate the cumulative effect of the
most effective mutations in helix 1, a triple mutant was con-
structed (V23A/H30A/F37A). Finally, Lys-181 was also mu-
tated to Glu in order to evaluate the effect of an opposite charge
at this position. The hPRL mutants are designated by a letter
referring to the mutated residue, followed by a number refer-
ring to its position, followed by a letter referring to the substi-
tute residue (i.e. R16A designates the analog in which Arg-16 is
replaced with Ala). The 16 preselected residues are shown in
Fig. 1.
Production and Purification Yields
The yield of overproduced protein was about the same for all
16 hPRL analogs as for native hPRL (6150 mg/liter) (35).
Routinely, ;30 mg of purified monomeric hPRL can be recov-
ered per liter of culture. Similar amounts of monomer were
recovered for all the hPRL mutants, which indicates a behavior
similar to that of native hPRL during renaturation.
Structural Characterization of hPRL Mutants
Since all residue changes reported in this study affect regu-
lar secondary structures (
a
-helices), each mutant was first
structurally characterized by circular dichroism and chroma-
tography on a molecular sieve to assess its proper folding. The
isoelectric point was also determined for each mutant.
Isoelectric Point—The major isoform of purified recombinant
hPRL exhibits a pI of 6.2 (21, 35). Introduction or removal of
charged residues alters the net protein charge. Accordingly, the
pI values of hPRL mutants R16A, R176A, K181A, K181E, and
R192A were 0.2– 0.3 units lower than normal (data not shown),
thus confirming at protein level the presence of the mutations.
Circular Dichroism—hPRL has an
a
-helix content of 45 6
5% (21, 26, 28) as determined by circular dichroism. In agree-
ment with the spectrum of native hPRL, all mutants produced
in this study displayed the typical curve of all
a
-proteins, with
two minima at 222 and 208 nm and a maximum at 195 nm. The
calculated helicities are reported in Table I. They are all in the
range of 45 65%, suggesting no significant alteration of the
overall secondary structure content.
Apparent Molecular Mass—The apparent molecular mass of
each mutant was estimated from its retention time on a high
pressure molecular sieve. The calculated apparent molecular
masses are reported in Table I. In agreement with the theoret-
ical molecular mass of hPRL (23 kDa), the estimated molecular
masses of all the mutants are in the range of 22 62 kDa; they
correlate with the data obtained by CD analysis.
FIG.1.Selection of putative binding determinants in helices 1
and 4. The three-dimensional model of hPRL (27) is shown with the
four helices and the second part of loop 1 (figure drawn by MOLSCRIPT
(50)). Putative binding determinants were selected on the basis of two
criteria. First, residues located on the exposed faces of the helices 1 and
4 were listed. Then, among this set of amino acids, we considered only
those whose side chain orientations were compatible with an involve-
ment in the binding site 1 pocket. The side chains of the 16 residues that
were studied by mutagenesis are colored in green (helix 1) and blue
(helix 4).
TABLE I
Structural analysis of hPRL mutants
The helical content of each alanine substitution mutant was meas-
ured by circular dichroism and calculated at 222 nm according to Chen
et al. (38). The apparent molecular mass of each mutant was estimated
by gel filtration as described under “Experimental Procedures.” The
calculated apparent molecular masses are presented.
hPRL mutants Helicity Apparent molecular mass
% kDa
Native hPRL 46.1 21.0
Helix 1
R16A 44.3 20.7
V23A 41.3 20.9
S26A 45.7 20.9
H30A 41.7 21.5
S34A 41.1 21.0
F37A 42.7 21.0
S38A 45.0 21.3
Helix 4
Y169A 41.9 20.0
H173A 46.7 22.2
R176A 45.1 23.6
H180A 40.5 21.1
K181A 50.6 22.2
K181E 46.6 23.6
N184A 40.4 21.1
Y185A 45.8 21.1
L188A 48.4 21.1
R192A 39.0 23.6
V23A/H30A/F37A 43.4 23.6
Prolactin-binding Site 1 14355
Since no significant alteration of the global structure was
detected by either procedure, we conclude that any alteration of
the biological properties (see below) reflects the functional in-
volvement of the mutated residue rather than an unexpected
effect of the mutation on protein folding.
Biological Analysis of hPRL Mutants
We have previously reported that recombinant native hPRL
stimulates Nb2 cells as efficiently as pituitary-purified hPRL,
with half-maximal growth at ;200 pg of hPRL/ml (ED
50
) (26).
Therefore, recombinant wild-type hPRL was used as a refer-
ence for estimating the bioactivity of all the hPRL mutants.
Nb2 cells contain ;12,000 PRL receptors/cell (43). As described
earlier (21, 26, 28), Nb2 cells were also used in binding assays
of hPRL mutants. Typical Nb2 cell proliferation and binding
curves are represented in Fig. 2. Binding and cell proliferation
data are summarized in Fig. 3.
When alanine was substituted for Val-23, His-30, or Phe-37
of helix 1, binding of hPRL to the Nb2 PRLR decreased signif-
icantly, with the affinity dropping to only 26 65, 30 610, or 21
66% of the reference value, respectively (Fig. 3A). The mito-
genic activity of these mutants toward Nb2 cells was reduced
accordingly (26 69% for V23A, 39 61% for H30A, and 44 65%
for F37A). A less marked affinity change was observed when
alanine replaced Arg-16 (101 619%), Ser-26 (48 615%),
Ser-34 (66 65%), or Ser-38 (52 612%); this tallies with the
mitogenic potency of these mutants (73 612% for R16A, 46 6
3% for S26A, 97 623% for S34A, and 94 67% for S38A) (Fig.
3A). These data suggest that Val-23, His-30, and Phe-37 are
binding determinants of hPRL. By comparison with mutations
in loop 1 (26) or in helix 4 (see below), the helix 1 mutations
have but a limited effect since the most effective among them,
F37A, results in a 5-fold reduction of binding. We thus con-
structed a hPRL mutant carrying the three most effective mu-
tations, namely V23A/H30A/F37A. Although this variant dis-
played lesser biological activity than any of the single mutants
(15 61% mitogenic activity), the effects of these mutations did
not appear to be additive, indicating a limited involvement of
helix 1 in binding site 1 of hPRL.
In helix 4, six mutations were found to significantly alter
binding: Tyr-169 (18 67%), His-173 (30 64%), Arg-176 (20 6
3%), His-180 (8 61%), Lys-181 (3.7 61.2%), and Tyr-185 (23 6
5%). Accordingly, the mitogenic activities of these mutants
were also markedly reduced: 9.1 62.4% for Y169A, 13 61.5%
for H173A, 5.2 60.5% for R176A, 6.2 62% for H180A, 1.3 6
0.2% for K181A, and 8.1 61.4% for Y185A (Fig. 3B). When
Asn-184, Leu-188, or Arg-192 was replaced with alanine, the
effect was weak (mitogenic activity ranged from 63 to 104%),
although binding of the L188A analog was more significantly
reduced (34 64%). Tyr-169, His-173, Arg-176, His-180, Lys-
181, and Tyr-185 were thus identified as binding determinants
within helix 4. To evaluate the importance of a positive charge
at position 181 (Lys-181 is the strongest binding determinant
of hPRL), we replaced this Lys residue with Glu. The biological
activity of the K181E mutant was ;10 times lower than that of
the K181A mutant, indicating that a positive charge is required
at this position.
DISCUSSION
Structure-based Prediction of Putative Binding Residues—A
few years ago, Luck et al. (29, 32, 44) reported the effects of
point mutations on the mitogenic activity of bPRL. Since no
structure was available for any PRL at that time, the residues
to be mutated were selected mainly on the basis of sequence
comparisons between members of the PRL/GH family. In many
cases, point mutations either proved ineffective (44) or were
assumed to affect biological properties solely as a result of
altered global protein folding (32). Consequently, only a very
few residues, such as Arg-177, could be clearly identified as
functionally required for bPRL bioactivity (29).
To circumvent the lack of a three-dimensional structure of
PRL, we have recently constructed a three-dimensional model
of hPRL (27) based on the crystallographic structure of porcine
GH, the first structure of a member of the PRL/GH family that
has been determined experimentally (30). Thanks to this
model, we were able to formulate hypotheses concerning the
interaction between PRL and its receptor, notably with regard
to the location of both binding sites and to the residues that
form them (27).
Analysis of sequence-structure-function relationships in
PRL led us to propose that binding site 1 involves the pocket
delimited by helix 1, helix 4, and loop 1 (26 –28). In agreement,
mutational analysis of loop 1 clearly demonstrated the involve-
ment of this region in bioactivity (26). To confirm our hypoth-
esis, we decided to further characterize binding site 1 by scan-
ning the two remaining regions, namely helices 1 and 4. As
reported earlier, helix 1 is involved in both binding sites since
residues facing helix 3 are involved in binding site 2, whereas
residues facing helix 4 are predicted to be part of binding site 1
FIG.2. A, competition curves for the displacement of
125
I-labeled
native hPRL by unlabeled native hormone and the K181A mutant. The
figure represents a typical experiment, in which nonspecific binding
was 22% of B
0
. The curves presented in this figure are taken from the
same experiment and are presented as percentages of specific binding.
Each point is the average of duplicate measurements; maximal dispar-
ity between duplicate values is 7% of specific binding. All the hPRL
mutants were tested in at least three independent experiments. B,
mitogenic activity toward Nb2 cells of native hPRL and the K181A
mutant. The relative mitogenic potency of each mutant was estimated
as the amount of native versus mutant hPRL required to produce
half-maximal proliferation of Nb2 cells (ED
50
). Each mutant was tested
at least three times in duplicate. A typical experiment is presented.
Prolactin-binding Site 114356
(21, 27). Therefore, we chose in this study to mutate only those
residues that point toward the binding site 1 pocket. Helix 4, on
the other hand, is at the center of binding site 1; there were
more residues in this region (10 residues) to be investigated in
order to assess their involvement in the protein’s biological
properties.
Structural and Biological Analysis of hPRL Mutants—Be-
cause all the selected residues are predicted to be located on
exposed faces of helices, they should not affect protein folding.
Accordingly, our structural analyses of the various mutants
failed to detect any significant alteration of the global protein
conformation. Although CD analysis and estimation of the ap-
parent molecular masses are probably not sufficiently sensitive
methods for detecting subtle and local structural changes, it
should be stressed that by combining these methods, we have
previously been able to identify misfolded hPRL mutants and
to eliminate them from our study (21, 26). Moreover, we know
of no reported structurally disruptive alanine substitution of
any residue located on an exposed face of PRL or GH, with the
sole exception of the cysteines involved in disulfide bonds (21,
24, 26, 42).
This study confirms the assumed involvement of both helices
1 and 4 in binding site 1 of hPRL. As previously observed for
hGH (Refs. 24, 45, and 46; for review, see Ref. 47), binding site
1 is centered on helix 4 since this segment contains not only the
greatest number of binding determinants, but also those whose
replacement with alanine is the most detrimental to both bind-
ing and mitogenic activity. The most effective mutations in
helix 1 (V23A and F37A) cause only a 5-fold reduction of the
biological activity, compared with the 100-fold decrease in
FIG.3.Bioactivity and affinity for the PRLR of the different hPRL mutants. The mitogenic activity and the affinity for the Nb2 receptor
were determined as described under “Experimental Procedures” and shown in Fig. 2 (Aand B). The values corresponding to wild-type hPRL are
arbitrary set at 100%. Aand Bshow mutations performed in helices 1 and 4, respectively. In each panel, the hPRL mutants are shown in three
groups: first, those that have not significantly affected biological properties; second, those that have slightly decreased potency, although mutated
residues cannot be considered as strong binding determinants; and third, mutants for which both binding and mitogenic activity are significantly
altered, indicating that mutated residues are involved in receptor binding.
Prolactin-binding Site 1 14357
binding when Lys-181 is replaced with alanine. Even when the
three most effective mutations in helix 1 are combined (V23A,
H30A, and F37A), the mitogenic activity is diminished only
6-fold; this suggests a limited involvement of this helical seg-
ment in the interaction with the receptor. Considering that a
.2-fold reduction of both binding and mitogenic activity re-
flects a significant functional involvement of an amino acid, we
have identified 12 residues (referred to as “binding determi-
nants”) in receptor-binding site 1 of hPRL (Figs. 4 and 5):
Val-23, His-30, and Phe-37 in helix 1 (this work); His-59, Pro-
66, and Lys-69 in loop 1 (26); and Tyr-169, His-173, Arg-176,
His-180, Lys-181, and Tyr-185 in helix 4 (this work). Further-
more, Luck et al. (29), using the same bioassay, found that
mutating Arg-177 in bPRL drastically alters the bioactivity of
the hormone (reducing it to 1.1% of the reference value). Since
an Arg residue is found at this position in all PRLs, it is most
likely that this residue is also a major binding determinant of
hPRL.
Some hPRL mutants, such as S26A, S34A, S38A, and L188A,
display lesser binding, but normal to slightly altered mitogenic
activity. This might reflect the “spare receptor” phenomenon,
in which maximal biological activity occurs at submaximal
receptor occupancy. In the Nb2 system, maximal cell growth
has been reported to occur at 35% of maximal binding (42).
Alanine substitution of Lys-187 is reported to halve the mito-
genic activity of bPRL, but since this position remained almost
insensitive to other mutations (replacement with Leu, Asn, or
Arg) (29), it seems unlikely that Lys-187 is a major determi-
nant of receptor binding. Finally, Luck et al. (29, 32) reported
that mutation of Arg-21 or Tyr-28 to various amino acids re-
duces the effect on Nb2 cells by a factor of 2–5. Our three-
dimensional structural model of hPRL (27) suggests that the
side chains of both these residues point outside binding site 1;
we anticipate that they belong to binding site 2, which involves
the facing sides of helices 1 and 3 (21, 27). For these various
reasons, we do not consider any of the residues just mentioned
to belong to binding site 1. Fig. 4 shows the spatial distribution
of the 13 determinants of binding site 1 of hPRL. Although all
potential binding determinants were selected on the basis of
our three-dimensional model, we cannot rule out the involve-
ment of other residues not tested in this study, although it does
appear unlikely.
Binding site 1 of hPRL contains both hydrophobic (Phe-37,
Tyr-169, and Tyr-185) and hydrophilic (Lys-69, Arg-176, Arg-
177, and Lys-181) residues. The importance of charged resi-
dues in hormone-receptor binding has been emphasized in a
comprehensive energetic study of cytokine-receptor interac-
tions, showing complementary electrostatic potentials on the
binding surfaces of the two interacting proteins (31). In the
case of hPRL, we have demonstrated the critical role of charged
residues by replacing Lys-181, the strongest binding determi-
nant of hPRL, with a negatively charged glutamic acid residue.
The K181E mutant was biologically 10 times less active than
the K181A mutant (the former displayed only 0.3% of the
activity of wild-type hPRL), suggesting that introducing a neg-
ative charge where a positive one is required is detrimental to
efficient receptor docking. Luck et al. (29) accordingly reported
FIG.4. Distribution of binding determinants on hPRL. The
three-dimensional model of hPRL (27) is shown with binding site 1
facing the viewer (see Fig. 1 for details). The side chains of the residues
identified as strong determinants of hPRL binding to the PRLR are
colored in green (helix 1), blue (helix 4), and red (loop 1). The side chain
of Arg-177 (29) is also represented.
FIG.5.Distribution of the determinants on hPRL for binding
to the PRLR and on hGH for binding to the PRLR and GHR.
Amino acid sequences forming binding sites 1 of hPRL and hGH are
aligned. Numbers above and below the sequences correspond to hPRL
and hGH, respectively. The first line represents the binding determi-
nants on hPRL for binding to the PRLR (Ref. 26 and this study).
Identification of Arg-177 as an important residue is from Luck et al.
(29). The second line refers to binding determinants on hGH for binding
to the PRLR, identified by Cunningham and Wells (42) using the
hPRLbp; and the third line represents the determinants on hGH for
binding to the hGHR, identified by means of the hGHbp (24).
Prolactin-binding Site 114358
that replacing Arg-177 with an alanine or a glutamate also
decreases bPRL bioactivity to 1.1 and 0.3% of the reference
value, respectively. Finally, Clarckson and Wells (46) proposed,
on the basis of an energy analysis of the hGH-hGHbp interface,
that electrostatic interactions might contribute to determining
the binding specificity; this is also possible in the case of PRL
(see below).
Comparison of Binding Sites 1 of hPRL and hGH—It is
usually assumed that homologous proteins exert a common
activity through identical or very similar mechanisms. In the
present context, hPRL and hGH might thus be expected to bind
by the same mechanism to the lactogen receptor. The available
data indicate otherwise. First, whereas tight binding of hGH to
the hPRLbp requires mediation by a zinc ion, hPRL binding to
the hPRLbp is zinc-independent (48). Second, as shown in Fig.
5, hGH and hPRL clearly appear to bind to the PRLR via
different sets of amino acids (Refs. 26 and 42 and this work).
For example, Lys-69, Tyr-169, and His-180 play a major role in
hPRL binding, whereas their hGH counterparts (Arg-64, Tyr-
160, and Asp171) can be mutated without significantly affect-
ing binding to the hPRLbp (Table II). In contrast, Ile-58, Ser-
62, Glu-65, and Arg-183 are required in hGH, while their hPRL
counterparts (Leu-63, Glu-67, Glu-70, and Arg-192) can be
mutated without compromising receptor binding. Even when
topologically equivalent residues are binding determinants for
both hormones, they do not appear to be equally important
(Table II). One of the rare similarities between binding of hGH
and hPRL to the PRLR is the involvement of two basic resi-
dues: Arg-176 and Arg-177 in hPRL and their topological equiv-
alents (Arg-167 and Lys-168) in hGH (see Fig. 5). In hGH, both
residues are considered specificity determinants, meaning that
they are crucial to hGH binding to the PRLR, but not to its
binding to the GHR (42, 47, 49). As Arg-176 and Arg-177 in
hPRL are also among the strongest binding determinants (Ta-
ble II), one would expect these residues to be a characteristic
requirement for PRLR binding, in agreement with the proposed
role of charged residues in determining the binding specificity
(46). The disruptive effect of mutating Arg-167 (in hGH), how-
ever, has been partly linked to an indirect alteration of the
zinc-binding pocket conformation (Table II) (42), so the role of
this arginine is likely to differ from that of Arg-176 (in hPRL)
since receptor binding to PRL is zinc-independent (48). The
same applies to His-30 in hPRL and its counterpart (His-21) in
hGH since the former is a real binding determinant in hPRL,
while the latter is involved only in zinc chelation in hGH
(42, 49).
Among the features common to the interaction of hGH with
the hGHbp (18) and with the hPRLbp (49) are the major con-
tacts involving two Trp residues found in both receptors (Trp-
104 and Trp-169 in the hGHR and Trp-74 and Trp-139 in the
hPRLR (Refs. 18 and 49; for a review, see Ref. 47). In the
hGH-hGHR interaction, these Trp residues are buried in a
hydrophobic environment formed by the alkyl portions of Lys-
172 and Thr-175 surrounded by Asp-171 and Phe-176 (46), in
keeping with earlier findings that these residues are among
those accounting for the majority of the free energy of the
hormone-receptor interaction (45). In hPRL, the topologically
equivalent amino acids are Lys-181, Asn-184, His-180, and
Tyr-185. With the exception of Asn-184, these residues are also
strong binding determinants in hPRL (Table II), suggesting
analogous interactions of helix 4 amino acids with Trp-74 and
Trp-139 of the PRLR. As these two Trp residues are not found
in the other cytokine receptors, it is likely that the network of
hormone-receptor contacts involving these Trp residues is a
characteristic feature of the interactions between PRL/GH hor-
mones and their receptors.
Altogether, this study thus confirms our earlier hypothesis
that hPRL and hGH bind to the PRLR via mechanisms with
different requirements at the molecular/residue level (26, 27).
In contrast, structural analysis of the binding sites (27) led us
to propose that there is a closer parallel between the mecha-
nisms by which hPRL and hGH bind to their respective recep-
tors. These interactions do indeed share some common fea-
tures, such as the non-involvement of zinc ions (48) and a
similar distribution of strong binding determinants (for exam-
ple, His-59, Lys-69, His-180, and Tyr-185 in hPRL and their
respective equivalents (Phe-54, Arg-64, Asp-171, and Phe-176)
in hGH) (see Table II).
This work completes our picture of lactogen receptor-binding
site 1 of hPRL. Combined with our previous work on binding
site 2 (21), it provides a global view of the interaction between
this important lactogenic hormone and its receptor.
TABLE II
Relative importance of binding determinants identified in
hGH and hPRL
The binding affinities of mutants in which the binding determinants
were mutated to alanine are expressed as percentages of the affinity of
the wild-type hormone. The first and last columns correspond to the
aligned sequences of hGH and hPRL, respectively (see Fig. 5). All
binding determinants for the three interactions hGH-hGHbp (second
column; data from Cunningham and Wells (24)), hGH-hPRLbp (third
column; data from Ref. 42), and hPRL-PRLR Nb2 (fourth column; data
from Goffin et al. (26) and this study) are indicated. When no value is
available for hPRL, results obtained for the bPRL mutants in the Nb2
proliferation assay are indicated and marked with an asterisk (29, 32,
44). The hGH-hPRLbp interaction is mediated by zinc chelation. In the
third column, values in parentheses indicate the effects of mutations in
the presence of EDTA, i.e. in the absence of zinc chelation (42). It
appears that the effects of mutating His-18, His-21, Glu-174, and, to a
lesser extent, Glu-167 are due mainly to alteration of zinc binding
and/or pocket shape. Although binding of the L188A hPRL mutant is
reduced by 33% (fourth column), this residue is not considered a strong
determinant since its mitogenic activity was reduced to a much lesser
extent (see text).
hGH hGH-hGHbp hGH-hPRLbp hPRL-rPRLR hPRL
helix 1
Phe-10 17 100 Phe-F19
Met-14 45 26 Val-23
Ala-17 48 Ser-26
His-18 62 <1(72) 68* His-27
His-21 303 1(135) 30 His-30
Phe-25 159 14 66 Ser-34
Tyr-28 21 Phe-37
Loop 1
Phe-54 23 71 37 His-59
Glu-56 24 125 127 Ser-61
Ile-58 6668 Leu-63
Phe-61 30 Phe-66
Ser-62 36 9 94 Glu-67
Asn-63 30 23 87* Asp-68
Arg-64 555 8Lys-69
Glu-65 170 40 110 Glu-70
Glu-66 48 90 150 Gln-71
Gln-68 19 83 130 Gln-73
Lys-70 41 67 Lys-75
Ser-71 50 36 Asn-76
Helix 4
Tyr-160 71 18 Tyr-169
Tyr-164 28 48 30 His-173
Arg-167 133 <0.2 (3) 20 Arg-176
Lys-168 91 61*Arg-177
Asp-171 14 91 8His-180
Lys-172 7<0.5 (3) 3 Lys-181
Glu-174 455 <0.3 (77) Asp-183
Thr-175 64369 Asn-184
Phe-176 6423Tyr-185
Arg-178 21446* Lys-187
Ile-179 37 55 33 Leu-188
Arg-183 48 38 121 Arg-192
Val-185 22 62 Ile-194
Prolactin-binding Site 1 14359
Acknowledgments—We thank Drs. C. Houssier and A. Taquet for
help with CD analysis. We thank Drs. R. Matagne and C. Grandfils for
lending the Coulter counter apparatus and Dr. J. Smal for lending the
g
-counter. We appreciate the critical reading of the manuscript by Dr.
P. A. Kelly. We also thank M. Lion for technical assistance and G.
Guillaume for constructing the mutated DNA for K181A and K181E.
We are grateful to Drs. K. Hoffmann and J. Lamotte for help with the
three-dimensional model figures.
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Prolactin-binding Site 114360
