(Received for publication, June 5, 1995; and in revised form, December 13, 1995)
From the
Five recombinant analogues of bovine placental lactogen (bPL) ((bPL(S184H), bPL(S187A), bPL(S187F), bPL(T188F), bPL(T188F,I190F)) were prepared, expressed in Escherichia coli, and purified to homogeneity. Circular dichroism analysis revealed no or minor structural changes, except in bPL(T188F,I190F). Binding and biological activities of bPL(T188F,I190F) were almost completely abolished, whereas bPL analogues mutated at position 187 retained their full activity. Point mutation T188F resulted in selective modification; binding to somatogenic receptors, their extracellular domains (ECDs), and to bPLR in the endometrium as well as somatogenic receptor-mediated biological activities were reduced or abolished, whereas binding to lactogenic receptors, their ECDs, and subsequent biological activity was fully or almost fully retained. This selective modification most likely results from a steric hindrance induced by a bulky Phe-188 chain of bPL which interacts with the Arg-43 of the human or Leu-43 of the non-human GHRs. Point mutation S184H abolished the interaction with hGHR, most likely due to the unfavorable charge-charge interaction, possibly accompanied by steric hindrance between Arg-43 of the receptor and the newly introduced His-184 and possible interference with the putative interaction between the alkyl portion of Thr-188 and Lys-185 of bPL with Trp-104 of hGHR. In contrast, bPL(S184H) retained its capacity to interact with nonhuman GHRs. Decrease in the biological activity of bPL(S184H) was also observed in two lactogenic receptor-mediated bioassays most likely due to the elimination of the intermolecular hydrogen bond of Ser-184 with a side chain of Tyr-127, which appears in all lactogenic receptors.
Bovine placental lactogen (bPL) ()has been purified
from term placental homogenates (1) and from isolated secretory
granules obtained from binucleate cells of fetal cotyledon (2) . The native 31 to 33 kDa bPL has at least five isoelectric
variants which are in part due to heterogeneity of the attached
oligosaccharides, and to as yet unidentified modifications. The gene
for bPL has been cloned and expressed with high efficiency in Escherichia coli and the recombinant bPL has been purified to
homogeneity(3) . The predicted mature bPL has 200 residues and
the primary sequence exhibits 50% and 23% homology to bovine prolactin
(bPRL) and growth hormone (bGH), respectively(3) . In
comparison with bGH and hGH, bPL has 12-13 additional amino acids
at the N-terminal portion of the molecule and, in common with mammalian
PRLs, it has a third disulfide bond located in this N-terminal region.
Deglycosylation of native bPL had no effect on PRL-like mitogenic
activity in an Nb
lymphoma cell proliferation assay in
which bPL was equally potent to human (h)GH, bPRL, and ovine (o)PRL,
and exhibited only slightly reduced binding to bGH
receptors(4) . Recombinant bPL was also equally potent to hGH
or oGH in somatogenic receptor-mediated 3T3-L1 or 3T3-F442A
preadipocyte bioassays(5, 6) . However, in a
homologous lactogenic receptor-mediated bioassay using bovine mammary
gland explants, we found that the native bPL is also as potent as hGH
or bPRL(7) . Binding experiments to various microsomal
fractions revealed that bPL binds with high affinity to prolactin
receptors and to somatogenic
receptors(2, 4, 5) . In addition, we have
documented that bPL binds also to unique receptors in bovine
endometrium(8) . Thus bPL is an unique hormone which may
transduce its activity through three different receptors.
Structure-function relationship studies of bPL have been conducted
in our laboratory by successive truncation of its N-terminal
domain(9, 10) . Assuming structural similarity to
porcine GH (11) these mutations were aimed to remove amino
acids beyond or at the beginning of the putative first -helix.
Results of these studies (9, 10) indicated that
similarly to
hGH(12, 13, 14, 15, 16) ,
bPL can be selectively modified so that particular biological
activities are changed while others remain relatively unaffected.
Information obtained from mutated analogues of hGH indicates that
not only the N-terminal domain(12, 13, 17) ,
but also the C-terminal domain and the non-helical sequence intervening
between the first and the second -helix participate in receptor
binding(18) . Publications concerning mutation of this domain
in hGH(18, 19, 20) , implicate its importance
in the binding to somatogenic receptors as well to lactogenic
receptors(21) . In the present work several mutations in an
analogous region of bPL were incorporated. The rationale behind
creating these mutations was based mainly on the findings using alanine
scanning mutagenesis of hGH (18, 19, 20) and
the structural analysis of hGH:hGHR-ECD complex(22) . One of
the most dramatic changes was obtained with the E174A mutation of hGH
which resulted in a 4-fold increase in affinity toward somatogenic
receptor with a simultaneous 356-fold decrease in affinity for
lactogenic receptor, and the D171A mutation which acted in the opposite
way. Asp-171 and Trp-175 of hGH have been found to participate in the
formation of hydrogen bonds with Arg-43 of hGHR-ECD, stabilizing the
complex(22) . These mutations are found in a portion of the
sequence DKVET(171-175). The corresponding amino acids of bPL are
most likely SKIST(184-188). To evaluate the importance of these
amino acids in bPL action, we prepared five analogues with changes in
these or neighboring residues. In most cases we mutated the respective
residues to phenylalanine in order to increase the hydrophobicity and
to introduce a large side chain. In one case (position 184), we
preferred mutation to histidine since this amino acid occupies the
corresponding position in all nonhuman GHs(23) .
Figure 1:
Binding of I-hGH to
hGHR-ECD or bPRLR-ECD and binding of
I-bPL to rbPRLR-ECD
or rPRLR-ECD. Competitive binding was determined by simultaneous
addition of bPL (
), bPL(S184H) (
), bPL(S187A) (
),
bPL(S187F) (
), bPL(T188F) (
), and bPL(T188F,I190F) (
).
Nonspecific binding was obtained by the addition of 2000 ng/tube hGH or
bPL, respectively. The actual specific binding was 22, 12, 27.6, and
17%, respectively.
Figure 2:
Binding of I-hGH to
homogenates of Nb
lymphoma cells or intact IM-9 cells,
binding of
I-bGH to bovine liver microsomal fraction, and
binding of
I-bPL to bovine endometrial microsomal
fraction. Competitive binding was determined by simultaneous addition
of bPL (
), bPL(S184H) (
), bPL(S187A) (
), bPL(S187F)
(
), bPL(T188F) (
), and bPL(T188F,I190F) (
).
Nonspecific binding was obtained by the addition of 2000 ng/tube hGH or
bGH or bPL, respectively. The actual specific binding was 31, 5.5, 5,
and 7%, respectively.
Figure 3:
Gel filtration chromatography of hGHR-ECD
and its complexes with bPL (A), bPL(S184H) (B),
bPL(T188F) (C), and bPL(T188F,I190F) (D) on a
Superdex 75 HR 10/30 column. Protein complexes were
preincubated in 1:1, 1:2, and 1:5 bPL or bPL analogue:hGHR-ECD molar
ratios. The concentration of hGHR-ECD was constant (15 µM)
and the concentrations of bPL or bPL analogues varied from 3 to 15
µM. Protein mixtures were incubated for 15 min at room
temperature in TNM buffer and then 200-µl aliquots were applied to
the column. The column was developed at 0.5 ml/min. Ordinate,
absorbance at 280 nm; abscissa, time of elution in minutes.
The retention times of bovine serum albumin (67 kDa), hGHR-ECD (28
kDa), and bPL (23 kDa) were 16.98, 21.25, and 22.42 min, respectively.
For further details, see text.
Complex formation between bPL analogues and rabbit and rPRLR-ECDs was monitored by gel chromatography using constant hormone and variable ECD concentrations ( Fig. 4and Fig. 5). These results showed that increase in the ECD to hormone ratio from 1:1 to 2:1 (Fig. 4A and 5A) resulted in doubling of the peak area, thus confirming our previous observations that in both cases bPL forms a 1:2 complex with each R-ECD(27, 37) . Increase of the ECD:analogue molar ratio to 3:1 did not further increase the size of the complex and the excess of the ECDs were observed. Analysis of the retention times of the eluted peaks indicated, however, that at initial 0.5:1 and 1:1 bPL:PRLR-ECD ratios a 1:2 complex and excess bPL were observed rather than a 1:1 complex. The smaller size of the bPL peak results from its almost 3-fold lower specific extinction coefficient at 280 nm. The small difference in retention times in experiments with rabbit and rPRLR-ECDs originates from using two columns with slightly different bead volumes and thus is irrelevant to an interpretation of the results. Three bPL analogues, bPL(S184H), bPL(S187A), and bPL(S187F), not shown in the figure yielded results identical to those of the unmodified hormone. In contrast, bPL(T188F) and bPL(T188F,I190F) retained their ability to form a 1:2 complex with rbPRLR-ECD (Fig. 5, B and C) but formed only a 1:1 complex with rPRL-ECD (Fig. 4, B and C).
Figure 4:
Gel filtration chromatography of rPRLR-ECD
and its complexes with bPL (A), bPL(T188F) (B), and
bPL(T188F,I190F) (C) on a Superdex 75 HR
10/30 column. Protein complexes were preincubated in 1:0.5, 1:1, 1:2,
and 1:3 bPL or bPL analogue:rPRLR-ECD molar ratios, using a constant
(1.2-1.3 µM) concentration of bPL or bPL analogue
and increasing the concentration of rPRLR-ECD. The column was developed
at 1 ml/min. The retention times of bovine serum albumin (67 kDa),
rPRLR-ECD (25.6 kDa), and bPL (23 kDa) were 9.41, 11.37, and 11.69 min,
respectively. For other details see the legend to Fig. 3.
Figure 5:
Gel filtration chromatography of
rbPRLR-ECD and its complex with bPL (A), bPL(T188F) (B), and bPL(T188F,I190F) (C) on a
Superdex 75 HR 10/30 column. Protein complexes were
preincubated in 1:0.5, 1:1, 1:2, and 1:3 bPL or bPL analogue:rbPRLR-ECD
molar ratios, using a constant (3.2-4.4 µM) amount
of bPL or bPL analogue and increasing concentrations of rbPRLR-ECD. The
column was developed at 1 ml/min. The retention times of bovine serum
albumin (67 kDa), rbPRLR-ECD (22 kDa), and bPL (23 kDa) were 10.03,
12.78, and 12.54 min, respectively. For other details see the legend to Fig. 3.
We
have recently shown by gel filtration experiments that at 1.6
µM concentration of both reagents of bPL forms a weak 1:1
complex with bPRLR-ECD. Lowering the reagent concentrations by
2-16-fold led to its progressive dissociation(26) .
Similar results performed at 3.3 µM concentration were
obtained in the present work with bPL(S184H), bPL(S187A), bPL(S187F),
and bPL(T188F), although the dissociation of the later occurred already
at relatively higher absolute concentrations (not shown). Since I-bPL binds very poorly in this system, and the K
of the bPL-bPRLR complex could not be calculated
from homologous binding experiments, its affinity toward bPRLR-ECD was
evaluated from the IC
values using
I-hGH as
a ligand. The K
of the hGH:bPRLR-ECD calculated
from the data in Fig. 1was 2.7 nM in agreement to the
previously reported value of 2.07 nM(26) , and the
relative IC
of bPL was
20-fold higher than that of
hGH. These results fully explain and support our interpretation of gel
filtration experiments. In contrast, no complexes could be detected
between bPL(T188F,I190F) and bPRLR-ECD, even at a 3-fold excess of the
latter (not shown).
Figure 6:
Biological activity of bPL and its
analogues in five in vitro bioassays: -casein synthesis
in HC11 cells, Nb
-11C lymphoma cell proliferation,
antimitogenic somatogenic receptor-mediated activity in 3T3-F442A
preadipocytes as shown by curves. bPL (
), bPL(S184H) (
),
bPL(S187A) (
), bPL(S187F) (
), bPL(T188F) (
), and
bPL(T188F,I190F) (
). IGF-I secretion from a primary culture of rat
hepatocytes and
-casein production in rabbit mammary gland explant
are shown by bars; bPL (
), bPL(S184H) (&cjs2089;),
bPL(S187A) (&cjs2110;), bPL(S187F) (&cjs2113;), bPL(T188F) (&cjs2090;),
bPL(T188F,I190F) (
). The original blot of
-casein synthesis
is shown at the upper right: bPL (A), bPL(S184H) (B), bPL(S187A) (C), bPL(S187F) (D),
bPL(T188F) (E), and bPL(T188F,I190F) (F).
CD analysis of the bPL analogues revealed preservation of
their secondary and, most likely, tertiary structure with the exception
of the double-mutated bPL(T188F,I190F) analogue. Thus, any functional
changes resulting from these mutations probably do not originate from
an overall structural change but rather from disruption of local
hormone-receptor contacts. In contrast, the overall -helix content
in bPL(T188F,I190F) and exposure of Tyr-189 to the solvent were reduced
and the environment of both Trp was changed. In hGH, the side chain of
the corresponding residue (Leu-177) is completely buried as an integral
part of the four-helix bundle core(22) . These differences most
likely account for the altered structure of this analogue that results
in almost complete loss of receptor binding and biological activities (Table 2). Yet this drastically modified analogue could bind to
respective receptors and exhibit some biological activity when its
concentration was increased 10-1000-fold over that of unmodified
bPL.
Modification of Ser-187 to either Ala or Phe minimally affects
its binding properties or biological activities. Ser-187 corresponds to
Glu-174 in hGH (Table 3), whose mutation to Ala dramatically
changes its somatogenic/lactogenic receptor specificity, mainly by
reducing the binding to lactogenic receptor(20) . This
difference may be attributed to the fact that lactogenic effects of hGH
are strongly dependent on Zn and the Glu-174 of hGH
is part of the Zn
binding site(20) .
Two
other mutations resulted, however, in specific modifications of binding
properties and biological activity (Table 2). Analogue bPL(T188F)
lost the ability to bind to human somatogenic receptors, to bovine
liver microsomal fraction, and most of its ability to bind to the
endometrial microsomal fraction which most likely contains unique bPL
receptors(8) . By comparison, its ability to bind to lactogenic
receptors was either unchanged (intact Nb cells, bovine
PRLR-ECD) or reduced (rat and rbPRLR-ECDs). The reduced ability of
bPL(T188F) to bind to human somatogenic receptor was also reflected by
its inability to form a 1:2 complex with hGHR-ECD and the formation of
a weak 1:1 complex only. However, the ability of this analogue to form
complexes with ECDs of various PRLRs was unchanged or only slightly
modified. In parallel, its somatogenic receptor-mediated biological
activity was reduced 20-fold in rat hepatocytes and 12-fold in mouse
3T3-F442A preadipocytes, whereas its lactogenic receptor-mediated
activity was either unaffected (Nb
-11C cells and rabbit
mammary gland explants) or 2.5-fold reduced (HC11 cells).
Selective
modification also occurred with the S184H mutation leading to a
100-200-fold decrease in binding to human, but not other,
somatogenic receptors. Conversely, its ability to bind to lactogenic or
bPL receptors remained unchanged (bovine endometrial, intact
Nb-11C cells, rat, and bPRLR-ECD) or was only slightly
reduced (rbPRLR-ECD). Yet, despite the drastically reduced ability to
bind to intact IM-9 cells or hGHR-ECD, bPL(S184H) at micromolar
concentration was capable of forming 1:2 complexes with hGHR-ECD, as
evidenced by gel filtration experiments. At the same time, the
somatogenic rat or mouse receptor-mediated bioactivity of bPL(S184H)
was unchanged. Some decrease, however, was observed in the biological
activity mediated through lactogenic receptors in Nb
-11C or
HC11 cells. Reduced biological activity of bPL(S184H) in
Nb
-11C cells was not, however, paralleled by a
corresponding change in binding to intact cells, indicating that
binding properties of hormones or their analogues are not always
indicative of biological potency. When interpreting these results, one
needs to bear in mind the fact that the gel filtration experiments were
performed at reagent concentrations (ECD and hormone) that were
100-1000-fold higher than those used in competition binding
experiments. Since dilution of the complex to concentrations close to
its K
values obviously leads to dissociation,
results of our previous dimerization experiments should be interpreted
with
caution(24, 25, 37, 38, 39, 40) ,
as should those by others(41, 42) performed with
micromolar concentrations of the hormones and ECDs. One should also
consider to what extent these results reflect binding that occurs under
physiological conditions.
The present results enabled us to compare
bPL's analogues binding to membrane-embedded receptors in IM-9
human lymphocytes and Nb cells to the corresponding
recombinant soluble R-ECDs. In IM-9 cells and hGHR-ECD the different
analogues yielded almost identical results, but bPL(T188F) exhibited
reduced affinity to soluble rPRLR-ECD, and full affinity to
membrane-embedded receptors in Nb
-11C cells. These results
imply that in contrast to hGHR-ECD, the shedding of PRLR-ECDs may cause
some structural changes that may affect their binding properties as
suggested previously (37) .
Although the three-dimensional
structure of bPL has yet to be resolved, sequence comparison suggests
high homology to hGH, in which both Thr-175 and Asp-171 are located at
the outer surface of the fourth -helix(22) . The
corresponding amino acids in bPL are most likely Thr-188 and Ser-184.
In hGH, Thr-175 O
and Asp-171 O
form
intermolecular hydrogen bonds with Arg-43 N
and
Arg-43 N
of hGHR-ECD(22) . These contacts,
and in particular that of Thr-175, seem to be most critical for
high-affinity complex formation, as exemplified by the facts that its
mutation to Ala reduced the affinity by over 2 kcal/mol and that it was
highly (75%) conserved after sorting for high-affinity mutants that
bind to hGHR-ECD(43, 44) . Our present results support
the hypothesis that Thr-188 of bPL indeed occupies the Thr-175 position
of hGH. Its mutation to a bulky Phe residue most likely interferes with
complex formation with either soluble hGHR-ECD or hGHR embedded in
intact IM-9 cells. The area of hPRLRs (Lys-17 and Glu-18, hPRLR-ECD
numbering) that corresponds to the first contact area of hGHR binding
site one (Arg-43 and Glu-44, hGHR-ECD numbering) is located in almost
the same region, as recently evidenced by a three-dimensional analysis.
However, in contrast to its interaction with hGHR-ECD, Thr-175 of hGH
does not participate in the formation of intermolecular hydrogen bonds
with these residues of hPRLR-ECD(21) . It should be noted that
positions 17 and 18 in rat(45) , rabbit(46) , and
bovine (26) are also occupied by Lys and Glu. The side chain of
hGH Thr-175 and most likely also that of bPL's Thr-188 residue
are buried in the hormone-PRLR interface(21) . However, since
bPL(T188F) retains its biological activity in Nb
cells and
rabbit mammary gland explants which are mediated through lactogenic
receptors, we conclude that the buried area is most likely large enough
to contain the benzene ring of Phe. These structural features explain
why the modification of bPL(T188F) is selective and leads to a loss in
its somatogenic activity only.
In contrast to bPL(T188F), bPL(S184H)
retained its ability to bind to nonhuman somatogenic receptors and to
exhibit full somatogenic receptor-mediated biological activity in rat
hepatocytes and 3T3-F442A preadipocytes, but almost completely lost its
ability to bind to hGHR-ECD or to GHR in intact IM-9 human lymphocytes.
Ser-184 in bPL most likely corresponds to Asp-171 in hGH, which is
implicated in the binding to hGHR-ECD and hPRLR-ECD through formation
of respective hydrogen bonds with Arg-43 N2 of hGHR-ECD (22) or with Tyr-127 O
of hPRLR-ECD(21) . Such an
interaction most likely does not exist or is modified in nonhuman GHs,
in which position 171 is occupied by His(23) , and between
GHR-ECDs of rabbit(47) , rat (48, 49) ,
mouse(50) , cow(51) , and chicken (52) in which
position 43 is occupied by Leu. Therefore, position Asp-171 in hGH and
Arg-43 in hGHR seem to be highly important for acquiring species
specificity. Although Asp-171 0
of hGH forms
intermolecular hydrogen bonds with Arg-43 N
of hGHR,
its apparent contribution is much less than that of hGH's
Thr-175(44) , thus giving potential explanation as to why bPL
exhibits binding specificity toward hGHR, despite the Ser-184. The fact
that hGH(D171A) retains almost full ability to interact with hGHR-ECD (20) supports this assumption. In contrast, mutation to a
larger side chain residue, such as in bPL(S184H) or His-171 in nonhuman
GHs, not only prevents formation of the hydrogen bond (which anyhow
does not play major role in interaction with the receptor) but also
most likely interferes with the interaction of the alkyl portion of
hGH's Thr-175 and Lys-172 with Trp-104 of hGHR which is critical
to complex formation(22, 44) .
Human GH(D171A) has
been reported to have a 7.1-fold lower affinity toward
hPRLR-ECD(20) , most likely because of disrupted interaction
with Tyr-127 (hGHR-ECD numbering) or Tyr-98 (hPRLR-ECD numbering),
which is one of nine intermolecular hydrogen bonds that stabilize the
hGH-hPRLR-ECD interaction(21) . Position Tyr-98 (hPRLR-ECD
numbering), is also occupied by Tyr in rat(45) ,
rabbit(46) , and bovine (53) PRLRs, but it is unclear
whether Ser-184 of bPL also forms a parallel hydrogen bond. Results
concerning the lactogenic receptor-mediated biological activity of
bPL(S184H) were not consistent. Whereas in Nb and HC11
cells an 2-3-fold decrease was observed, the activity in rabbit
mammary gland explants was unchanged. This reduced activity was not
always paralleled by reduced affinity toward soluble or
membrane-embedded PRLRs. These results are difficult to explain and may
be related to minor structural differences between various PRLRs, as
exemplified by our studies with their soluble
ECDs(24, 25, 26, 27, 37) .