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Institute of Biochemistry, Food Science and Nutrition (D.H., A.G.), Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot 76100, Israel; Unite d’Endocrinologie Moleculaire (J.D.), Unite de Virologie et Immunologie Moleculaire (J.G.), Institut National de la Recherche Agronomique, 78352 Jouy-en-Josas Cedex, France; and Animal Science Division and Searle (N.R.S., J.B., R.E.M.), c/o Monsanto Co., Department of Molecular Biology, St. Louis, Missouri 63198
Address all correspondence and requests for reprints to: Arieh Gertler, Institute of Biochemistry, Food Science and Nutrition, Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot 76100, Israel. E-mail: gertler{at}agri.huji.ac.il
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In vivo biological effects of recombinant bPL in dairy cows have been recently investigated, indicating its possible effect in mammogenesis, energy balance during pregnancy, and support of fetal growth (for review see 14 . The in vivo effect of bPL and oPL in rat suggest potent somatogenic activity (15, 16). The in vivo effect in ewes and lambs is less clear, due to the limited amount of experimental data on this aspect (17, 18).
One possible way of studying the specific activity of bPL is to
prepare analogs in which either the lactogenic or somatogenic activity
of the hormone has been selectively modified. The first of these were
aimed at successive truncation of bPL’s N-terminal domain (12, 19).
Assuming structural similarity to porcine (p) GH (20), these mutations
were aimed at removing amino acids beyond or at the beginning of the
putative first 
-helix. Results of these studies indicated that bPL
can be selectively modified such that particular biological activities
are changed while others remain relatively unaffected, although the
effect was only partial. The more recent point-mutated bPL(T188F)
exhibited such selective modification. Its binding to full-size
somatogenic receptors, or their ECDs, and to bPLR in the endometrium,
as well as somatogenic-receptor-mediated biological activities, were
reduced or abolished, whereas binding to lactogenic receptors or their
ECDs, and subsequent biological activity was either fully or almost
fully retained (8). Another possible target for selective mutations is
Lys 73. This residue corresponds to Arg 64 in hGH, which has been
identified as important in the interaction with hGHR-ECD (21), but not
with hPRLR-ECD (22). To evaluate the importance of this residue in bPL,
three recombinant analogs were prepared and tested: bPL (K73A) and bPL
(K73F) in which a small side-chain residue or bulky side-chain residue
were introduced, respectively, and bPL (K73D), in which the positive
charge was replaced by a negative one.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Construction of bPL-analog expression vectors
Synthetic genes for each bPL analog were constructed using PCR
technology, with a GeneAmp PCR Reagent Kit (Perkin Elmer, Norwalk, CT).
Briefly, synthetic oligonucleotides (primers) were used to generate a
double-stranded DNA that contained the mutation(s) of interest from a
template, pMON3401 (24), in addition to restriction-enzyme sites for
cloning. The forward primer encoded an NcoI
restriction-enzyme site and an initiator methionine codon immediately
upstream of the first mature codon (alanine) of bPL. The reverse primer
encoded the mutation(s) of interest, as well as a HindIII
restriction site and TAA termination codon immediately after the final
codon (cysteine). The PCR products were purified using a Wizard PCR kit
(Promega, Madison, WI) and digested with NcoI and
HindIII restriction enzymes before ligation to parental
vector pMON3401 using T4 DNA ligase. The resulting plasmids were
sequenced using a Sequenase DNA Sequencing Kit (USB, Cleveland, OH) and
the positive constructs were passaged through Escherichia
coli strain LE392 before transformation of MON105 (26).
Expression, refolding, and purification of bPL analogs
Escherichia coli MON105 cells transformed with
the expression plasmids containing the bPL variant genes were incubated
in 500 ml of Terrific Broth (TB) medium (27) by shaking at 300 rpm at
37 C in 2-liter flasks to an A600 of 0.9, after which
nalidixic acid (25 mg/flask) was added. The cells were incubated an
additional 4 h, harvested by 5-min centrifugation at 10,000
x g, decanted, and then frozen at -20 C. Over 95% of the
bPL protein was found in the inclusion bodies that were prepared as
described previously (19). The inclusion-body pellet containing the bPL
analogs was solubilized in 600 ml of 4.5 M urea buffered
with 10 mM Tris base. The pH was increased to 11.3 with
NaOH, cysteine was added to 0.1 mM, the clear solution was
stirred at 4 C for 48 h and then dialyzed for 48 h against
5 x 10 l of 10 mM Tris-HCl, pH 9. The solution
was then loaded at 120 ml/h onto a Q-Sepharose column (2.6 x 7
cm), preequilibrated with 10 mM Tris-HCl, pH 9.0 at 4 C.
Elution was carried out using a discontinuous NaCl gradient in the same
buffer at a rate of 120 ml/h, and 5-ml fractions were collected.
Protein concentration was determined by absorbance at 280 nm, and
monomer content by gel-filtration chromatography on a Superdex 75
column.
SDS-PAGE
SDS-PAGE was carried out according to Laemmli (28) using 15%
gels. Gels were stained with Coomassie Brilliant Blue R.
Circular dichroic (CD) spectra
CD spectra were collected at 4 C in a Jasco J-500C
spectropolarimeter using either 0.2- or 0.5-mm cylindrical cells. The
spectropolarimeter was routinely calibrated with
D-10-(+)-camphor sulfonic acid at 290 nm. The CD spectra
(average of four scans) were baseline-corrected and converted to mean
residue molar ellipticity ([
]) and analyzed by a
least-squares-fitting procedure (29). Additionally, the -helix
content was estimated by the magnitude of [] at 222 nm (30). The
absorbance was used to estimate the protein concentration for CD
analysis using the method of Gill and von Hippel (31). A sample of
myoglobin was analyzed in parallel with the bPL samples and was found
to agree with literature values (
80% -helix, 29 .
Determination of monomer content and complex formation
HPLC gel-filtration chromatography on a Superdex 75 HR 10/30
column was performed with 200-µl aliquots of
Q-Sepharose-column-eluted fractions, freeze-dried samples dissolved in
H2O or complexes between the soluble recombinant GHR- or
PRLR-ECDs and bPL or bPL analogs, using methods described previously
(8).
Binding experiments
Binding to intact IM-9 human lymphocytes, Nb2 rat lymphoma-cell
homogenate, bovine liver-gland microsomal fractions, and soluble
hGHR-ECD and rat and rabbit PRLR-ECD was carried out as described
previously (8, 12, 23, 24).
Coupling of bPL or bPL analogs to a CM-dextran matrix via amino
groups
The bPL analogs were covalently linked according to Johnsson
et al. (32). HBS was injected at 5 µl/min and activation
with 0.05 M EDC/NHS in HBS was carried out for 7–8 min.
The analogs were then injected at a concentration of 100 µg/ml in 10
mM Na-acetate buffer, pH 5.4, yielding 1000–2000 resonance
units (RU) of immobilized hormone. Nonreacted sites were blocked with
an 8-min injection of 1 M ethanolamine hydrochloride at pH
8.5. Binding capacities were checked by repeated injections of 5
µM R-ECDs in HBS. Immobilized bPL or its analogs could be
regenerated over 50 runs with 4.5 M MgCl2
pulses (1–2 min).
Kinetic measurements of R-ECD:hormone interactions
All experiments were performed at a flow rate of 5 µl/min in
HBS at 25 C. Once the hormone being tested was covalently immobilized
through amino-group coupling, serial dilutions of each R-ECD were
injected for 6 min, and then washed out for 10 min before regeneration.
Because the recombinant R-ECDs had been lyophilized with Na-bicarbonate
buffers at a salt:protein ratio of 1:2, bulk refractive indexes varied
with sample dilution, and these variations were corrected for by
injecting the same dilutions into flow cells in which unrelated ligands
had been immobilized.
Data analysis and calculation of kinetic constants
BIAcore incorporated software (BIA Evaluation and BIA
Simulation) allowed us to: 1) fit experimental curves with 1:1 or 1:2
association/dissociation models and calculate the probabilities of each
being the most accurate representation of reality; 2) calculate kinetic
constants with SDs. Reverse verification of calculated data
were performed by simulating the interaction assuming a variable
relative occupation of the two sites.
In vitro bioassays
Three in vitro bioassays in which the signal was
transduced through lactogenic receptors were performed: rat
Nb2-11C lymphoma-cell-proliferation bioassay (33) and
ß-casein production in a mouse HC-11 mammary cell line,
nontransfected or transfected with full-size rbPRLR (8). Two in
vitro bioassays in which the signal was transduced through
somatogenic receptors were based on the proliferation of FDC-P1 cells
transfected with rabbit (clone FDC-P1-B9) or human (clone FDC-P1-D11)
GHRs (34, 35). Cells cultured in RPMI-1640 medium supplemented with 5%
FCS and hGH (100 ng/ml) were washed in PBS, resuspended in RPMI-1640
medium supplemented with 5% horse serum at a concentration of 50,000
cells/ml, and plated (one ml/well) in 24-well plates. The bPL or its
analogs were then added and the cells were grown for an additional
48 h. Cell growth was determined by counting the cells with a
Coulter counter (Coulter Electronics Inc., Hialeah, FL), and the number
of doublings was calculated as described previously (33).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) shows that over 50% of the
protein was eluted with 0.15 M NaCl. Every third tube was
analyzed for monomer content and fractions containing >98% pure
monomer (tubes 26–44) were pooled, dialyzed against NaHCO3
(1:3 salt:protein ratio), and lyophilized. This fraction was further
used for binding and biological studies. Fractions eluted with 0.4
M NaCl consisted mainly of oligomers (not shown). SDS-PAGE
of the pooled monomeric fraction, performed with and without
ß-mercaptoethanol, revealed only one band with a molecular mass of 23
kDa (not shown). The oligomeric fraction eluted at 0.4 M
NaCl also yielded mostly 23-kDa band, indicating that the oligomers
were formed by noncovalent interactions. Purification profiles of
bPL(K73A) and bPL(K73D) were very similar to that of bPL (K73F) (not
shown).
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-helix content (Table 1) revealed values close to (and within
experimental error) those of bPL, indicating proper refolding.
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). Binding to intact IM-9 human
lymphocytes yielded similar results, although in this case the binding
ability of bPL(K73D) was almost totally abolished (Fig. 2B). Binding of
[125I]bPL to homologous somatogenic receptors in a
microsomal fraction from bovine liver (Fig. 2C) also showed bPL(K73D)
to be totally inactive, and the ability of bPL(K73F) and bPL(K73A) to
compete with the labeled ligand as compared with wild-type bPL was
reduced 13- and 25-fold, respectively.
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, D–G). Homologous bPRLR-ECD or bovine mammary gland microsomal
fractions were not used because the complex formed with bPL is very
unstable due to rapid dissociation (13, 36), and therefore its
existence can hardly be detected by standard methods. Competition
curves of both bPL(K73A) and bPL(K73F) binding to rPRLR-ECD were
similar to that of bPL, whereas the binding ability of bPL(K73D) was
reduced by approximately 30-fold. In contrast, the binding abilities of
bPL(K73A), bPL(K73F) and bPL(K73D) to homogenate of rat Nb2
lymphoma cells were only slightly reduced (Fig. 2E). Binding of
bPL(K73A), bPL(K73F), and bPL(K73D) to rbPRLR-ECD was reduced by 2-,
5-, and 8-fold, respectively, as compared with bPL (Fig. 2F) and to the
microsomal fraction from rabbit mammary gland by 3-, 4- and 6-fold,
respectively (Fig. 2G).
Gel filtration experiments
The stoichiometries of interaction between hGHR-ECD or
rPRLR-ECD and the bPL analogs were studied by preparing the respective
complexes at increasing ECD:hormone ratios, while maintaining a
constant concentration (2 µM) of the latter. In contrast
to the binding experiments, both bPL(K73A) and bPL(K73F) were capable
of dimerizing hGHR-ECD, similarly to bPL (Fig. 3, A–D). This observation is most likely
related to the fact that the binding experiments, in contrast to the
gel filtration experiments, were performed at pM to
nM concentrations, which favor the dissociation of weak
complexes. In contrast to these analogs, bPL(K73D) formed only weak 1:1
complex (Fig. 3B). These assumptions are based on a comparison of
complex-peaks sizes and retention times. The effect of absolute
concentration on complex formation was further analyzed at a 2:1
hGHR-ECD:bPL (or bPL analog) ratio by decreasing the absolute
concentrations of both components 4- to 64-fold. Results of this test
(Table 2) supported those of the binding
experiments, indicating that complex formation is
concentration-dependent in a analog-specific manner. Dilution of the
bPL:hGHR-ECD complex from 2.0 to 0.03 µM had only a minor
effect on its retention time, indicating a high-affinity complex. In
contrast, dilution-dependent increases in retention time values were
observed with both bPL(K73A) and bPL(K73F), and even more strikingly
with bPL(K73D). In the latter analog, even at 2.0 µM the
complex was hardly detected. Note that during the course of gel
filtration, the injected material undergoes a 5- to 10-fold dilution.
Thus, with weak complexes, partial dissociation occurs gradually,
resulting in a widening and shifting of the complex peak to higher
retention-time values, rather than in the appearance of separate peaks
for the complex and its components. Interaction of rPRLR-ECD with bPL
and bPL analogs indicated that at 2 µM, the wild-type bPL
and its analogs retain full ability to form 2:1 complexes (Fig. 3, E–H). Complexes with wild-type bPL, bPL(K73A) and bPL(K73F) remained
stable, even when their concentrations were decreased to 0.03
µM, whereas the complex with bPL(K73D) underwent partial
or even full dissociation (Table 2).
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and Table 3). Nevertheless, kinetic analysis
revealed a very good fit in all cases to the two-site model and a poor
fit to the one-site model, even in the latter case. The same situation
also appeared to exist in the interaction of bPL(K73A), bPL(K73F) and
bPL(K73D) with different R-ECDs. Thus, the lack of apparent 1:2
stoichiometry does not necessarily indicate its absence, but rather
shows that the 1:2 complex is extremely transient, undergoing rapid
dissociation to the 1:1 form. Table 3 summarizes the effect of
mutations on the kinetic and thermodynamic constants of the
hormone:R-ECD interactions. The effect was highly selective. In the
case of bPRLR-ECD, all three mutations increased the affinity of site 1
but had little effect on site 2. In contrast, all three mutations
caused an up to 8-fold decrease in the affinity of site 1 toward rabbit
and rat PRLR-ECDs. An even larger decrease (12- to 80-fold in the case
of rbPRLR-ECD and 6 to 12-fold in the case of rPRLR-ECD) in the
affinity of site 2 was found. In both cases the decrease in site 2
affinity resulted from the elevated koff values, whereas
the effect on kon was small and in-consistent.
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In vitro biological activity
Somatogenic-receptor-mediated biological activity of all
three bPL analogs was severely impaired. In the experimental system
using FDC-P1 cells transfected with hGHR (9D11 cell line), the
biological activity of both bPL(K73A) and bPL(K73F) was reduced over
30-fold, and that of bPL(K73D) was completely lost (Fig. 5A). Similar results were also obtained
in a cell line transfected with rbGHR (cell line 3B9) although in that
case, the activities of bPL(K73A) and bPL(K73F) were reduced to a
lesser extent (Fig. 5B).
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). In other
lactogenic-receptor-mediated bioassays, the bioactivity was also
totally or almost totally retained. In HC-11 cells (Fig. 6A) at a
concentration of 2 nM, the activity of the analogs was 25
to 40% lower than that of bPL. At 20 nM, the results were
similar, except for bPL(K73F), the activity of which equalled that of
bPL. In HC-11 cells transfected with rbPRLR, the activities of both
bPL(K73F) and bPL(K73D) were similar to that of bPL, whereas the
activity of bPL(K73A) was lower (Fig. 6B). It should be noted that the
quantitation of the results in these bioassays was based on scanning of
the immunologically detected casein band on a ligand blot, a method
that involves rather high experimental error.
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Discussion |
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In binding experiments to intact cells, cell homogenate or microsomal fractions, mutations of K73 led to a remarkable decrease in binding to somatogenic receptors, whereas binding to the lactogenic receptor changed only slightly. The most dramatic effect was exhibited by bPL(K73D), whereas a lesser reduction was seen in the competitive ability of mutations bPL(K73A) and bPL(K73F). In most cases, binding to soluble PRLR-ECDs and to membrane-embedded receptors did not change, with the exception of bPL(K73D), whose binding ability to rPRLR-ECD was drastically decreased. This decreased ability was also demonstrated in the gel-filtration: while rPRLR-ECD formed stable 2:1 complexes with analogs bPL(K73A) and bPL(K73F) that did not dissociate upon dilution, the corresponding complex with bPL (K73D) was unstable. These differences were seen in the interaction with hGHR-ECD as well. At 2 µM, the analogs bPL(K73A) and bPL(K73F) formed a rather stable 2:1 complex that dissociated upon dilution, whereas the bPL(K73D) analog formed only a weak 1:1 complex that was detectable only at that molar concentration.
A detailed kinetic analysis by SPR was performed because neither the gel filtration nor the binding experiments provided a sufficient understanding of all the changes in binding properties. The advantage of the kinetic analysis stems from its ability to deduce the stoichiometry of interaction, even in transient ligand:receptor interaction, in addition to permitting kinetic calculations (kon and koff) for each of the two hormone’s binding sites. Because the kinetic analysis and simulation revealed that, in all cases, the analogs form a homodimeric complex with R-ECDs, that model was used to calculate the kinetic constants. Excluding the interaction with bPRLR-ECD in which the mutations increased the affinity of site 1 of the hormone, the affinities of both sites 1 and 2 were reduced in all other instances. This reduction originated mainly from the destabilization of the complex, as evidenced by the increased koff values. This was particularly true in the case of rat and rabbit PRLR-ECDs for the interaction with site 2, whereas the interaction with site 1 was affected to a lesser degree. On other hand, in hGHR-ECD, both sites were affected almost equally.
Data presented in this paper raise fundamental questions about the connection between the binding of bPL and its analogs to somatogenic or lactogenic receptors and the biological activity transduced as a result of this interaction. Two main features were observed: 1) the changes in binding properties to a soluble R-ECD were not always paralleled by corresponding changes in the binding to membrane-embedded receptors; 2) in spite of the reduced affinity toward both somatogenic and lactogenic receptors, dramatic reduction in biological activity mediated was observed in events mediated through somatogenic receptors. In contrast, in events mediated through lactogenic receptors, the activity was barely or not at all affected.
To provide a possible explanation for the first finding, we have to remember that, in general, the affinity toward soluble receptors (except for rbPRLRs) is less than that toward membrane-embedded receptors. Thus, the discrepancy may result from two reasons: 1) an additional as yet unidentified protein or glycolipid stabilizes the hormone-receptor interaction; or 2) the hormone-induced dimerization of the R-ECD in the full-size receptor subsequently leads to interactions in the cytosolic domain to further stabilize the complex. Modification in phosphorylation and dephosphorylation events of the cytosolic domain should be considered as potentially responsible for such stabilization.
The second finding may be explained in view of our recent suggestion that transient dimerization of PRLRs, lasting a few seconds or less, is sufficient to elicit full biological response (13). This assumption was supported by the finding that after the homodimeric complex is formed, receptor-associated JAK2 or other kinases are instantly activated by mutual transphosphorylation, forming docking sites for other downstream proteins (37, 38). Once this occurs, the receptor dimers are no longer needed. This hypothesis is likely to be true for biological events induced through both lactogenic and somatogenic receptors. However, quantitative differences in the duration of dimer existence may be important. Indeed, the results of the SPR experiments, in which the mutations caused rather similar changes in binding properties, suggest that the persistence of the homodimer life required for the activation of GHR must be longer than for PRLR. This conclusion may be related to the fact that in lactogenic receptors JAK2 kinase is constitutively associated with the receptor, whereas, in somatogenic receptors, the receptor:JAK2 association occurs subsequently to the interaction with the hormone (39).
Although the three-dimensional structure of bPL is not known, it is probably similar to that of hGH due to the high homology in primary structures, as well as to functional similarities. This comparison suggests that the K73 of bPL occupies a position parallel to that of R64 in hGH. K73 at this position is also found in other somatogenic and lactogenic hormones such as bGH, oPL, bovine, rat, and human PRLs, but not in hPL, in which this place is occupied by methionine. Structural data indicate that R64 of hGH interacts in two ways with the hGHR-ECD: its methylene groups are in van der Waals contact with W169 of the receptor at a distance of 3.8–4 A and its guanido group forms a salt bridge/hydrogen bond to D164 of the receptor at a distance of 2.8–2.9 A. It should be emphasized that K73 can exhibit the same interactions with both W169 and D164. In contrast, in the hGH:hPRLR-ECD complex, only the first of these interactions occurs. This is because the shift in the C-terminal receptor domain as compared with that of the hGHR-ECD has moved D134 of the hPRLR-ECD (analogous to D164 of the hGHR-ECD) away from R64 to a distance of about 4.0 A, making a direct, tight interaction impossible (A. M. de Vos, personal communication). Therefore, mutation of bPLs K73 to A, F, or D would abolish the salt bridge/hydrogen bond to D164 of the receptor, resulting in a total loss or at least a remarkable decrease in biological activity mediated through the somatogenic receptor. Several pieces of hGH mutagenesis data support this conclusion: 1) R64A in hGH reduced hGHR-ECD binding 16- to 21-fold, but hPRLR-ECD binding only 1.8-fold, whereas R64K increased hGH binding to the hGHR-ECD by 3-fold. (40, 41); 2) the double hGH mutation (R64 M/E56D) reduced hGHR-ECD binding 30-fold, but hPRLR-ECD binding only 2.1-fold (41); 3) the D164A mutation of hGHR-ECD decreased its ability to bind hGH 12.3-fold (42); 4) in hPL, which binds only very weakly to hGHR-ECD, mutation of M64R increased binding by 23-fold, while leaving binding to hPRLR-ECD unaffected (43).
In conclusion, we have shown that preparation of a selectively modified analog of bPL is feasible and have proposed a possible structural and functional explanation for this selectivity. Preparation of other selectively modified analogs is now in progress.
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Acknowledgments |
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Footnotes |
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Received March 19, 1997.
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), bPL(K73D) (
),
bPL(K73F) (
) and bPL(K73A) (
).
