1The Ohio State Biochemistry Program, 2Department of Veterinary Biosciences and 3Department of Biochemistry, The Ohio State University, 1925 Coffey Road, Columbus, OH 43210, USA
4 To whom correspondence should be addressed. E-mail: brooks.8{at}osu.edu
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Abstract |
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Keywords: growth hormone/human/prolactin/rat
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Introduction |
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Class 1 of this protein family contains these three lactogenic hormones and others ligands including erythropoietin, IL-6 and leptin. They share a long four-helix bundle structure with an upupdowndown orientation of the four main helices (Figure 1). The first and last pairs of the main helices are connected by long segments that may contain small helices, frequently called mini-helices (Wells and de Vos, 1996). Structures are available for hGH either free from the receptor (PDB# 1HGU) (Chantalat et al., 1995
) (Figure 1), or bound to either one extracellular domain of the hPRL receptor (hPRLbp) (PDB# 1BP3) (Somers et al., 1994
). Currently, only one structure is available for hPRL (PDB# 1N9D) (Keeler et al., 2003
); it is a structure not associated with a receptor. These structures and other heterotrimeric hormone/receptor complexes show a similar pattern: two unique surfaces of the ligand bind the receptors. Functional epitopes of hPRL receptor-binding sites are documented between helices 1 and 4 plus the loop connecting helices 1 and 2 for the first receptor-binding site (Goffin et al., 1992
; Kinet et al., 1996
) and the groove that lies between helices 1 and 3 for the second receptor-binding site (Goffin et al., 1994
, 1996
) (Figure 1). With the limited mutagenesis performed on hPRL, it is unlikely that the full cadre of ligand residues participating in receptor binding has been identified.
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hGH is found in the pituitary in two forms created by alternative gene splicing (Lewis et al., 1978; Estes et al., 1990
) of the mRNA of the hGH-N gene. A 190 residue 22 kDa hGH and a 175 residue 20 kDa hGH lacking amino acids 3246 are the respective mature translation products. Many of these deleted residues are found in the sequence that is structured by binding to the hPRLbp. Pituitary isolates of 20 kDa hGH retain somatotrophic activity but have a reduced lactogenic activity (Tsunekawa et al., 1999
), supporting our previous report of the requirement of F44 for lactogenic activity in hGH (Peterson and Brooks, 1997
).
We have recently identified a hGH motif, external to the two lactogenic receptor-binding sites and required for actions mediated through the hPRL receptor, but not for those mediated through the hGH receptor (Duda and Brooks, 1999, 2003
). The motif contains residues in mini-helix 1, the N-terminal portion of helix 4, and the portion of helix 2 that is C-terminal to P89. Individual replacement of these hydrophobic residues with hydrophilic species disrupts the hydrophobic packing of this motif and functionally uncouples site 1 from site 2. A similar, although not identical, coupling motif also exists in hPRL (Sivaprasad and Brooks, unpublished results). Phenylalanine 44 (F44) of hGH is located in mini-helix 1, is a member of this motif, and is required for actions mediated by the hPRL receptor, but not the somatotrophic activity in hGH (Peterson and Brooks, 1997
). Deletion of F44 reduces the lactogenic activity of hGH by 218-fold while reducing the somatotrophic activity by <3-fold. Replacement of F44 with either alanine or leucine provided partial replacement of lactogenic activity. Changes in the apparent affinities discerned in [125I]hGH membrane-binding studies for the PRL receptor paralleled changes in activity.
In this report we demonstrate that the effect on lactogenic activity of deleting F44 from hGH is similar to the deletion of residues 3246, as occurs in the 20 kDa form of hGH. These structural changes have little or no effect on somatotrophic activity. These data demonstrate the central importance of F44 in this section of hGH as a motif critical for hGH activity. In contrast, the deletion of the homologous F50 from hPRL had little effect on lactogenic activity. Despite a low sequence homology between these sections of hGH and hPRL (Figure 2), deletion of the corresponding residues 4152 from hPRL (4152 hPRL) produced a protein with a profound reduction in lactogenic activity. These data indicate that the section of both hGH and hPRL from the C-terminus of helix 1 to the disulfide bond at either C53 (hGH) or C58 (hPRL) are important motifs for lactogens, but they require distinct structural features to initiate activity through the PRL receptor.
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Materials and methods |
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An f1 origin of replication was inserted into the pT7-7 plasmid (kindly provided by S.Tabor, Harvard Medical School, Boston, MA, USA) generating the pT7-7f() phagemid. The negative strand was used for cloning, ssDNA production, and expression. Escherichia coli strains DH5, RZ1032 and BL21(DE3) were used for cloning, production of ssDNA, and protein expression, respectively. This expression system and the cloning of hGH, hPRL and rPRL has been previously described in detail (Maciejewski et al., 1995
; Peterson et al., 1999
). Mutagenesis was performed by the Kunkel method (Kunkel et al., 1991
) from wild-type plasmids that contained an N-terminal methionine codon (termed residue #0 in this work) using primers to create the following mutant proteins:
3241 hGH,
F50 hPRL,
4152 hPRL and
F50 rat PRL (rPRL).
Expression, purification and characterization of recombinant hGHs and hPRLs
Proteins were expressed from our pT7-7 phagemid in the BL21(DE3) strain of E.coli. Each protein was extracted, folded and purified by DEAE-cellulose chromatography as previously described (Peterson et al., 1999). Proteins were evaluated for size and purity by SDS-containing 15% polyacrylamide gel electrophoresis under reducing and non-reducing conditions. Proper protein folding was confirmed from absorption and fluorescence spectra that were collected at 20°C in 10 mM Tris pH 8.2, 150 mM NaCl. Expressed proteins included: wild-type and
3246 hGHs, wild-type,
F50 and
4152 hPRLs, plus wild-type and
F50 rPRLs.
Biological assays
FDC-P1 cells expressing the hGH receptor were a gift from Genentech Inc. (South San Francisco, CA, USA). Nb-2 rat lymphoma cells expressing the rat PRL receptor were obtained from P.Gout (The Cancer Control Agency of British Columbia, Vancouver, BC, Canada) (Tanaka et al., 1980). Hormone doseresponse curves were obtained as previously described (Peterson and Brooks, 1997
). Protein concentrations were measured by the bicinchoninic acid (BCA) protein assay (Smith et al., 1985
). The ED50s of the doseresponse curve were calculated by a four-parameter fit calculation (Munson and Rodbard, 1980
).
[125I]hGH prolactin receptor-binding assays
hGH was iodinated with Iodogen (Pierce Chemical Co., Rockford, IL, USA) and carrier-free [125I] iodine to a specific activity between 21 and 66 µCi/µg. Binding reactions contained membranes from 2 x 106 Nb-2 cells and between 1.0 and 1.9 ng of [125I]hGH and various concentrations of recombinant hormones in 700 µl of Fisher's media supplemented with 0.5% bovine albumin, 25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM ZnSO4, 1 mM PMSF and 10 µg/ml aprotinin. Binding reactions were allowed to approach equilibrium by incubation for 20 h at room temperature. The membranes were subsequently collected by centrifugation and the associated [125I]hGH measured. Non-specific binding was measured in a set of tubes with 10 µg of hGH added to the binding reaction. Non-specific binding was subtracted from all tubes and data from these competition studies were used to calculate the relative affinities.
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Results |
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The complete nucleic acid sequence of hGH and hPRL confirmed the appropriate nucleic acid sequence for each of the proteins used in this study. Recombinant proteins were prepared with final yields between 10 and 40 mg/l of fermentation. Recombinant proteins were >95% pure as judged by SDS-containing 15% polyacrylamide gel electrophoresis run under reducing conditions (Figure 3). Wild-type hGH, hPRL and rPRL co-migrated with pituitary isolates of these hormones, while 3246 hGH or
4152 hPRL ran at an appropriate smaller molecular weight as judged from the molecular weight standards. The gel position of proteins with a single residue deleted (
50 hPRL and
50 rPRL) could not be discerned by electrophoretic methods to be smaller than the parent protein.
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Comparison of hGH and 3246 hGH by spectroscopy showed that the deletion of the 15 residues produced significant stresses within the structure of hGH (Figure 4).
3246 hGH had significantly increased absorption when compared to wild-type hGH. When normalized to the absorbance at 277 nm the absorbance spectra showed that
3246 hGH had a much greater increase in absorbance in the 250 nm region than in the 280 nm region. Fluorescence studies using a 285 nm excitation showed a large reduction in the emission spectrum and a 2 nm red shift. These results indicated that deletion of residues 3246 resulted in a significant stress in the folded structure. These changes suggest that 20 kDa hGH has increased stress on the disulfide bonds, most likely the large disulfide loop (residues 58174) that is close to the deleted sequence.
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In contrast to hGH, the UV absorption and fluorescence spectra of F50 and
4152 hPRLs were quite similar to those of wild-type hPRL (Figure 6). Deletion of either resides 4152 or F50 produced a slight reduction in the absorbance in the 250 nm region, with small changes in the 280 nm region. The lack of signal in the 340 nm region indicated the absence of aggregation-associated light scatter. The fluorescence spectra showed a general increase in the signal with
4152 hPRL and a further increase in the signal for
F50 hPRL. Data normalized at 340 nm show that
4152 hPRL is red-shifted by
2 nm.
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Deletion of F50 in rPRL produced a protein with optical properties that are very similar to wild-type rPRL; both absorbance and fluorescence spectra essentially overlay (data not shown). In the Nb-2 bioassay wild-type rPRL had an ED50 approximately twice that of the wild-type hPRL. Deletion of F50 from rPRL increased the ED50 6-fold (Table III).
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Discussion |
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Removal of 15 residues from this section of hGH produces the 20 kDa form of the protein where activity through the PRL receptor is reduced 251-fold with a modest 2.6-fold reduction in somatotrophic activity. The losses of lactogenic activity associated with the removal of either F44 or residues 3246 (218- versus 251-fold reductions, respectively) are comparable. Thus, in hGH, F44 is the critical element in this motif and its removal can account for the reduced lactogenic activity of 20 kDa hGH.
Deletion of F50 from either hPRL or rPRL had only a modest effect on the activities of these hormones. Deletion of this residue had little effect on the structure of the protein in that the absorbance and fluorescence spectra were essentially unchanged. Thus, it appears that in PRLs the presence of F50 is relatively unimportant for biological activity. When the sequences for hGH and hPRL are aligned (Figure 2), the sequence homologous to residues 3246 of hGH is residues 4152 of hPRL. When these residues are removed, the activity of hPRL is reduced by >14 000-fold. This is in contrast to the situation in hGH where deletion of either F44 or residues 3246 produced similar and smaller (200-fold) changes in lactogenic activity. Therefore, this section of hPRL is critical for hPRL activity and the residues required for this activity do not include F50. F50 is also not critical in rPRL indicating that the importance of F50 is similar from PRLs of several species.
Evaluation of the sequences of hGH and hPRL suggest that they have evolved from a common ancestral gene (Kawauchi et al., 1990) and have retained a low sequence homology (Figure 2). During this evolution several of the major mechanisms by which these ligands bind and activate the hPRL receptor have been retained, including: receptor dimerization, ordered binding of receptors by the ligand, and the general structure of the ligands including the regions in which sites 1 and 2 are located. Despite these conserved general properties of the mechanism for lactogenic stimulation of target cells, many of the details differ between lactogenic hormones, including: affinity for the lactogenic receptor and biological potency within the same assay system. These differences must be based on structural differences and the mechanisms by which they perform their function. Although this region of these proteins is essential for lactogenic activities, the more subtle functional differences may be based on their divergent sequences. Thus, this diversity may represent an opportunity to fine-tune the functions of these proteins.
In all lactogens, helices 1 and 4 are connected most directly by a disulfide bond between cysteines 53 and 165 or between cysteines 58 and 174 in hGH or hPRL, respectively. Reduction or elimination of this disulfide bond essentially eliminates activity (Doneen et al., 1979; Luck et al., 1992
). C53 and C58 are located at one end of the mini-helix 1 region located immediately to the C-terminus of helix 1. Therefore, helices 1 and 4 are tethered to each other by this amino acid sequence and the helices' spatial articulation is at least in part defined by this sequence. When site 1 is bound to a PRL receptor, the articulation of helices 1 and 4 would be further restricted due to the binding-induced helix formation in this sequence. Binding a PRL receptor places F44 of hGH between Y160 and Y164 by promoting the formation of mini-helix 1 (Somers et al., 1994
; Chantalat et al., 1995
) that initiates a change of conformation propagated by functionally coupling residues (Duda and Brooks, 2003
) that are largely contained in two hydrophobic clusters (HC1 and HC2). This structure appears to be a switch mechanism for lactogenic activity, but not relevant for somatotrophic activity. With this mechanism it is not surprising that either elimination or replacement of F44 or the larger surrounding sequence have similar effects.
In hPRL the functional coupling of the two receptor-binding surfaces appears to be different. Removal of F50 from the loop does not influence activity and would only have a modest effect on mobility in this region of PRL. Removal of residues 4152 severely restricts the movement of helix 1 relative to helix 4 by reducing the length of the sequence linking them. This suggests that the structural interaction of F50 is dissimilar in hPRL. The pocket at the top of helix 4 (Y169 and H173) is less hydrophobic in hPRL than in hGH (Y160 and Y164) and may be less likely to form a hydrophobic cluster. Because only the removal of residues 4152 reduced the activity of hPRL, we anticipate that the restricted articulation of helices 1 and 4 is the primary mechanism for reduction of activity in hPRL.
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Acknowledgments |
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References |
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Received February 12, 2004; revised April 29, 2004; accepted May 21, 2004.
Edited by Andreas Kungl
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