PEDS Advance Access originally published online on July 13, 2004
Protein Engineering Design and Selection 2004 17(5):417-424; doi:10.1093/protein/gzh051
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Different elements of mini-helix 1 are required for human growth hormone or prolactin action via the prolactin receptor
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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Human growth hormone (hGH) and prolactin (hPRL) have a low sequence homology, but both bind and activate hPRL receptors. hGH also binds hGH receptors. hGH has 22 and 20 kDa forms; residues 32–46 have been deleted by alternative RNA splicing to create the smaller form. hGH requires F44 for activity through the hPRL receptor, but not for activity through the hGH receptor. The deletion of F44 from hGH has the same effect as removal of residues 32–46 (
200-fold loss in activity), indicating the importance of F44 in hGH when activating the hPRL receptor. In contrast, when the homologous F50 is deleted from hPRL little or no activity is lost, indicating that this highly conserved phenylalanine is not required for the action of hPRL. Deletion of residues 41–52 (a non-conserved sequence homologous to residues 32–46 of hGH) reduced the activity of hPRL by >14 000-fold. This region is essential for the biological activity of hPRL. As these two proteins have evolved from a common ancestor, they have retained the requirement for this region but need different structural elements to activate hPRL receptors. Such diversity represents an opportunity to fine-tune hormone activity.
Keywords: growth hormone/human/prolactin/rat
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Introduction |
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Growth hormone (GH), prolactin (PRL) and placental lactogen (PL) are a subfamily of a large 2-class cytokine superfamily of proteins (Kossiakoff and de Vos, 1998
). The amino acid sequences of hGH and hPL are similar (85% homology) while the sequence homology of hPRL to the other two proteins is substantially lower (
23% homology) (Niall, 1971; Sherwood et al., 1971; Shome and Parlow, 1977). In humans, each of these three proteins can bind hPRL receptors and promote a variety of physiological actions (Bern and Nicoll, 1968; Kelly et al., 1984).
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 up–up–down–down 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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The binding of hPRLbp to these ligands is an ordered (Hooper et al., 1993
; Gertler et al., 1996) and highly cooperative process. Functional linkages (Wyman and Gill, 1990) between distant residues in ligands have been repeatedly documented (see Johnson and Akers, 1995) and in the case of hGH, structural comparisons of receptor-bound and receptor-free ligand show that binding of the first hPRLbp induces a conformation change (compare PDB# 1HGU with PDB#s 1A22 or 1BP3). These structures reveal that upon receptor binding at site 1, the section of hGH between the C-terminus of helix 1 (approximately Ala34) and Cys53 (that forms the large disulfide loop) plus the terminal portion of helix 3 (P89 to N99) become more ordered. A short helix, frequently called mini-helix 1, is formed in this first section of hGH when bound to a receptor protein. The receptor-free hPRL structure does not show a small helix in this area, and it is unknown if a helix forms when bound by hPRLbp.
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 32–46 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 32–46, 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 41–52 from hPRL (
41–52 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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Plasmids and bacterial strains
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: 32–41 hGH, F50 hPRL, 41–52 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 32–46 hGHs, wild-type, F50 and 41–52 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 dose–response 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 dose–response 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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Preparation and characterization of recombinant hGHs and hPRLs
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 32–46 hGH or 41–52 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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Spectroscopy and biological activity of human growth hormones
Comparison of hGH and 32–46 hGH by spectroscopy showed that the deletion of the 15 residues produced significant stresses within the structure of hGH (Figure 4). 32–46 hGH had significantly increased absorption when compared to wild-type hGH. When normalized to the absorbance at 277 nm the absorbance spectra showed that 32–46 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 32–46 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 58–174) that is close to the deleted sequence.
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In the FDC-P1 somatotrophic bioassay deletion of residues 32–46 decreased the ED50 by
2.6-fold (Figure 5A and Table I). In contrast, in the Nb-2 biological assay for PRL activity, deletion of these residues reduced the ED50 by 251-fold (Figure 5B and Table II). The ED50s for wild-type hGH in the FDC-P1 and Nb-2 assays were 0.2 and 20 pM, respectively. The affinity for the PRL receptor in Nb-2 cells, as determined by competitive radioligand-binding assays (Table II) was reduced from a Kd of 0.36 nM for wild-type hGH to 17.6 nM for the for 32–46 hGH, an 50-fold loss of affinity.
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Spectroscopy and biological activity of human prolactins
In contrast to hGH, the UV absorption and fluorescence spectra of F50 and 41–52 hPRLs were quite similar to those of wild-type hPRL (Figure 6). Deletion of either resides 41–52 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 41–52 hPRL and a further increase in the signal for F50 hPRL. Data normalized at 340 nm show that 41–52 hPRL is red-shifted by 2 nm.
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Deletion of F50 from hPRL reduced the activity by
2.7-fold from that of the wild-type protein (Figure 7 and Table III) in the Nb-2 biological assay. This reduction in activity is much smaller than hGH where a homologous deletion of F44 resulted in a 218-fold loss in activity (Peterson and Brooks, 1997). Deletion of residues 41–52 from hPRL created a protein analogous to the 20 kDa form of hGH. 41–52 hPRL activity in the Nb-2 bioassay was reduced by 14 375-fold from the activity of wild-type hPRL. This reduction in activity is 50-fold greater than that for the homologous deletion of residues 32–46 in hGH that resulted in only a 251-fold reduction. The maximal stimulation (Figure 7, y-axis) of Nb-2 cell proliferation by 41–52 hPRL was significantly less than either wild-type or F50 hPRLs.
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Spectroscopy and biological activity of rat prolactins
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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The sequence of amino acids between the C-terminus of helix 1 and the first cysteine of the large disulfide loop in both hGH and hPRL form a functional motif required for activity initiated at the PRL receptor, but not for somatotrophic activity. Our work demonstrates that the residues required for activation of the PRL receptor differ when comparing hGH with either hPRL or rPRL. Phenylalanine 44 in hGH or F50 in hPRL or rPRL is conserved in all lactogenic hormones but is absent in non-primate GHs, family members with only somatotrophic activities. Deletion of this residue or diminution of its hydrophobic character in hGH reduces its activity via the PRL receptor by up to 218-fold (Peterson and Brooks, 1997
). Addition of a phenylalanine to bovine GH in this position was one of two steps required to provide this somatotrophic hormone with full lactogenic activity (Peterson and Brooks, 2000). Surprisingly, deletion of F50 in hPRL or rPRL results in only a 2–6-fold reduction of activity, indicating that F50 is less important in activating the PRL receptor than in hGH. Deletion of this phenylalanine had little effect on either the absorbance or fluorescence spectra of hGH, hPRL or rPRL, indicating little gross structural perturbation. Therefore, phenylalanine in this position appears to play a specific role in the local structure of hGH important for both binding and activation of the PRL receptor, but not required for hPRL action. 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 32–46 (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 32–46 of hGH is residues 41–52 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 32–46 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 41–52 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 41–52 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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Pituitary isolates of hGH, hPRL and rPRL were kindly provided by the National Hormone and Pituitary Program, Dr A.Parlow, Director (http://www.humc.edu/hormones/) supported by the National Institutes of Health. Supported by grant DK 56117 from the National Institutes of Health, US Department of Health and Human Services.
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Received February 12, 2004; revised April 29, 2004; accepted May 21, 2004.
Edited by Andreas Kungl
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