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• W2164468235 abstract "Competitive antagonists of the human prolactin (hPRL) receptor are a novel class of molecules of potential therapeutic interest in the context of cancer. We recently developed the pure antagonist Del1-9-G129R-hPRL by deleting the nine N-terminal residues of G129R-hPRL, a first generation partial antagonist. We determined the crystallographic structure of Del1-9-G129R-hPRL, which revealed no major change compared with wild type hPRL, indicating that its pure antagonistic properties are intrinsically due to the mutations. To decipher the molecular bases of pure antagonism, we compared the biological, physicochemical, and structural properties of numerous hPRL variants harboring N-terminal or Gly129 mutations, alone or combined. The pure versus partial antagonistic properties of the multiple hPRL variants could not be correlated to differences in their affinities toward the hPRL receptor, especially at site 2 as determined by surface plasmon resonance. On the contrary, residual agonism of the hPRL variants was found to be inversely correlated to their thermodynamic stability, which was altered by all the Gly129 mutations but not by those involving the N terminus. We therefore propose that residual agonism can be abolished either by further disrupting hormone site 2-receptor contacts by N-terminal deletion, as in Del1-9-G129R-hPRL, or by stabilizing hPRL and constraining its intrinsic flexibility, as in G129V-hPRL. Competitive antagonists of the human prolactin (hPRL) receptor are a novel class of molecules of potential therapeutic interest in the context of cancer. We recently developed the pure antagonist Del1-9-G129R-hPRL by deleting the nine N-terminal residues of G129R-hPRL, a first generation partial antagonist. We determined the crystallographic structure of Del1-9-G129R-hPRL, which revealed no major change compared with wild type hPRL, indicating that its pure antagonistic properties are intrinsically due to the mutations. To decipher the molecular bases of pure antagonism, we compared the biological, physicochemical, and structural properties of numerous hPRL variants harboring N-terminal or Gly129 mutations, alone or combined. The pure versus partial antagonistic properties of the multiple hPRL variants could not be correlated to differences in their affinities toward the hPRL receptor, especially at site 2 as determined by surface plasmon resonance. On the contrary, residual agonism of the hPRL variants was found to be inversely correlated to their thermodynamic stability, which was altered by all the Gly129 mutations but not by those involving the N terminus. We therefore propose that residual agonism can be abolished either by further disrupting hormone site 2-receptor contacts by N-terminal deletion, as in Del1-9-G129R-hPRL, or by stabilizing hPRL and constraining its intrinsic flexibility, as in G129V-hPRL. Human (h) 4The abbreviations used are: h, human; PRL, prolactin; GH, growth hormone; PL, placental lactogen; PRLR, prolactin receptor; GHR, GH receptor; ECD, extracellular domain; r, rat; o, ovine; WT, wild type; FCS, fetal calf serum; PBS, phosphate-buffered saline; r.m.s.d., root mean square deviation; Ni-NTA, nickel-nitrilotriacetic acid; SPR, surface plasmon resonance; PDB, Protein Data Bank. prolactin receptor (PRLR) antagonists are a new class of potential drugs developed to target prolactin (PRL)-sensitive pathologies that cannot be treated with current inhibitors of the production of hPRL by the pituitary (1Goffin V. Touraine P. Culler M.D. Kelly P.A. Nat. Clin. Pract. Endocrinol. Metab. 2006; 2: 571-581Crossref PubMed Scopus (52) Google Scholar). These include dopamine-resistant prolactinomas (i.e. pituitary tumors of PRL-secreting cells), as well as breast cancer, prostate cancer, and benign prostate hyperplasia, in which evidence for the tumor growth-promoting actions of autocrine PRL has been emerging within the past decade (2Goffin V. Bernichtein S. Touraine P. Kelly P.A. Endocr. Rev. 2005; 26: 400-422Crossref PubMed Scopus (163) Google Scholar, 3Clevenger C.V. Furth P.A. Hankinson S.E. Schuler L.A. Endocr. Rev. 2003; 24: 1-27Crossref PubMed Scopus (453) Google Scholar). In view of the ubiquitous expression pattern of the PRLR (4Bole-Feysot C. Goffin V. Edery M. Binart N. Kelly P.A. Endocr. Rev. 1998; 19: 225-268Crossref PubMed Scopus (1612) Google Scholar), these indications are not necessarily exhaustive and may be extended to other pathologies that remain to be identified (or better characterized) with respect to the involvement of locally produced PRL in their etiology. Because of their mechanism of action involving competition with endogenous hPRL for receptor binding, competitive hPRLR antagonists need to be used in molar excess compared with endogenous hPRL. Therefore, the most promising compounds are anticipated to be those that are devoid of any residual agonistic properties, even at high concentration (5Bernichtein S. Kayser C. Dillner K. Moulin S. Kopchick J.J. Martial J.A. Norstedt G. Isaksson O. Kelly P.A. Goffin V. J. Biol. Chem. 2003; 278: 35988-35999Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). We recently developed an antagonist exhibiting such unique properties, referred to as Del1-9-G129R-hPRL. In contrast to other antagonists developed to date (for reviews see Refs. 2Goffin V. Bernichtein S. Touraine P. Kelly P.A. Endocr. Rev. 2005; 26: 400-422Crossref PubMed Scopus (163) Google Scholar, 6Goffin V. Tallet E. Jomain J.B. Kelly P.A. Recent Patents on Endocrine, Metabolic & Immune Drug Discovery. 2007; 1: 41-52Crossref Google Scholar), Del1-9-G129R-hPRL was shown to be devoid of residual agonism in every cell or animal model in which it has been tested to date (5Bernichtein S. Kayser C. Dillner K. Moulin S. Kopchick J.J. Martial J.A. Norstedt G. Isaksson O. Kelly P.A. Goffin V. J. Biol. Chem. 2003; 278: 35988-35999Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 7Ma F.Y. Anderson G.M. Gunn T.D. Goffin V. Grattan D.R. Bunn S.J. Endocrinology. 2005; 146: 5112-5119Crossref PubMed Scopus (69) Google Scholar, 8Ma F.Y. Grattan D.R. Goffin V. Bunn S.J. Endocrinology. 2005; 146: 93-102Crossref PubMed Scopus (43) Google Scholar, 9Eyal O. Jomain J.B. Kessler C. Goffin V. Handwerger S. Biol. Reprod. 2007; 76: 777-783Crossref PubMed Scopus (43) Google Scholar, 10Diogenes A. Patwardhan A.M. Jeske N.A. Ruparel N.B. Goffin V. Akopian A.N. Hargreaves K.M. J. Neurosci. 2006; 26: 8126-8136Crossref PubMed Scopus (106) Google Scholar, 11Dagvadorj A. Collins S. Jomain J.B. Abdulghani J. Karras J. Zellweger T. Li H. Nurmi M. Alanen K. Mirtti T. Visakorpi T. Bubendorf L. Goffin V. Nevalainen M.T. Endocrinology. 2007; 148: 3089-3101Crossref PubMed Scopus (110) Google Scholar). As highlighted by its name, Del1-9-G129R-hPRL is a hPRL core protein containing two modifications: deletion of the nine N-terminal residues, and substitution of Gly129 for an Arg (5Bernichtein S. Kayser C. Dillner K. Moulin S. Kopchick J.J. Martial J.A. Norstedt G. Isaksson O. Kelly P.A. Goffin V. J. Biol. Chem. 2003; 278: 35988-35999Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). The rationale for engineering this variant was based on previous structure-function studies performed in the Goffin laboratory. First, as demonstrated for all members of the PRL/growth hormone (GH)/placental lactogen (PL) family (2Goffin V. Bernichtein S. Touraine P. Kelly P.A. Endocr. Rev. 2005; 26: 400-422Crossref PubMed Scopus (163) Google Scholar), substitution of the conserved helix 3 Gly for an Arg was shown to drastically impair the agonistic properties of hPRL while maintaining its ability to bind to the PRLR (12Goffin V. Struman I. Mainfroid V. Kinet S. Martial J.A. J. Biol. Chem. 1994; 269: 32598-32606Abstract Full Text PDF PubMed Google Scholar, 13Goffin V. Kinet S. Ferrag F. Binart N. Martial J.A. Kelly P.A. J. Biol. Chem. 1996; 271: 16573-16579Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 14Kinet S. Bernichtein S. Kelly P.A. Martial J.A. Goffin V. J. Biol. Chem. 1999; 274: 26033-26043Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Second, the sole G129R mutation appeared to be insufficient to completely abolish the ability to activate the PRLR, as highlighted in sensitive cell bioassays (12Goffin V. Struman I. Mainfroid V. Kinet S. Martial J.A. J. Biol. Chem. 1994; 269: 32598-32606Abstract Full Text PDF PubMed Google Scholar, 15Bernichtein S. Jeay S. Vaudry R. Kelly P.A. Goffin V. Endocrine. 2003; 20: 177-190Crossref PubMed Scopus (41) Google Scholar) or in transgenic mice overexpressing this variant. 5V. Rouet, C. Kayser, and V. Goffin, unpublished observations. Third, mutations of the N terminus, which is interestingly the most divergent region within the PRL/GH/PL family, were shown to slightly modulate the properties of hPRL (16Bernichtein S. Jomain J.B. Kelly P.A. Goffin V. Mol. Cell. Endocrinol. 2003; 208: 11-21Crossref PubMed Scopus (19) Google Scholar). Although the combination of the N terminus deletion and the G129R mutation was successful in achieving the goal of generating a pure PRLR antagonist, the mechanism underlying these unique properties remained poorly understood. Although structural studies of the hGH·hGHR complex have clearly identified the helix 3 Gly pocket as a critical characteristic of binding site 2 (17De Vos A.M. Ultsch M. Kossiakoff A.A. Science. 1992; 255: 306-312Crossref PubMed Scopus (2029) Google Scholar, 18Fuh G. Cunningham B.C. Fukunaga R. Nagata S. Goeddel D.V. Wells J.A. Science. 1992; 256: 1677-1680Crossref PubMed Scopus (573) Google Scholar), the roles of the structurally equivalent region (Gly129), and even more of the N terminus, in receptor binding and activation have remained largely speculative for hPRL. Ultimately, this lack of information about the molecular bases of our best antagonist hampers its improvement through knowledge-assisted strategies. The aim of this work was to elucidate the importance of these two hPRL modifications at the molecular level, using a combination of structural, biophysical, and biological approaches. First, to identify the structural characteristics underlying its pure antagonistic properties, we determined the three-dimensional structure of Del1-9-G129R-hPRL by x-ray diffraction. Second, we generated numerous hPRL variants harboring single or combined modifications at the N terminus (deletion/elongation) and the Gly129 residue (different substitutions), to be compared with the prototype G129R-hPRL and Del1-9-G129R-hPRL variants. These mutants were analyzed for their (residual) agonistic and antagonistic properties in cell bioassays, and for their thermodynamic stability and structural integrity. Finally, we designed an appropriate methodology to determine independently the affinities of binding sites 1 and 2 of all the variants by surface plasmon resonance. Culture media, fetal calf serum (FCS), geneticin (G-418), trypsin, and glutamine were purchased from Invitrogen. Luciferin and cell lysis buffer were from Promega (Madison, WI), and luciferase activity was measured in relative light units using a Lumat LB 9501 (Berthold, Nashua, NH). IODO-GEN was purchased from Sigma, and carrier-free Na125I was obtained from GE Healthcare. Oligonucleotides were from Eurogentec (Liège, Belgium). All immobilization reagents used for surface plasmon resonance experiments were purchased from Biacore (Uppsala, Sweden). Optimization of crystallization conditions was performed in Linbro plates from Hampton. Chemicals were purchased from Sigma, VWR (Fontenay-sous-Bois, France), or Merck. Expression plasmids encoding WT hPRL, G129R-hPRL, and Del1-9-G129R-hPRL were available from previous studies (5Bernichtein S. Kayser C. Dillner K. Moulin S. Kopchick J.J. Martial J.A. Norstedt G. Isaksson O. Kelly P.A. Goffin V. J. Biol. Chem. 2003; 278: 35988-35999Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 12Goffin V. Struman I. Mainfroid V. Kinet S. Martial J.A. J. Biol. Chem. 1994; 269: 32598-32606Abstract Full Text PDF PubMed Google Scholar). They were used as templates for generating the other mutants of this study, using the QuikChange II mutagenesis kit from Stratagene (La Jolla, CA). Sequences of forward and reverse (complementary) primers are given in Table 1, with mutated codons underlined. The same primers were used for generating Nter-hPRL and Nter-G129R-hPRL plasmids using, respectively, WT hPRL and G129R-hPRL encoding plasmids as templates. For all steps, we strictly followed the recommendations of the manufacturer. After transformation, Escherichia coli BL21(DE3) colonies were analyzed for their DNA content; plasmids were sequenced to verify the presence of the expected mutations.TABLE 1Mutagenesis primershPRL variantsPrimersG129PForward, 5′-CAAACGGCTTCTAGAGGCCATGGAGCTGATAGTC-3′Reverse, 5′-GACTATCAGCTCCATGGCCTCTAGAAGCCGTTTG-3′G129DForward, 5′-CAAACGGCTTCTAGAGGACATGGAGCTGATAGTC-3′Reverse, 5′-GACTATCAGCTCCATGTCCTCTAGAAGCCGTTTG-3′G129FForward, 5′-CAAACGGCTTCTAGAGTTCATGGAGCTGATAGTC-3′Reverse, 5′-GACTATCAGCTCCATGAACTCTAGAAGCCGTTTG-3′G129LForward, 5′-CAAACGGCTTCTAGAGCTGATGGAGCTGATAGTC-3′Reverse, 5′-GACTATCAGCTCCATCAGCTCTAGAAGCCGTTTG-3′G129NForward, 5′-CAAACGGCTTCTAGAGAACATGGAGCTGATAGTC-3′Reverse, 5′-GACTATCAGCTCCATGTTCTCTAGAAGCCGTTTG-3′G129YForward, 5′-CAAACGGCTTCTAGAGTACATGGAGCTGATAGTC-3′Reverse, 5′-GACTATCAGCTCCATGTACTCTAGAAGCCGTTTG-3′G129VForward, 5′-CAAACGGCTTCTAGAGGTCATGGAGCTGATAGTC-3′Reverse, 5′-GACTATCAGCTCCATGACCTCTAGAAGCCGTTTG-3′Nter and Nter-G129RForward, 5′-GAGATATACATATGGCACAGCATCCACCATACTGTCCCGGCGGGG-3′Reverse, 5′-CCCCGCCGGGACAGTATGGTGGATGCTGTGCCATATGTATATCTC-3′ Open table in a new tab Plasmids encoding the extracellular domain (ECD) of human or rat (r) PRLR were generated by PCR amplification using plasmids containing the full-length receptor cDNA (19Boutin J.M. Edery M. Shirota M. Jolicoeur C. Lesueur L. Ali S. Gould D. Djiane J. Kelly P.A. Mol. Endocrinol. 1989; 3: 1455-1461Crossref PubMed Scopus (238) Google Scholar, 20Boutin J.M. Jolicoeur C. Okamura H. Gagnon J. Edery M. Shirota M. Banville D. Dusanter-Fourt I. Djiane J. Kelly P.A. Cell. 1988; 53: 69-77Abstract Full Text PDF PubMed Scopus (452) Google Scholar); sequences encoding residues 1-210 of WT PRLR were inserted at SpH1-BamHI sites into the pQE-70 expression plasmid containing a His6 tag at the C-terminal end (Qiagen, Courtaboeuf, France). Subcloning constraints led to the addition of four amino acids just before the His tag (Gly211-Ser212-Arg213-Ser214 for hECD, and Arg211-Ser212-Arg213-Ser214 for rECD) as described (21Teilum K. Hoch J.C. Goffin V. Kinet S. Martial J.A. Kragelund B.B. J. Mol. Biol. 2005; 351: 810-823Crossref PubMed Scopus (83) Google Scholar). Recombinant WT hPRL, hPRL variants, and PRLR ECDs were overexpressed in 0.5-1-liter cultures of E. coli BL21(DE3) and purified as described previously (22Paris N. Rentier-Delrue F. Defontaine A. Goffin V. Lebrun J.J. Mercier L. Martial J.A. Biotechnol. Appl. Biochem. 1990; 12: 436-449PubMed Google Scholar), with minor modifications. Briefly, when the A600 of bacterial cultures reached 0.7-0.9, overexpression was induced using 2 mm isopropyl 1-thio-β-d-galactopyranoside for 4 h (A600 = 2-2.5 after 4 h). Cells were broken using high pressure (French press). Proteins were overexpressed as insoluble inclusion bodies, which were solubilized in 8 m urea (5 min at 55 °C and then 2 h at room temperature) and refolded by continuous dialysis (72 h, 4 °C) against 100 volumes of 25 mm NH4HCO3, pH 8.6. Solubilized proteins were centrifuged and then loaded onto a HiTrap Q anion exchange column (GE Healthcare) equilibrated in 25 mm NH4HCO3, pH 8.6. Prolactin (WT and variants) and receptor ECDs were eluted along a NaCl gradient (0-500 mm), and the major peak was collected, quantified, and kept frozen until use. Purity of the various hPRL variant/receptor ECD batches was >95% as judged from SDS-PAGE analysis. Initial crystallization screening was performed in 96-well sitting drop crystallization plates (Greiner Bio-One) using a Cybi-Disk robot from Cybio. Crystallization screens were set up using several commercially available high throughput crystallization screening kits (Hampton Research). Crystals appeared as needle clusters after 1 week at 18 °C in several conditions (29Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13914) Google Scholar, 36Glezer A. Soares C.R. Vieira J.G. Giannella-Neto D. Ribela M.T. Goffin V. Bronstein M.D. J. Clin. Endocrinol. Metab. 2006; 91: 1048-1055Crossref PubMed Scopus (73) Google Scholar, 41Bernat B. Pal G. Sun M. Kossiakoff A.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 952-957Crossref PubMed Scopus (44) Google Scholar) of the MemFac crystallization kit. The initial crystals were refined using standard techniques, which lead to isolated needles too thin to be collected. A second screening was performed by mixing 75% of the optimized crystallization solution with 25% of the crystal screen kits from Hampton. Larger needles appeared in five new conditions. After manual optimization in Linbro plates using the hanging drop method, only one led to diffracting crystals. The corresponding reservoir was composed of 100 mm Tris-HCl, pH 8.5, 675 mm K2HPO4, 45 mm (NH4)2PO4, 50 mm LiSO4, 7.5% glycerol, 8% PEG4000. The drop was formed by mixing 1.5 μl of protein at 10 mg/ml with 1.5 μl of reservoir. The largest final crystal size was 40 × 40 × 200 μm3. Best diffracting crystals were flash-frozen in liquid nitrogen without previous soaking. A complete data set at 2.6 Å resolution was obtained on the ID29 beam line at ESRF (Grenoble, France). The diffraction images were reduced, scaled, and merged with programs MOSFLM and SCALA (23Potterton E. Briggs P. Turkenburg M. Dodson E. Acta Crystallogr. Sect. D. Biol. Crystallogr. 2003; 59: 1131-1137Crossref PubMed Scopus (1080) Google Scholar, 24No Collaborative Computational Project Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar). The intensities were then converted to the structural factor amplitudes with TRUNCATE (23Potterton E. Briggs P. Turkenburg M. Dodson E. Acta Crystallogr. Sect. D. Biol. Crystallogr. 2003; 59: 1131-1137Crossref PubMed Scopus (1080) Google Scholar, 24No Collaborative Computational Project Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar). A summary of the crystallographic data and refinement statistics is given in Table 2. The crystals belonged to space group I4 with unit cell dimensions of a = b = 122.59 Å, c = 28.68 Å. The Matthews coefficient (2.25) and solvent content analysis (∼42%) indicated the presence of one antagonist molecule in the asymmetric unit.TABLE 2X-ray diffraction data collection and refinement statistics for Del1-9-G129R-hPRLData collectionSpace group14Unit cell parameter (Å)a = b = 122.59, c = 28.68Resolution (Å)85.00-2.60 (2.74-2.60)aValues in parentheses are for the highest resolution shell.Total no. of reflections15,583 (2216)No. of unique reflections6433 (905)Completeness (%)93.8 (93.1)RmergebRmerge = ∑h∑j|〈I〉h - Ih,j|/∑h∑jIh,j, where 〈I〉h is the mean intensity of symmetry equivalent reflections.0.080 (0.235)I/σ(I)12.1 (5.5)Multiplicity2.4 (2.4)RefinementResolution (Å)20.0-2.7 (2.77-2.7)No. of reflections5139 (375)R/Rfree (%)c∑|Fobs - Fcalc|/∑Fobs. The formula for Rfree is the same as that for R, except it is calculated with a portion of the structure factors that had not been used for refinement.21.1/30.2 (29.0/35.6)Free R value test set size (%)/count11/632Mean B value Å233.8Correlation coefficient Fo - Fc/free0.919/0.831Total no. of residues/water191/48r.m.s.d.Bond lenghts (Å)0.012Bond angles (°)1.391Ramachandran plot (%)Most favored86.0Allowed8.9a Values in parentheses are for the highest resolution shell.b Rmerge = ∑h∑j|〈I〉h - Ih,j|/∑h∑jIh,j, where 〈I〉h is the mean intensity of symmetry equivalent reflections.c ∑|Fobs - Fcalc|/∑Fobs. The formula for Rfree is the same as that for R, except it is calculated with a portion of the structure factors that had not been used for refinement. Open table in a new tab The crystal structure of the antagonist was solved by molecular replacement using the program PHASER (25Read R.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 1373-1382Crossref PubMed Scopus (789) Google Scholar, 26Storoni L.C. McCoy A.J. Read R.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 432-438Crossref PubMed Scopus (1103) Google Scholar, 27McCoy A.J. Grosse-Kunstleve R.W. Storoni L.C. Read R.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 2005; 61: 458-464Crossref PubMed Scopus (1602) Google Scholar), with the structure of oPL extracted from the 2:1 complex structure of oPL/rPRLR (Protein Data Bank code 1F6F) used as search model. A PHASER search with the WT hPRL structure resolved by NMR (Protein Data Bank code 1RW5) used as search model also yielded the right solution, but the PHASER statistics were not convincing. Refinement of the structure was carried out through multiple cycles of manual rebuilding using the program O (28Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar) and refinement using Refmac5 (29Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13914) Google Scholar), resulting in a final model with an R factor of 21.1% and an Rfree factor of 30.2%. The refined structure was validated by the program PROCHECK (30Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). Figure panels showing three-dimensional structures were generated using the PyMOL Molecular Graphics System (DeLano Scientific). The refined structure of the antagonist and that of WT hPRL (PDB code 1RW5) (21Teilum K. Hoch J.C. Goffin V. Kinet S. Martial J.A. Kragelund B.B. J. Mol. Biol. 2005; 351: 810-823Crossref PubMed Scopus (83) Google Scholar) were both subjected to all-atom normal mode analysis, using the web-based servers HingeMaster (31Flores S. Echols N. Milburn D. Hespenheide B. Keating K. Lu J. Wells S. Yu E.Z. Thorpe M. Gerstein M. Nucleic Acids Res. 2006; 34: D296-D301Crossref PubMed Scopus (125) Google Scholar) and NOMAD-Ref (32Lindahl E. Azuara C. Koehl P. Delarue M. Nucleic Acids Res. 2006; 34: W52-W56Crossref PubMed Scopus (293) Google Scholar). Far-UV CD spectra as well as temperature melts of all hPRL variants were recorded on a Jasco 810 spectropolarimeter. Temperature melts were performed in 0.01 m NaH2PO4, pH 7.4, using a water-jacketed cuvette of 1-cm light path length, from 37 to 95 °C, and the change in signal followed at 222 nm. Far-UV CD spectra were run at 37 °C from 250 to 190 nm, with a 1-cm path length. However, because of high absorbance from buffer, no signal below 197 to 195 nm could be interpreted. Protein concentrations were set at ∼1-2 μm for the temperature melts and far-UV CD spectra. Unfolding was assumed to follow a two-state transition. Circular dichroism thermal scans were fitted to the Gibbs-Helmholz equation as described (33Oliveberg M. Vuilleumier S. Fersht A.R. Biochemistry. 1994; 33: 8826-8832Crossref PubMed Scopus (82) Google Scholar), using Gnuplot. Because of the high melting temperatures, which result in the lack of sufficient post-transition regions, it was assumed that the unfolded state was unchanged by the mutations. The slope of the post-transition region was therefore extrapolated for each mutant protein from the well determined post-transition of G129P-hPRL. The results of the fits are melting temperature (Tm) and change in enthalpy on unfolding at Tm (ΔH(Tm)). Immobilization of PRLR ECD—As a first approach, the ECDs (human or rat) were covalently coupled in a random orientation, through their solvent-accessible primary amine groups, to the carboxymethylated dextran matrix of a CM5 sensor chip, using a Biacore 2000 instrument and the amine coupling kit (Biacore), according to manufacturer's instructions. Briefly, each flow cell, equilibrated at a flow rate of 5 μl/min in phosphate-buffered saline (PBS, pH 7.4, supplemented with 0.005% Tween 20), was activated for 12 min with an NHS-EDC solution (50 mm N-hydroxysuccinimide and 200 mm N-ethyl-N′-(3 dimethylaminopropyl) carbodiimide), followed by an injection of the ECD of interest (100 nm) in 10 mm sodium acetate, pH 4.5. The surface was finally deactivated for 12 min with 1 m ethanolamine, pH 8.5. We routinely immobilized 500-1500 resonance units (1 resonance unit ∼1 pg·mm-2) of ECD on three flow cells out of four on the sensor chip. The fourth, treated only by NHS-EDC and ethanolamine, was used as a reference surface. In a second approach, the ECDs were covalently coupled in an oriented fashion onto a nitrilotriacetic acid-derivatized NTA sensor chip, as described (34Willard F.S. Siderovski D.P. Anal. Biochem. 2006; 353: 147-149Crossref PubMed Scopus (18) Google Scholar). Briefly, each flow cell, equilibrated at a flow rate of 5 μl/min in PBS supplemented with 0.005% Tween 20 and 50 μm EDTA, was sequentially loaded with 500 μm NiCl2 for 4 min and activated with NHS-EDC for 2 min. The His6-tagged ECD (100 nm in PBS/Tween/EDTA) was injected until 500-2,000 resonance units were captured by the Ni-NTA moieties. The surface was finally deactivated by a 4-min ethanolamine injection, followed by a 2-min injection of EDTA 0.35 m. One flow cell was used as a reference surface, after activation by NiCl2 and NHS-EDC, and deactivation by ethanolamine and EDTA. Real Time Binding Assays—All the binding assays were performed at 25 °C at a flow rate of 20 μl/min, in running buffer (25 mm NH4HCO3, pH 8.6, 150 mm NaCl, 0.005% Tween 20). For site 1 characterization, 5-8 different concentrations (ranging from 0.54 to 350 nm) of the hPRL variants were injected for 12 min onto the PRLR surfaces, followed by a 10-min dissociation period. At the end of each cycle, the sensor chip was regenerated by two 1-min injections of 2 m MgCl2 (followed by a 1-min injection of EDTA 0.35 m for Ni-NTA chips only). For site 2 characterization, the PRLR surface was first saturated with each hPRL variant (350 nm). Five concentrations of ECD (ranging from 1.7 to 350 μm) were then injected for 2 min on the ECD·PRL complexes, followed by a 5-min dissociation period. Control experiments showed the absence of detectable ECD-ECD interaction in the absence of hormone (not shown). Data Analysis—All the association and dissociation profiles were double-referenced using the Scrubber 2.0 software. The site 1 binding curves were globally analyzed with a nonlinear least squares algorithm implemented in the BIAevaluation 4.1 software (Biacore), using single-exponential functions of time (Langmuir monovalent binding model). The site 2 binding curves were subjected to steady-state analysis. Kinetic parameters (kon and koff) and equilibrium dissociation constants (Kd) were determined based on at least two experiments. Binding affinities of hPRL variants were determined using cell homogenates of HEK 293 cells stably expressing the human PRLR (so-called HL-5 clone), following a procedure described previously (14Kinet S. Bernichtein S. Kelly P.A. Martial J.A. Goffin V. J. Biol. Chem. 1999; 274: 26033-26043Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Briefly, hPRL was iodinated using IODO-GEN, and binding assays were performed overnight at room temperature using 150-300 μg of cell homogenate protein in the presence of 20,000-30,000 cpm 125I-labeled hPRL and increasing concentrations of unlabeled competitor (WT or mutated hPRL). Results presented are representative of at least three independent experiments performed in duplicate. The relative binding affinities of hPRL variants were calculated with respect to that of WT hPRL based on their IC50. The biological properties of hPRL variants were analyzed using two homologous bioassays that we recently developed for human lactogens (15Bernichtein S. Jeay S. Vaudry R. Kelly P.A. Goffin V. Endocrine. 2003; 20: 177-190Crossref PubMed Scopus (41) Google Scholar). The transcriptional assay involved HEK 293 cells stably expressing the hPRLR and the lactogenic hormone-response element-luciferase reporter gene (HL-5 clone); it was used to determine the antagonistic properties by competing a fixed concentration (9 nm) of WT hPRL with increasing amounts of each variant (14Kinet S. Bernichtein S. Kelly P.A. Martial J.A. Goffin V. J. Biol. Chem. 1999; 274: 26033-26043Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 15Bernichtein S. Jeay S. Vaudry R. Kelly P.A. Goffin V. Endocrine. 2003; 20: 177-190Crossref PubMed Scopus (41) Google Scholar, 35Bernichtein S. Kinet S. Jeay S. Madern M. Martial J.A. Kelly P.A. Goffin V. Endocrinology. 2001; 142: 3950-3963Crossref PubMed Scopus (36) Google Scholar). A proliferation assay was also performed to determine the residual agonistic activity of the antagonists, using Ba/F-03 cells stably expressing the human PRLR (referred to as Ba/F-LP cells); this assay has been shown to be much more sensitive than the HL-5 clone to reveal low level agonistic effects (5Bernichtein S. Kayser C. Dillner K. Moulin S. Kopchick J.J. Martial J.A. Norstedt G. Isaksson O. Kelly P.A. Goffin V. J. Biol. Chem. 2003; 278: 35988-35999Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 15Bernichtein S. Jeay S. Vaudry R. Kelly P.A. Goffin V. Endocrine. 2003; 20: 177-190Crossref PubMed Scopus (41) Google Scholar, 36Glezer A. Soares C.R. Vieira J.G. Giannella-Neto D. Ribela M.T. Goffin V. Bronstein M.D. J. Clin. Endocrinol. Metab. 2006; 91: 10" @default.
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