title: Human growth hormone: 45-kDa isoform with extraordinarily stable interchain disulfide links has attenuated receptor-binding and cell-proliferative activities.
abstract: BACKGROUND
  Human growth hormone (hGH) is a complex mixture of molecular isoforms. Gaps in our knowledge exist regarding the structures and biological significances of the uncharacterized hGH molecular variants. Mercaptoethanol-resistant 45-kDa human growth hormone (MER-45 kDa hGH) is an extraordinarily stable disulfide-linked hGH homodimer whose biological significance is unknown.
  
  
  OBJECTIVE
  To elucidate the pharmacokinetic abilities of dimeric MER-45-kDa hGH to bind to GH and prolactin (PRL) receptors and to elucidate its abilities to stimulate cell proliferation in lactogen-induced and somatogen-induced in vitro cell proliferation bioassays.
  
  
  METHODS
  The binding of MER-45-kDa hGH to GH and PRL receptors was tested in radioreceptor assays (RRAs). Competitive displacements of [(125)I]-bovine GH from bovine liver membranes, [(125)I]-ovine PRL from lactating rabbit mammary gland membranes and [(125)I]-hGH from human IM-9 lymphocytes by unlabelled GHs, PRLs or dimeric MER-45-kDa hGH were evaluated. The abilities of dimeric MER-45-kDa hGH to stimulate proliferation of lactogen-responsive Nb2 lymphoma cells and to stimulate proliferation of somatogen-responsive T47-D human breast cancer cells were assessed by incubation of cells with GHs or PRLs and subsequently measuring growth using the MTS cell proliferation assay.
  
  
  RESULTS
  Dimeric MER-45-kDa hGH, compared to monomeric hGH, had reduced binding affinities to both GH and prolactin receptors. In a bovine liver GH radioreceptor assay its ED(50) (197.5 pM) was 40.8% that of monomeric hGH. In a human IM-9 lymphocyte hGH RRA its ED(50) (2.96 nM) was 26.2% that of monomeric hGH. In a lactating rabbit mammary gland prolactin RRA its ED(50) (3.56 nM) was 16.8% that of a monomeric hGH. Dimeric MER-45-kDa hGH, compared to monomeric hGH, had a diminished capacity to stimulate proliferation of cells in vitro. In a dose-response relationship assessing proliferation of Nb2 lymphoma cells its ED(50) (191 pM) was 18.0% that of monomeric hGH. While monomeric hGH stimulated a 2.2-fold proliferation of T47-D human breast cancer cells above vehicle control, dimeric MER-45-kDa hGH was unable to stimulate the cells to proliferate and slightly inhibited their proliferation to 77.6% that of control.
  
  
  CONCLUSIONS
  The topological arrangement of monomeric hGHs to form an unusually stable disulfide-linked dimer markedly diminishes hGH's binding affinities to both GH and PRL receptors and also drastically attenuates its ability to stimulate proliferation of cells in vitro.
authors.affiliation: Irma Lerma Rangel College of Pharmacy, Texas A&M Health Science Center, Kingsville, Texas 78363, USA.
authors.forename: Juan J
authors.initials: JJ
authors.lastname: Bustamante
authors.forename: Alexei L
authors.initials: AL
authors.lastname: Grigorian
authors.forename: Jesus
authors.initials: J
authors.lastname: Muñoz
authors.forename: Roberto M
authors.initials: RM
authors.lastname: Aguilar
authors.forename: Lisa R
authors.initials: LR
authors.lastname: Treviño
authors.forename: Andrew O
authors.initials: AO
authors.lastname: Martinez
authors.forename: Luis S
authors.initials: LS
authors.lastname: Haro
mesh_terms: D000818:Animals; D001665:Binding Sites; D002417:Cattle; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D005260:Female; D019382:Human Growth Hormone / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008214:Lymphocytes / Q000378:metabolism; D011388:Prolactin / Q000378:metabolism; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D011981:Receptors, Prolactin / Q000378:metabolism
chemical_list: D020033:Protein Isoforms; D011981:Receptors, Prolactin; D019382:Human Growth Hormone; D011388:Prolactin
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