title: Solubilization and characterization of a lactogenic receptor from human placental chorion membranes.
abstract: Prolactin has a wide range of actions, including osmoregulation and the control of mammary gland development and lactation. These effects are mediated through a high-affinity cell surface receptor, which has been well characterized in a number of animal tissues. The molecular characteristics of the human receptor are unknown, however. The present studies were initiated, therefore, to determine the binding and molecular characteristics of the lactogenic receptor of human placental chorion membranes. Subcellular fractionation studies showed that the bulk of the receptor sedimented in the microsomal fraction at 45,000gav. Endogenous ligand was dissociated from the receptor with 3.5 M MgCl2 or 0.05 M acetate buffer (pH 4.8) with preservation of binding activity. The microsomal receptor bound human growth hormone (hGH), human prolactin (hPRL), ovine prolactin (oPRL), and human placental lactogen (hPL) but not non-primate growth hormones, indicating a narrow specificity for lactogenic hormones. The binding was only partially reversible in agreement with the known binding kinetics of animal lactogenic receptors. The receptor was solubilized with 45% yield from the microsomes using 16 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulphonate (CHAPS) detergent-250 mM NaCl, and the binding activity was fully restored by a two-fold dilution in the binding reaction to reveal a KD of 0.8 nM for hGH and a binding capacity of 200 fmol of specifically bound hGH per mg of microsomal protein. Gel filtration chromatography indicated the minimum molecular weight of the ligand-receptor complex was approximately 60,000 daltons, and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of covalently cross-linked 125I-hGH-receptor complexes revealed a molecular size of 58,000 daltons. When account was taken of the contribution of the ligand, a molecular weight of 36,000 for the receptor's binding domain was obtained. These data indicate that the chorion lactogenic receptor has very similar binding and molecular characteristics to the lactogenic receptors from other mammalian species. Chorion membranes are thus a convenient source of material for the further purification and characterization of the human lactogenic receptor.
authors.affiliation: Garvan Institute of Medical Research, St. Vincent's Hospital, Sydney, New South Wales, Australia.
authors.forename: C J
authors.initials: CJ
authors.lastname: Ormandy
authors.forename: C L
authors.initials: CL
authors.lastname: Clarke
authors.forename: R L
authors.initials: RL
authors.lastname: Sutherland
mesh_terms: D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism; D002793:Cholic Acids; D002823:Chorion / Q000378:metabolism* / Q000648:ultrastructure; D002850:Chromatography, Gel; D003720:Densitometry; D003902:Detergents; D005260:Female; D013006:Growth Hormone / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D008861:Microsomes / Q000378:metabolism; D010928:Placental Lactogen / Q000378:metabolism*; D011247:Pregnancy; D011388:Prolactin / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018000:Receptors, Peptide; D012965:Sodium Chloride; D012995:Solubility
chemical_list: D002793:Cholic Acids; D003902:Detergents; D011956:Receptors, Cell Surface; D018000:Receptors, Peptide; C028202:placental lactogen receptor; D015636:Magnesium Chloride; D012965:Sodium Chloride; D011388:Prolactin; D013006:Growth Hormone; D010928:Placental Lactogen; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate