The growth hormone (GH)-binding protein cloned from human IM-9 lymphocytes modulates the down-regulation of GH receptors by 22- and 20-kilodalton human GH in IM-9 lymphocytes and the biological effects of the hormone in Nb2 lymphoma cells.
The cDNA coding for the 246-amino acid long N-terminal extracellular portion of the human (h) GH receptor, corresponding to the circulating GH-binding protein (hGHBP), was cloned by polymerase chain reaction from human IM-9 lymphocytes. The cDNA sequence was identical to that reported for human liver and placenta and demonstrated alternative splicing of exon 3. The protein with the exon 3-encoded domain was expressed and secreted in glycosylated form from baby hamster kidney (BHK) cells, purified to homogeneity, and sequenced; the amino acid sequence was identical to that predicted from liver cDNA. The cloned hGHBP competed in a dose-dependent fashion for binding of 125I-labeled 22-kilodalton (kDa) hGH, and at higher concentrations for binding of 125I-labeled 20-kDa hGH, to IM-9 lymphocytes. hGHBP decreased the association rate of [125I]hGH to the cells without decreasing the dissociation rate. hGHBP blocked the down-regulation of GH receptor in IM-9 cells by both 22- and 20-kDa hGH. hGHBP also blocked the binding of [125I]hGH to PRL receptors on Nb2 lymphoma cells and the effect of the hormone on thymidine incorporation. Binding of both 22- and 20-kDa hGH to the binding protein was demonstrated directly by immunoprecipitation with monoclonal antibody 263. The present work thus establishes the identity of the IM-9 human GHBP from those of liver and placenta, and demonstrates its ability to bind both 22- and 20-kDa hGH with good affinity and to block their biological actions mediated though both somatogenic and lactogenic receptors. The modulation of receptor down-regulation by the BP may be a relevant facet of its physiological role.
Hansen
B S
BS
Biopharmaceutical Division, Novo Nordisk A/S, Gentofte, Denmark.
Hjorth
S
S
Welinder
B S
BS
Skriver
L
L
De Meyts
P
P
D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000494:pharmacology*; D003001:Cloning, Molecular; D004247:DNA / Q000096:biosynthesis; D015536:Down-Regulation / Q000187:drug effects*; D013006:Growth Hormone / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D008214:Lymphocytes / Q000378:metabolism*; D008223:Lymphoma / Q000378:metabolism* / Q000473:pathology; D015335:Molecular Probes / Q000235:genetics; D008969:Molecular Sequence Data; D011986:Receptors, Somatotropin / Q000187:drug effects / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D015335:Molecular Probes; D011986:Receptors, Somatotropin; D013006:Growth Hormone; D004247:DNA; C051815:somatotropin-binding protein