TY - JOUR

T1 - Nuclear translocation and retention of growth hormone

AU - Mertani, Hichem C

AU - Raccurt, Mireille

AU - Abbate, Aude

AU - Kindblom, Jenny

AU - Törnell, Jan

AU - Billestrup, Nils

AU - Usson, Yves

AU - Morel, Gérard

AU - Lobie, Peter E

PY - 2003/7

Y1 - 2003/7

N2 - We have previously demonstrated that GH is subject to rapid receptor-dependent nuclear translocation. Here, we examine the importance of ligand activation of the GH-receptor (GHR)-associated Janus kinase (JAK) 2 and receptor dimerization for hormone internalization and nuclear translocation by use of cells stably transfected with cDNA for the GHR. Staurosporine and herbimycin A treatment of cells did not affect the ability of GH to internalize but resulted in increased nuclear accumulation of hormone. Similarly, receptor mutations, which prevent the association and activation of JAK2, did not affect the ability of the hormone to internalize or translocate to the nucleus but resulted in increased nuclear accumulation of GH. These results were observed both by nuclear isolation and confocal laser scanning microscopy. Staurosporine treatment of cells in which human GH (hGH) was targeted to the cytoplasm (removal of secretion sequence) or to the nucleus (addition of the nuclear localization sequence of SV40 large T antigen) resulted in preferential accumulation of hGH in the nucleus. We further investigated the requirement of receptor dimerization for GH nuclear translocation using the non-receptor-dimerizing hGH antagonist, hGH-G120R, conjugated to fluorescein isothiocyanate. Confocal laser scanning microscopy demonstrated efficient internalization of both hGH and hGH-G120R but lack of nuclear translocation of hGH-G120R. Thus, we conclude that activation of JAK2 kinase and the subsequent tyrosine phosphorylation is not required for nuclear translocation of GH but is pivotal for the removal of the hormone from the nucleus, and that GH translocates into the nucleus in a GHR dimerized-dependent fashion.

AB - We have previously demonstrated that GH is subject to rapid receptor-dependent nuclear translocation. Here, we examine the importance of ligand activation of the GH-receptor (GHR)-associated Janus kinase (JAK) 2 and receptor dimerization for hormone internalization and nuclear translocation by use of cells stably transfected with cDNA for the GHR. Staurosporine and herbimycin A treatment of cells did not affect the ability of GH to internalize but resulted in increased nuclear accumulation of hormone. Similarly, receptor mutations, which prevent the association and activation of JAK2, did not affect the ability of the hormone to internalize or translocate to the nucleus but resulted in increased nuclear accumulation of GH. These results were observed both by nuclear isolation and confocal laser scanning microscopy. Staurosporine treatment of cells in which human GH (hGH) was targeted to the cytoplasm (removal of secretion sequence) or to the nucleus (addition of the nuclear localization sequence of SV40 large T antigen) resulted in preferential accumulation of hGH in the nucleus. We further investigated the requirement of receptor dimerization for GH nuclear translocation using the non-receptor-dimerizing hGH antagonist, hGH-G120R, conjugated to fluorescein isothiocyanate. Confocal laser scanning microscopy demonstrated efficient internalization of both hGH and hGH-G120R but lack of nuclear translocation of hGH-G120R. Thus, we conclude that activation of JAK2 kinase and the subsequent tyrosine phosphorylation is not required for nuclear translocation of GH but is pivotal for the removal of the hormone from the nucleus, and that GH translocates into the nucleus in a GHR dimerized-dependent fashion.

KW - Active Transport, Cell Nucleus

KW - Amino Acid Sequence

KW - Animals

KW - CHO Cells

KW - Cell Nucleus

KW - Cricetinae

KW - Cytoplasm

KW - DNA, Complementary

KW - Dimerization

KW - Enzyme Inhibitors

KW - Enzyme-Linked Immunosorbent Assay

KW - Gene Deletion

KW - Growth Hormone

KW - Janus Kinase 2

KW - Ligands

KW - Microscopy, Confocal

KW - Molecular Sequence Data

KW - Mutagenesis

KW - Protein-Tyrosine Kinases

KW - Proto-Oncogene Proteins

KW - Rats

KW - Receptors, Cell Surface

KW - Receptors, Somatotropin

KW - Staurosporine

KW - Transfection

U2 - 10.1210/en.2002-221121

DO - 10.1210/en.2002-221121

M3 - Journal article

C2 - 12810575

VL - 144

SP - 3182

EP - 3195

JO - Journal of Clinical Endocrinology and Metabolism

JF - Journal of Clinical Endocrinology and Metabolism

SN - 0013-7227

IS - 7

ER -