Disulfide Linkage of Growth Hormone (GH) Receptors (GHR) Reflects GH-induced GHR Dimerization

The growth hormone (GH) receptor (GHR) binds GH in its extracellular domain and transduces activating signals via its cytoplasmic domain. Both GH-induced GHR dimerization and JAK2 tyrosine kinase activation are critical in initiation of GH signaling. We previously described a rapid GH-induced disulfide linkage of GHRs in human IM-9 cells. In this study, three GH-induced phenomena (GHR dimerization, GHR disulfide linkage, and enhanced GHR-JAK2 association) were examined biochemically and immunologically. By using the GH antagonist, G120K, and an antibody recognizing a dimerization-sensitive GHR epitope, we demonstrated that GH-induced GHR disulfide linkage reflects GH-induced GHR dimerization. GH, not G120K, promoted both GHR disulfide linkage and enhanced association with JAK2. Measures that diminished GH-dependent JAK2 and GHR tyrosine phosphorylation diminished neither GH-induced GHR disulfide linkage nor GH-enhanced GHR-JAK2 association. By using both transient and stable expression systems, we determined that cysteine 241 (an unpaired extracellular cysteine) was critical for GH-induced GHR disulfide linkage; however, GH-induced GHR dimerization, GHR-JAK2 interaction, and GHR, JAK2, and STAT5 tyrosine phosphorylation still proceeded when this cysteine residue was mutated. We conclude GH-induced GHR disulfide linkage is not required for GHR dimerization, and activation and GH-enhanced GHR-JAK2 association depends more on GHR dimerization than on GHR and/or JAK2 tyrosine phosphorylation.

[1]  J. Schlessinger,et al.  Platelet-derived growth factor (PDGF)-induced disulfide-linked dimerization of PDGF receptor in living cells , 1991, Molecular and cellular biology.

[2]  G. Yancopoulos,et al.  LIFR beta and gp130 as heterodimerizing signal transducers of the tripartite CNTF receptor. , 1993, Science.

[3]  J. Jansson,et al.  Mode of action of pituitary growth hormone on target cells. , 1985, Annual review of physiology.

[4]  M. Shibuya,et al.  The Dimerization Property of Glutathione S-Transferase Partially Reactivates Bcr-Abl Lacking the Oligomerization Domain* , 1996, The Journal of Biological Chemistry.

[5]  O. Silvennoinen,et al.  Identification of JAK2 as a growth hormone receptor-associated tyrosine kinase , 1993, Cell.

[6]  J. Ihle.  The Janus protein tyrosine kinase family and its role in cytokine signaling. , 1995, Advances in immunology.

[7]  K. Arai,et al.  SR alpha promoter: an efficient and versatile mammalian cDNA expression system composed of the simian virus 40 early promoter and the R-U5 segment of human T-cell leukemia virus type 1 long terminal repeat , 1988, Molecular and cellular biology.

[8]  J. Nickoloff,et al.  Site-directed mutagenesis of virtually any plasmid by eliminating a unique site. , 1992, Analytical biochemistry.

[9]  J. Cleveland,et al.  Structure of the murine Jak2 protein-tyrosine kinase and its role in interleukin 3 signal transduction. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[10]  K. Yasukawa,et al.  IL-6-induced homodimerization of gp130 and associated activation of a tyrosine kinase. , 1993, Science.

[11]  H. Green,et al.  Spontaneous heritable changes leading to increased adipose conversion in 3T3 cells , 1976, Cell.

[12]  J. Schwartz,et al.  Molecular mechanism of growth hormone action. , 1996, Annual review of physiology.

[13]  T. Wagner,et al.  Glycine 119 of bovine growth hormone is critical for growth-promoting activity. , 1991, Molecular endocrinology.

[14]  D. Leroith,et al.  Growth hormone stimulates the formation of a multiprotein signaling complex involving p130(Cas) and CrkII. Resultant activation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK). , 1998, The Journal of biological chemistry.

[15]  R. Mandler,et al.  Growth hormone-dependent tyrosine phosphorylation of a GH receptor-associated high molecular WEIGHT protein immunologically related to JAK2. , 1998, Biochemical and biophysical research communications.

[16]  J. Roth,et al.  Binding of 125I-human growth hormone to specific receptors in human cultured lymphocytes. Characterization of the interaction and a sensitive radioreceptor assay. , 1974, The Journal of biological chemistry.

[17]  I. Sakai,et al.  Regions of the JAK2 Tyrosine Kinase Required for Coupling to the Growth Hormone Receptor (*) , 1995, The Journal of Biological Chemistry.

[18]  D. Goeddel,et al.  Rational design of potent antagonists to the human growth hormone receptor. , 1992, Science.

[19]  A. Ożyhar,et al.  GST-Induced dimerization of DNA-binding domains alters characteristics of their interaction with DNA. , 1998, Protein expression and purification.

[20]  E. Stanley,et al.  Role of dimerization and modification of the CSF‐1 receptor in its activation and internalization during the CSF‐1 response. , 1991, The EMBO journal.

[21]  William I. Wood,et al.  Growth hormone receptor and serum binding protein: purification, cloning and expression , 1987, Nature.

[22]  P. Kelly,et al.  Grb10 Identified as a Potential Regulator of Growth Hormone (GH) Signaling by Cloning of GH Receptor Target Proteins* , 1998, The Journal of Biological Chemistry.

[23]  H. Okayama,et al.  High-efficiency transformation of mammalian cells by plasmid DNA. , 1987, Molecular and cellular biology.

[24]  S. Bass,et al.  The human growth hormone receptor. Secretion from Escherichia coli and disulfide bonding pattern of the extracellular binding domain. , 1990, The Journal of biological chemistry.

[25]  J. Bazan,et al.  Structural design and molecular evolution of a cytokine receptor superfamily. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[26]  A. Shaw,et al.  The Conserved Box 1 Motif of Cytokine Receptors Is Required for Association with JAK Kinases (*) , 1995, The Journal of Biological Chemistry.

[27]  J. A. Scarlett,et al.  Inhibitory effect of a growth hormone receptor antagonist (G120K-PEG) on renal enlargement, glomerular hypertrophy, and urinary albumin excretion in experimental diabetes in mice. , 1999, Diabetes.

[28]  L. Zhang,et al.  Domains of the growth hormone receptor required for association and activation of JAK2 tyrosine kinase. , 1994, The Journal of biological chemistry.

[29]  Jing Jiang,et al.  Involvement of the Src Homology 2-containing Tyrosine Phosphatase SHP-2 in Growth Hormone Signaling* , 1998, The Journal of Biological Chemistry.

[30]  I. Kerr,et al.  Activation of Jak2 catalytic activity requires phosphorylation of Y1007 in the kinase activation loop , 1997, Molecular and cellular biology.

[31]  Q. Sun,et al.  Identification of a Cys Motif in the Common β Chain of the Interleukin 3, Granulocyte-Macrophage Colony-stimulating Factor, and Interleukin 5 Receptors Essential for Disulfide-linked Receptor Heterodimerization and Activation of All Three Receptors* , 1998, The Journal of Biological Chemistry.

[32]  G. Goodall,et al.  Human interleukin-3 (IL-3) induces disulfide-linked IL-3 receptor alpha- and beta-chain heterodimerization, which is required for receptor activation but not high-affinity binding , 1996, Molecular and cellular biology.

[33]  K. Clauser,et al.  Dimerization of the extracellular domain of the human growth hormone receptor by a single hormone molecule. , 1991, Science.

[34]  M. Ultsch,et al.  Human growth hormone and extracellular domain of its receptor: crystal structure of the complex. , 1992, Science.