Intracellular processing of growth hormone receptors by adipocytes in primary culture

Roupas, Peter; Herington, Adrian C.

doi:10.1016/0303-7207(88)90037-8

Cited by 53 publications

(35 citation statements)

References 26 publications

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“…4). Dissociation of GH⅐GHR complexes does not occur at (endosomal) pH 5.5 (20), indicating that GH remains complexed to its receptor, independent of its intracellular routing, unless it is localized to the lysosome. In that case, the ligand as well as the receptor is rapidly degraded (21).…”

Section: Possible Role Of Proteasomes In Modulating Phosphatase Activmentioning

confidence: 99%

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The Signal Transduction of the Growth Hormone Receptor Is Regulated by the Ubiquitin/Proteasome System and Continues After Endocytosis

Santos

Kerkhof

Strous

2001

Journal of Biological Chemistry

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The growth hormone receptor (GHR) intracellular domain contains all of the information required for signal transduction as well as for endocytosis. Previously, we showed that the proteasome mediates the clathrin-mediated endocytosis of the GHR. Here, we present evidence that the proteasomal inhibitor MG132 prolongs the GH-induced activity of both GHR and JAK2, presumably through stabilization of GHR and JAK2 tyrosine phosphorylation. If proteasomal inhibitor was combined with ligand in an endocytosis-deficient GHR mutant, the same phenomenon occurred indicating that proteasomal action on tyrosine dephosphorylation is independent of endocytosis. Experiments with a GHRtruncated tail mutant (GHR-(1-369)) led to a prolonged JAK2 phosphorylation caused by the loss of a phosphatase-binding site. This raised the question of what happens to the signal transduction of the GHR after its internalization. Co-immunoprecipitation of GH⅐GHR complexes before and after endocytosis showed that JAK2 as well as other activated proteins are bound to the GHR not only at the cell surface but also intracellularly, suggesting that the GHR signal transduction continues in endosomes. Additionally, these results provide evidence that GHR is present in endosomes both in its full-length and truncated form, indicating that the receptor is down-regulated by the proteasome.

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Section: Possible Role Of Proteasomes In Modulating Phosphatase Activmentioning

confidence: 99%

“…In the presence of ligand, GHR endocytoses rapidly via clathrin-coated pits (20), and its degradation occurs at least partially within the lysosome (21). The ubiquitin system is required for ligand-induced GHR internalization (23).…”

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confidence: 99%

The Signal Transduction of the Growth Hormone Receptor Is Regulated by the Ubiquitin/Proteasome System and Continues After Endocytosis

Santos

Kerkhof

Strous

2001

Journal of Biological Chemistry

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“…On the other hand, simultaneous mutation of the four prolines abolished the capacity of the receptor to interact with and activate JAK2 and other signaling proteins (4,15,16). Thus, the proline-rich region is critical for GH signal transduction.After binding to its receptor, GH internalizes via clathrincoated pits and is degraded in lysosomes (17,18). This process is regulated by both the ubiquitin system and the proteasome (19 -22).…”

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confidence: 99%

Growth Hormone Receptor Ubiquitination, Endocytosis, and Degradation Are Independent of Signal Transduction via Janus Kinase 2

Santos

Broeke

Strous

2001

Journal of Biological Chemistry

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The ubiquitin-proteasome system is required in growth hormone receptor (GHR) endocytosis. For cytokine receptors, which lack intrinsic tyrosine kinase activity, signal transduction is initiated by the activation of a member of the Janus kinase (JAK) family. Previously, we have shown that GHR and JAK2 tyrosine (de) phosphorylation are regulated via the ubiquitin system. In this study, we examined the role of JAK2-mediated signal transduction in GHR internalization and downregulation. Mutation of the attachment site for JAK2, box-1, in the GHR cytoplasmic tail resulted in the complete absence of GHR and JAK2 phosphorylation. This modification did not alter the rate and extent of receptor-bound growth hormone internalization as compared with a functional GHR, nor did it change its turnover and transport to the plasma membrane. In addition, the receptor was still normally ubiquitinated and remained dependent on both an intact ubiquitin system and proteasomal action for its internalization. Thus, GHR ubiquitination, endocytosis, and degradation occur independently of GHR signal transduction via JAK2. We conclude that whereas endocytosis and degradation require the ubiquitin system, they are independent of GHR signal transduction. GH1 regulates important physiological processes such as growth, metabolism, and cellular differentiation. The actions of GH are mediated through activation of the GHR, a member of the cytokine/hematopoietin receptor superfamily with homologies defined in the extracellular domain and that lacks intrinsic tyrosine kinase activity in its intracellular domain (1-3). Upon GH binding, the dimerized complex associates with the tyrosine kinase JAK2, a member of the Janus family of cytosolic kinases (3-5). GH-dependent tyrosine phosphorylation of JAK2 itself, of GHR, and of other cellular proteins depends on the receptor's ability to activate JAK2 (3-9). Biochemical evidence has shown that JAK2 activation by the GHR is essential for activating the signal transducer and activator of transcription proteins, several proteins involved in the Ras/MAP kinase pathway, and the insulin receptor substrate proteins IRS-1 and IRS-2, which initiate the phosphatidylinositol 3-kinase pathway (4, 9, 10, 11). Thus far, only the GH-dependent effect on calcium entry appears to involve mechanisms independent of JAK2 activation (12). Because a C-terminally truncated receptor able to interact with and activate JAK2 was unable to activate specific signaling molecules such as signal transducer and activator of transcription 5 (13), activation of JAK2 alone is insufficient to elicit all of the responses to GH, suggesting that such proteins are unlikely to be direct JAK2 substrates.A proline-rich motif termed box-1 between amino acids 297 and 311 is conserved within members of the cytokine family and is required for the association of JAK2 with GHR as well as GH-dependent activation of JAK2 (1,4,14). Studies using mutated box-1 regions of the GHR have shown that this region is intermediate in GH-dependent association and acti...

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“…15,[27][28][29] Govers et al, 9) using a mutant GHR in which all cytoplasmic lysine residues were mutated to arginine, demonstrated that the conjugation of ubiquitin to a lysine residue in the intracellular domain of GHR was not necessary for the ligand-induced internalization. Our results also support this idea.…”

Section: Discussionmentioning

confidence: 99%

“…Similar results were obtained for the GHR turnover rates of cultured mouse cells (BALB/c 3T3; 1.25 h), 14) and cultured rat adipocytes (45 min). 15) We have previously described that the proteolytic cleavage of the extracellular domain of GHR at the cell surface results in the release of soluble growth hormone-binding protein (GH-BP) from IM-9 cells, 16) which is one of the mechanisms of hGHR degradation. Ba/F3-hGHR cells also constitutively released hGH-BPs, but the amount was very little.…”

Section: Human Ghr Degradation In the Ba/f3-hghr Cellsmentioning

confidence: 99%

Proteasomes Are Involved in the Constitutive Degradation of Growth Hormone Receptors.

Takagi

Saito

Sawada

2001

Biological & Pharmaceutical Bulletin

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Growth hormone receptor (GHR) is a member of the cytokine/hematopoietin receptor superfamily. Once the ligand growth hormone (GH) binds GHR, two GHRs are dimerized by a single GH molecule, which results in the recruitment and binding of JAK2 tyrosine kinase and the activation of intracellular signal transduction pathways.1) The activated complex is then internalized through clathrin-mediated endocytosis [2][3][4] and degraded in the endosome/lysosome pathway. 5,6) It was reported that ligand-induced rabbit GHR internalization and degradation in transfected CHO cells was dependent on the ubiquitin conjugation system. 7) Moreover, proteasome inhibitors have been shown to inhibit GH-induced endocytosis.8) However, it has been shown that ubiquitination of the GHR itself is not necessary for ligand-induced internalization.9) Thus, the role of ubiquitination and the proteasome system in ligand-induced GHR endocytosis is still obscure. Furthermore, there is no report on the role of proteasomes in the constitutive internalization and degradation of hGHRs.In this report, in order to clarify the mechanisms of the constitutive degradation of GHR, we investigated the effects of the proteasome inhibitors on the degradation of hGHR in hGHR-transfected murine Ba/F3 (Ba/F3-hGHR) cells. Ba/F3-hGHR cells were kindly provided by Mitsui Chemicals, Inc. Full-length hGHR cDNA was transfected into the mouse pro-B cell line Ba/F3 cells by electroporation, and the cells stably expressing hGHR were established. MATERIALS AND METHODS Reagents and Cell Line12) The cells were cultured in RPMI 1640 medium containing 1 mg/ml G418, 10% FCS, 50 mM 2-mercaptoethanol, 10% of a WEHI-3 cell culture supernatant as a source of interleukin-3 (IL-3) or 1.5 ng/ml recombinant mouse IL-3 (Pepro Tech EC, London, U.K.), and 60 mg/ml kanamycin.Immunoblotting of hGHRs and hGH-BPs Immunoblotting of hGHRs in the cell lysate and hGH-BPs in the culture medium using the GHRP2-88 antibody and 125 I-la-744

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Intracellular processing of growth hormone receptors by adipocytes in primary culture

Cited by 53 publications

References 26 publications

The Signal Transduction of the Growth Hormone Receptor Is Regulated by the Ubiquitin/Proteasome System and Continues After Endocytosis

The Signal Transduction of the Growth Hormone Receptor Is Regulated by the Ubiquitin/Proteasome System and Continues After Endocytosis

Growth Hormone Receptor Ubiquitination, Endocytosis, and Degradation Are Independent of Signal Transduction via Janus Kinase 2

Proteasomes Are Involved in the Constitutive Degradation of Growth Hormone Receptors.

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