Growth hormone promoted tyrosyl phosphorylation of growth hormone receptors in murine 3T3-F442A fibroblasts and adipocytes

Abstract

Because many growth factor receptors are ligand-activated tyrosine protein kinases, the possibility that growth hormone (GH), a hormone implicated in human growth, promotes tyrosyl phosphorylation of its receptor was investigated. 125I-Labeled human GH was covalently cross-linked to receptors in intact 3T3-F442A fibroblasts, a cell line which differentiates into adipocytes in response to GH. The cross-linked cells were solubilized and passed over a column of phosphotyrosyl binding antibody immobilized on protein A-Sepharose. Immunoadsorbed proteins were eluted with a hapten (p-nitrophenyl phosphate) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The eluate from the antibody column contained an Mr 134,000 125I-GH-receptor complex. A similar result was obtained when the adipocyte form of 3T3-F442A cells was used in place of the fibroblast form. O-Phosphotyrosine prevented 125I-GH-receptor complexes from binding to the antibody column, whereas O-phosphoserine and O-phosphothreonine did not. In studies of GH-promoted phosphorylation in 3T3-F442A fibroblasts labeled metabolically with [32P]Pi, GH was shown to stimulate formation of a 32P-labeled protein which bound to immobilized phosphotyrosyl binding antibodies. The molecular weight of 114,000 obtained for this protein is similar to that expected for non-cross-linked GH receptor. The Mr 114,000 phosphorylated protein could be immunoprecipitated with anti-GH antibody, indicating that GH remained noncovalently bound to this protein during absorption to and elution from the immobilized phosphotyrosyl binding antibody. Phosphoamino acid analysis after both limited acid hydrolysis and extensive base hydrolysis of the Mr 114,000 phosphoprotein confirmed the presence of phosphotyrosyl residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Nuclear Factor-κB (NF-κB) p65 Interacts with Stat5b in Growth Plate Chondrocytes and Mediates the Effects of Growth Hormone on Chondrogenesis and on the Expression of Insulin-like Growth Factor-1 and Bone Morphogenetic Protein-2

“…GH action in mammalian cells is mediated by binding to the transmembrane growth hormone receptor, thereby triggering increased association with and activation of Janus kinases (JAKs) (25)(26)(27), which in turn leads to the activation of signal transducers and activators of transcription (STATs) (28 -30), the phosphatidylinositol 3-kinase (PI3K)/Akt system (31,32), and the extracellular signal-regulated kinases 1 and 2 (ERKs 1 and 2) (33)(34)(35). Recent studies have demonstrated that GH stimulates IGF-1 gene transcription through the activation of Stat5b (36), and findings in experimental animals and in humans have shown that the absence or mutations of the Stat5b gene are associated with diminished postnatal growth, GH resistance, and reduced IGF-1 synthesis (15,37,38).…”

The role of STAT proteins in growth hormone signaling

“…The GH receptor was the ®rst member of the cytokine receptor family to be cloned (Leung et al, 1987) and to be recognized as associating with and activating a tyrosine kinase (Carter-Su et al, 1989;Endo et al, 1997;Foster et al, 1988;Wang et al, 1993). GH binding to its receptor is now known to recruit and activate the receptor-associated JAK2 that in turn phosphorylates tyrosines within itself and the GH receptor.…”

Negative Regulation of Growth Hormone Receptor/JAK2 Signaling by Signal Regulatory Protein α

“…The stock of murine 3T3-F442A fibroblasts was kindly provided by H. Green (Harvard University). 3T3-F442A cells and 293T cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 100 U of penicillin per ml, 100 µg of streptomycin per ml, 0.25 µg of amphotericin B per ml, 1 mM L-glutamine and 9% calf serum as described previously (17). Clonal cell lines co-expressing SIRPα1 and green fluorescent protein (GFP) or SIRP 4YF and GFP and pooled cell lines co-expressing prk5 and GFP, SIRPα1 and GFP, or SIRP 4YF and GFP were created.…”