Identification of Tyrosine Residues in the Intracellular Domain of the Growth Hormone Receptor Required for Transcriptional Signaling and Stat5 Activation

Abstract

The binding of growth hormone (GH) to its receptor results in its dimerization followed by activation of Jak2 kinase and tyrosine phosphorylation of the GH receptor itself, as well as Jak2 and the transcription factors Stat1, -3, and -5. In order to study the role of GH receptor tyrosine phosphorylation in intracellular signaling, we constructed GH receptors in which combinations of tyrosines were mutated to phenylalanines. We identified three tyrosine residues at positions 534, 566, and 627 that were required for activation of GH-stimulated transcription of the serine protease inhibitor (Spi) 2.1 promoter. Any of these three tyrosines is able to independently mediate GH-induced transcription, indicating redundancy in this part of the GH receptor. Tyrosine phosphorylation was not required for GH stimulation of mitogen-activated protein (MAP) kinase activity or for GH-stimulated Ca 2؉ channel activation since these pathways were normal in cells expressing a GH receptor in which all eight intracellular tyrosines were mutated to phenylalanines. Activation of Stat5 by GH was, however, abolished in cells expressing the GH receptor lacking intracellular tyrosines. This study demonstrates that specific tyrosines in the GH receptor are required for transcriptional signaling possibly by their role in the activation of transcription factor Stat5.Pituitary growth hormone (GH) 1 is the major regulator of postnatal growth (1). The actions of GH at the cellular level include direct mitogenic effects (2, 3), insulin-like and insulinantagonizing metabolic effects (4), as well as gene regulatory actions (5-7). All of these effects are initiated by the binding of GH to its receptor, which belongs to the cytokine receptor superfamily. Members of this family of receptors activate cytoplasmic tyrosine kinases of the Jak family, and these activated kinases are required for most receptor-initiated signaling pathways (8). The activated Jak2 kinase has been shown to phosphorylate several intracellular substrates including the GH receptor itself, as well as transcription factors of the Stat family (9 -11).Not only does GH activate the Jak/Stat pathway in which specific tyrosine phosphorylation of Stat1, -3, and -5 occurs in response to GH, resulting in dimerization, nuclear translocation, and binding to ␥-interferon activated sequence-like elements (11-13), but we and others have previously identified several alternative signaling pathways induced by the activated GH receptor. Stimulation and tyrosine phosphorylation of MAP kinase by GH have been studied both in cultured cells (14,15) and in vivo (16). In addition to activation of MAP kinase, its translocation to the nucleus has also been demonstrated. The activation of MAP kinase by GH is dependent upon the proline-rich box 1 domain of the GH receptor that presumably is directly involved in the binding of Jak2. When the box 1 domain is deleted or the prolines are mutated to alanines, the GH receptor is no longer able to mediate GHinduced MAP kinase activity. The functional role of MAP k...

Adaptation of pancreatic islet B-cells during the last third of pregnancy: regulation of B-cell function and proliferation by lactogenic hormones in rats

“…Several analyses of the growth hormone (GH) receptor in B-cell function have demonstrated that the proliferative signaling only requires a membrane proximal part of the receptor and activation of the tyrosine kinase Janus activation kinase 2 and the transcription factors STAT1 and STAT3 (signal transducers and activators of transcription) (25). On the other hand, the action of insulin secretion via the GH receptor requires the distal part of the receptor and activation of calcium uptake and STAT5 (26)(27)(28). Thus, GH (and PRL) may affect insulin secretion and B-cell proliferation via distinct signaling pathways.…”

The role of STAT proteins in growth hormone signaling

“…The current evidence suggests that STAT5 activation by GH requires phosphorylation of speci®c tyrosine residues within the GH receptor, thereby providing a high anity binding site for the SH2 domain in the STAT proteins. Speci®c tyrosines in porcine GH receptor (Y534, Y566 and Y627, equivalent to Y534, Y566 and Y626 in rat GH receptor) have been identi®ed as being required for GH-dependent tyrosyl phosphorylation of STAT5 and transcription of the Spi 2.1 promoter (Hansen et al, 1996;Wang et al, 1996). An additional tyrosine (487) has also been suggested as being required for tyrosyl phosphorylation of STAT5 .…”

“…GH induces the binding of STAT5 proteins to IFNg activated sequence (GAS)-like elements (GLE) in several dierent genes, including the GH-sensitive spi 2.1, Insulin 1 and p450 CYP3A 6 beta-hydroxylase genes (Bergad et al, 1995;Galsgaard et al, 1996;Hansen et al, 1996;Subramanian et al, 1995;. STAT5 mediates GH-dependent transcriptional activation of promoter-reporter constructs containing the STAT5-binding promoter elements of these genes (Bergad et al, 1995;Galsgaard et al, 1996;Hansen et al, 1996;Sotiropoulos et al, 1996;Subramanian et al, 1995;Wood et al, 1995).…”

Evolution of Receptors for Growth Hormone and Somatolactin in Fish and Land Vertebrates: Lessons from the Lungfish and Sturgeon Orthologues

“…Predicted signal peptides, transmembrane domains, N glycosylation sites, and other conserved residues/subdomains were boxed with the same color used in (a). Functional prolines in the box I motif (Goujon et al 1994) and tyrosines (Hansen et al 1996) are highlighted by gray CCCHHHHHEEEEEEECCCCCCCCCHHHHCCCCCCCCCEEEEEECCCCEEEEHHCHCCECCCCCCCCCEEEEEEEECCCCCCCCCEEEEEEHHHCHEEEEEEEHHCCCCEEEEEEECCCCCCCCCCC 309 309 309 309 309 305 305 305 309 309 309 309 318 310 285 282 285 283 280 282 284 289 285 286 286 287 287 287 287 285 287 294 286 294 291 289 293 293 289 289 291 293 303 310 296 297 297 297 297 183 183 183 183 183 179 179 179 183 183 183 183 192 184 159 156 159 157 154 156 158 163 159 160 160 161 161 161 161 159 161 168 160 168 165 163 167 167 163 163 165 167 177 184 170 171 171 171 171 a approximately 35% identity between GHRs of tetrapods and teleosts), the predicted secondary structures are all strikingly similar (Fig. 6a).…”