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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...