Regulation of Glucose Transport and c-fos and egr-1 Expression in Cells with Mutated or Endogenous Growth Hormone Receptors* TZY-WEN L. GONG†, DEBRA J. MEYER‡, JINFANG LIAO§, CHRISTINA L. HODGE, GEORGE S. CAMPBELL¶, XUEYAN WANG**, NILS BILLESTRUP, CHRISTIN CARTER-SU, AND JESSICA SCHWARTZ Department of Physiology (T.-W.L.G., J.L., G.S.C., X.W., C.C.-S., J.S.) and Program in Cellular and Molecular Biology (D.J.M., C.L.H., C.C.-S., J.S.), University of Michigan Medical School, Ann Arbor, Michigan 48109-0622; and Hagedorn Research Laboratory (N.B.), Gentofte, Denmark ABSTRACT To identify mechanisms by which GH receptors (GHR) mediate downstream events representative of growth and metabolic responses to GH, stimulation by GH of c-fos and egr-1 expression and glucose transport activity were examined in Chinese hamster ovary (CHO) cells expressing mutated GHR. In CHO cells expressing wild-type GHR (GHR 1– 638 ), GH stimulated the expression of c-fos and egr-1, and stimulated 2-deoxyglucose uptake, responses also mediated by en- dogenous GHR in 3T3-F442A cells. Deletion of the proline-rich box 1 of GHR (GHR P ) abrogated all of these responses to GH, indicating that box 1, a site of association of GHR with the tyrosine kinase JAK2, is crucial for these GH-stimulated responses. As the C-terminal half of the cytoplasmic domain of GHR is required for GH-stimulated calcium flux and for stimulation of spi-2.1 transcription, GHR lacking this sequence (GHR 1– 454 ) were examined. Not only did GHR 1– 454 mediate stimulation of c-fos and egr-1 expression and 2-deoxyglucose uptake, but they also mediated GH-stimulated transcriptional acti- vation via Elk-1, a transcription factor associated with the c-fos Se- rum Response Element. Thus, the C-terminal half of the cytoplasmic domain of GHR is not required for GH-stimulated c-fos transcription, suggesting that increased calcium is not required for GH-stimulated c-fos expression. In CHO cells lacking all but five N-terminal residues of the cytoplasmic domain (GHR 1–294 ), GH did not induce c-fos or egr-1 expression or stimulate 2-deoxyglucose uptake. Further, in 3T3- F442A fibroblasts with endogenous GHR, GH-stimulated c-fos ex- pression and 2-deoxyglucose uptake were reduced by the tyrosine kinase inhibitors herbimycin A, staurosporine, and P11. Herbimycin A and staurosporine inhibit JAK2 and tyrosyl phosphorylation of all proteins stimulated by GH, whereas P11 inhibits the GH-dependent tyrosyl phosphorylation of only some proteins, including extracellular signal regulated kinases ERK1 and -2, but not JAK2. Taken together, these results implicate association of GHR with JAK2 and GH-stim- ulated tyrosyl phosphorylation of an additional cellular protein in GH-stimulated glucose transport and c-fos and egr-1 expression. These studies also indicate that, in contrast to spi-2.1, the N-terminal half of the cytoplasmic domain of GHR is sufficient to mediate stim- ulation of c-fos and egr-1 expression and Elk-1 activation, supporting multiple mechanisms for GH signaling to the nucleus. (Endocrinology 139: 1863–1871, 1998) T HE DIVERSE effects of GH on cell growth, differentia- tion, and metabolism are thought to share early sig- naling pathways involving the GH receptor (GHR). The GHR is a single transmembrane protein with an extracellular bind- ing domain and a cytoplasmic domain that mediates signal- ing (1). For insight into GH signaling mechanisms, functions of the GHR have been dissected by analysis of GHR mutants in which the cytoplasmic domain has been truncated or mu- tated (2–10). A complex picture is emerging indicating that the N-terminal half of the cytoplasmic domain of GHR is sufficient to initiate some GH-stimulated events, such as stimulation of protein and lipid synthesis, but that the C-terminal half of the cytoplasmic domain is additionally required for other responses to GH, including activation of the spi-2.1 gene and stimulation of insulin synthesis (1). Ac- tivation of JAK2, via association with box 1 just proximal to the transmembrane domain of GHR (10, 11), is required for most, but not all, responses to GH identified to date, includ- ing tyrosyl phosphorylation of signaling molecules such as SHC, a component of the Ras-mitogen-activated protein ki- nase (MAPK) pathway (12–16), insulin receptor substrate-1 (IRS-1) and- 2 (17–19), and signal transducers and activators of transcription-1 (Stats), 1, -3, -5A, and -5B (8, 20 –22), as well as induction of spi-2.1 gene expression. Interestingly, stim- ulation of calcium flux by GH appears to be independent of JAK2, as it is reported to be mediated by GHR lacking a functional box 1 (4). The present study examines the ability of mutated and truncated GHR to mediate GH responses thought to be rep- Received August 15, 1997. Address all correspondence and requests for reprints to: Jessica Schwartz, Ph.D., Department of Physiology, University of Michigan Med- ical School, Ann Arbor, Michigan 48109-0622. E-mail: jeschwar@umich.edu. * This work was supported by research grants from the NSF (DCB8918289 and IBN9221667) and the Juvenile Diabetes Foundation (to J.S.), and NIH Grant RO1-DK-34171 (to C.C.-S. and J.S.). † Recipient of Postdoctoral Fellowship DK-08572 from the NIH. ‡ Supported by Predoctoral Traineeship in Cellular and Molecular Biology GM-07315 from the NIH, a Rackham Predoctoral Fellowship from the University of Michigan, and a Student Research Fellowship from The Endocrine Society. § Supported by Postdoctoral Fellowship DK-09293 from the NIH.  Supported by a Minority Graduate Fellowship from the NSF and a Rackham Merit Fellowship from the University of Michigan. ¶ Recipient of Postdoctoral Fellowship GM-14099 from the NIH. ** Recipient of a predoctoral fellowship from the American Diabetes Association, MI Affiliate, and a Dissertation Award from Rackham School of Graduate Studies, University of Michigan. 0013-7227/98/$03.00/0 Vol. 139, No. 4 Endocrinology Printed in U.S.A. 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