REGULATION OF GLUCOSE-TRANSPORT AND C-FOS AND EGR-1 EXPRESSION IN CELLS WITH MUTATED OR ENDOGENOUS GROWTH-HORMONE RECEPTORS

To identify mechanisms by which GH receptors (GHR) mediate downstream events representative of growth and metabolic responses to GH, stimula tion by GH of c-fos and egr-1 expression and glucose transport activit y were examined in Chinese hamster ovary (CHO) cells expressing mutate d GHR. In CHO cells expressing wild-type GHR (GHR(1-638)), GH stimulat ed the expression of c-fos and egr-1, and stimulated 8-deoxyglucose up take, responses also mediated by endogenous GHR in 3T3-F442A cells. De letion of the proline-rich box 1 of GHR (GHR(Delta P)) abrogated all o f these responses to GH, indicating that box 1, a site of association of GHR with the tyrosine kinase JAK2, is crucial for these GH-stimulat ed 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 examine d. Not only did GHR(1-454) mediate stimulation of c-fos and egr-1 expr ession and 2-deoxyglucose uptake, but they also mediated GH-stimulated transcriptional activation via Elk-l, a transcription factor associat ed with the c-fos Serum 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-te rminal residues of the cytoplasmic domain (GHR(1-294)), GH did not ind uce c-fos or egr-1 expression or stimulate 2-deoxyglucose uptake. Furt her, in 3T3-F442A fibroblasts with endogenous GHR, GH-stimulated c-fos expression and 2-deoxyglucose uptake were reduced by the tyrosine kin ase 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 phosp horylation of only some proteins, including extracellular signal regul ated kinases ERK1 and -2, but not JAK2. Taken together, these results implicate association of GHR with JAK2 and GH-stimulated tyrosyl phosp horylation 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 d omain of GHR is sufficient to mediate stimulation of c-fos and egr-1 e xpression and Elk-l activation, supporting multiple mechanisms for GH signaling to the nucleus.