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, 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 endogenous GHR in 3T3-F442A cells. Deletion of the proline-rich box 1 of GHR (GHR(deltaP)) 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 activation via Elk-1, a transcription factor associated 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-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 expression 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-stimulated 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 stimulation of c-fos and egr-1 expression and Elk-1 activation, supporting multiple mechanisms for GH signaling to the nucleus.
Gong
T W
TW
Department of Physiology, University of Michigan Medical School, Ann Arbor 48109-0622, USA.
Meyer
D J
DJ
Liao
J
J
Hodge
C L
CL
Campbell
G S
GS
Wang
X
X
Billestrup
N
N
Carter-Su
C
C
Schwartz
J
J
D016475:3T3 Cells / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D016466:CHO Cells; D006224:Cricetinae; D004268:DNA-Binding Proteins / Q000235:genetics*; D003847:Deoxyglucose / Q000378:metabolism*; D051766:Early Growth Response Protein 1; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017353:Gene Deletion; D005786:Gene Expression Regulation / Q000187:drug effects*; D016762:Genes, fos / Q000235:genetics*; D013006:Growth Hormone / Q000494:pharmacology*; D017874:Immediate-Early Proteins; D051379:Mice; D016296:Mutagenesis; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D051381:Rats; D011986:Receptors, Somatotropin / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins; D014157:Transcription Factors / Q000235:genetics*
D004268:DNA-Binding Proteins; D051766:Early Growth Response Protein 1; C486109:Egr1 protein, mouse; C486110:Egr1 protein, rat; D004791:Enzyme Inhibitors; D017874:Immediate-Early Proteins; D011986:Receptors, Somatotropin; D011994:Recombinant Proteins; D014157:Transcription Factors; D013006:Growth Hormone; D003847:Deoxyglucose; D011505:Protein-Tyrosine Kinases