Abstract
Glucocorticoids inhibit growth in children and antagonize the growth-promoting action of GH in peripheral tissues. Recently, they have been shown to decrease GH binding. In this study we examine the molecular mechanisms by which the glucocorticoid dexamethasone (DEX) and the phorbol ester phorbol myristate acetate (PMA) decrease cellular GH binding. In 3T3-F442A fibroblasts, DEX and PMA decrease the number of GH receptors (GHRs) capable of binding GH by 50% (t1/2 = 6 h) and 70% (t1/2 = 15 min), respectively. Neither appear to decrease the total number of cellular GHR. Rather, they appear to redistribute GHRs away from the plasma membrane or inactivate GHRs on the membrane such that they cannot bind GH. DEX and PMA also decrease GH-induced tyrosyl phosphorylation of GHR and JAK2 with a magnitude and time course correlating with that of inhibition of GH binding. DEX- and PMA-induced reductions of GH binding are also observed in a Chinese hamster ovary (CHO) cell line stably transfected with a rat liver GHR cDNA, further arguing that DEX and PMA act post-translationally on GHR. Using mutant GHRs stably expressed in CHO cells, amino acids 455-506 and tyrosines 333 and/or 338 of GHR were shown to be required for maximal DEX-induced inhibition of GH binding. DEX decreased GH binding to a GHR mutant F346A, which is reported to be deficient in ligand-induced internalization, suggesting that DEX decreases GH binding by a mechanism distinct from that of ligand-induced GHR internalization. PMA reduced GH binding to CHO cells expressing all GHR mutants tested. However, deletion of the C-terminal 132 amino acids decreased this effect, suggesting that at least one component of PMA action on GHR requires amino acids 507-638. These data suggest that distinct pathways mediate the effects of GH, DEX, and PMA on GHR number in the plasma membrane.
Highlights
Glucocorticoids inhibit growth in children and antagonize the growth-promoting action of GH in peripheral tissues
DEX- and phorbol myristate acetate (PMA)-induced reductions of GH binding are observed in a Chinese hamster ovary (CHO) cell line stably transfected with a rat liver GH receptors (GHRs) cDNA, further arguing that DEX and PMA act post-translationally on GHR
This study identifies distinct cytoplasmic domains of GHR reported to be required for DEX- and PMA-induced decreases in GH binding that are different from that required for ligand-induced internalization, suggesting that while GH, DEX, and PMA all appear to decrease GHR number in the plasma membrane by redistributing GHRs away from the plasma membrane, they may do so by different mechanisms
Summary
Glucocorticoids inhibit growth in children and antagonize the growth-promoting action of GH in peripheral tissues. The synthetic glucocorticoid dexamethasone (DEX) recently has been shown in this laboratory to decrease in 3T3-F442A fibroblasts GH-stimulated tyrosyl phosphorylation of microtubule-associated protein kinases, signal transducers, and activators of transcription (Stats) 1 and 3, JAK2, and GH receptor (GHR) [10]. This decrease in GHstimulated tyrosyl phosphorylation closely correlates with a decrease in the number of GH binding sites in the plasma membrane, suggesting that glucocorticoids decrease tissue sensitivity to GH at least in part by decreasing the number of GHRs available to GH.
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