Nuclear translocation and anchorage of the growth hormone receptor

Abstract

The extracellular domain of the rabbit growth hormone (GH) receptor has previously been shown to be associated with the nucleus. However, in this species the GH binding protein (BP) is derived by proteolytic cleavage of the full-length receptor, and thus distinction between the receptor and BP is difficult. The intracellular domain of the GH receptor is required for GH-stimulated function. Thus a direct nuclear function of GH would presumably require the receptor intracellular domain in the nucleus. We have therefore characterized the rat nuclear GH receptor and BP based on their distinct antigenic identity. We show, in vivo, that the full-length receptor is associated with the nucleus, including the respective subnuclear fractions (nucleoplasm, outer nuclear membranes, inner nuclear membranes, and chromatin). In vivo, the receptor is also subject to ligand-dependent nuclear translocation. Cellular transfection of rat GH receptor cDNA resulted in the appearance of nuclear binding sites for 125I-labeled human GH not present in the untransfected parental cell line (Chinese hamster ovary (CHO), buffalo rat liver). To determine which portion of the receptor was responsible for nuclear anchorage, we examined the binding of 125I-labeled human GH to whole nuclei isolated from CHO cells expressing the full-length receptor, a receptor in which 184 amino acids had been deleted from the carboxyl-terminal intracellular domain (CHO-454) and a receptor containing only 5 of 349 amino acids in the intracellular domain (CHO-294). Nuclear binding above the level of the untransfected parental cell line was detected only in CHO-638 and CHO-454 cells, suggesting that amino acids 294-454 of the receptor are necessary for nuclear anchorage. This observation was not due to membrane contamination, as the CHO-294 cells express a membrane-bound receptor that was not anchored in the nucleus. The full-length GH receptor in receptor cDNA-transfected cells is nucleocytoplasmic in the absence of ligand but is also subject to rapid ligand-dependent nuclear translocation. The presence of the intracellular domain of the GH receptor in the nucleus allows the possibility of a direct nuclear response to GH.