OR01-04 The Growth Hormone Receptor Interacts With Transcriptional Regulator, HMGN1, Upon GH-induced Nuclear Translocation

Abstract

Abstract Disclosure: L. Jain: None. M.H. Vickers: None. B. Jacob: None. M.J. Middleditch: None. D.A. Chudakova: None. A.R. Ganley: None. J.M. O’Sullivan: None. J.K. Perry: None. Numerous classical cell-surface receptors that were previously thought to signal exclusively at the cell-surface can also translocate to the nucleus upon ligand binding, with increasing evidence supporting a transcriptional role for examples such as the epidermal growth factor receptor. In addition to classic cell-surface growth hormone receptor (GHR)-mediated signaling, the GHR is rapidly imported into the nucleus of cells upon stimulation with growth hormone (GH). Nuclear GHR localization has been associated with increased cancer cell proliferation. However, whether there are transcriptional changes associated with nuclear GHR import remains to be determined. To investigate whether the GHR interacts with transcription regulators in the nucleus following nuclear translocation we used immunoprecipitation combined with mass spectrometry. GH-dependent phosphorylation of STAT5 was confirmed by western blotting in the human cell-lines RL95-2 (endometrial cancer cell-line), and MCF-10A (mammary epithelial cell-line). The GHR was found to rapidly translocate to the nucleus in both cell lines following GH treatment by immunofluorescence staining, with maximal localisation at 5-10 minutes (p<0.001; RL95-2). Combined immunoprecipitation-mass spectrometry analysis of RL95-2 whole cell lysates following GH stimulation (500 ng/mL, 10 mins) identified 40 novel GHR binding partners, including 3 transcriptional regulators (HMGN1, SUMO1 and DDX21). Gene expression analysis identified 416 genes that were differentially expressed following GH treatment (500 ng/mL, 90 mins). Intersection of differentially expressed genes with known gene targets of HMGN1 (ChIP-Atlas) identified an enrichment of HMGN1 target genes (p<0.001). Co-immunoprecipitation combined with western blot analysis confirmed that HMGN1 associated with the GHR in nuclear extracts. Our results suggest that GHR nuclear translocation might mediate GH actions via interaction with chromatin factors that then drive changes in specific downstream transcriptional programs. Presentation: Thursday, June 15, 2023