Effects of the Truncated Human Growth Hormone Receptor (GHR) on Downstream Signaling
Presentation Number: SUN-0630
Date of Presentation: June 22nd, 2014
Alona O Nakonechnaya*1, Christopher Pagano1 and Cynthia Gates Goodyer2
1McGill University, Montreal, QC, Canada, 2MUHC-MCH Rsrch Inst, Montreal, QC, Canada
Human growth hormone (GH) effects on target cells are exerted via a dimer of the growth hormone receptor (GHR) expressed on the cell membrane. Besides the full length form of the receptor (FL GHR), two truncated isoforms (TR GHR1-279 and TR GHR1-277) are produced from the same gene by alternative splicing at exon 9, resulting in receptors that bind GH but lack >97% of the intracellular domain. Relative amounts of the truncated isoforms are tissue-specific and can comprise up to 10% of the FL GHRmRNA produced (1,2). Following translation, TR GHRs are able to dimerize with the FL GHRs and are considered to serve as a dominant negative, as previously shown by inhibition of STAT5 phosphorylation (3). We have now examined the effect of TR GHR1-279 on JAK2, STAT5, SRC, ERK(1/2) and Akt pathways.
We transfected varying amounts (0-500ng) of a TR GHR1-279 expression vector in HEK293 cells followed by 250ng/ml GH stimulation for 0-60 min. We found that JAK2 and STAT5 activation by GH (0 vs. 10 min: p<0.01, p<0.001) was decreased to non-significance by 500 ng TR GHR1-279 in a dose-dependent manner. GH also induced phosphorylation (p) of ERK(1/2) (0 vs. 10 min: p<0.05) but, in this case, TR GHR1-279 had no effect. In contrast, GH decreased the high background levels of pSRC (0 vs. 10 min: p<0.01) and pAkt (10 vs. 30 min: p<0.05) in the wild-type HEK293 cells; the presence of TR GHR1-279 abolished the effect of GH on pSRC and pAkt levels were moderately altered.
To understand how the TR GHR1-279 was able to interfere with multiple signaling pathways, we crosslinked HEK293 cells following transfection with 0-500ng of the TR GHR1-279 expression vector and 250 ng/ml GH for 15 min and analyzed the components of complexes with and without DTT using gradient SDS gels. Without DTT, we identified high molecular weight complexes (180kD to >500kD) that immunoreacted with antibodies for FL GHR, TR GHR1-279, SRC and JAK2. With DTT, the complexes dissociated into the individual components.
These results suggest that the FL and TR GHRs as well as the initial signaling components are localized together in the plasma membrane. Surprisingly, neither JAK2 nor SRC activation could provide a rationale for unchanging GH effects on ERK(1/2) stimulation in the presence of increasing amounts of TR GHR1-279. Ongoing research is focused on alternative mechanisms of GHR pathway activation.
Nothing to Disclose: AON, CP, CGG