Abstract
Growth hormone receptor (GHR) is a cytokine receptor superfamily member that binds growth hormone (GH) via its extracellular domain and signals via interaction of its cytoplasmic domain with JAK2 and other signaling molecules. GHR is a target for inducible metalloprotease-mediated cleavage in its perimembranous extracellular domain, a process that liberates the extracellular domain as the soluble GH-binding protein and leaves behind a cell-associated GHR remnant protein containing the transmembrane and cytoplasmic domains. GHR metalloproteolysis can be catalyzed by tumor necrosis factor-alpha-converting enzyme (ADAM-17) and is associated with down-modulation of GH signaling. We now study the fate of the GHR remnant protein. By anti-GHR cytoplasmic domain immunoblotting, we observed that the remnant induced in response to phorbol ester or platelet-derived growth factor has a reliable pattern of appearance and disappearance in both mouse preadipocytes endogenously expressing GHR and transfected fibroblasts expressing rabbit GHR. Lactacystin, a specific proteasome inhibitor, did not appreciably change the time course of remnant appearance or clearance but allowed detection of the GHR stub, a receptor fragment slightly smaller than the remnant but containing the C terminus of the remnant (receptor cytoplasmic domain). In contrast, MG132, another (less specific) proteasome inhibitor, strongly inhibited remnant clearance and prevented stub appearance. Inhibitors of gamma-secretase, an aspartyl protease, also prevented the appearance of the stub, even in the presence of lactacystin, and concomitantly inhibited remnant clearance in the same fashion as MG132. In addition, mouse embryonic fibroblasts derived from presenilin 1 and 2 (PS1/2) knockouts recapitulated the gamma-secretase inhibitor studies, as compared with their littermate controls (PS1/2 wild type). Confocal microscopy indicated that the GHR cytoplasmic domain became localized to the nucleus in a fashion dependent on PS1/2 activity. These data indicate that the GHR is subject to sequential proteolysis by metalloprotease and gamma-secretase activities and may suggest GH-independent roles for the GHR.
Highlights
Growth hormone receptor (GHR) is a cytokine receptor superfamily member that binds growth hormone (GH) via its extracellular domain and signals via interaction of its cytoplasmic domain with JAK2 and other signaling molecules
We sought to determine whether MG132 affected the generation or fate of the GHR remnant that results from receptor proteolysis
Concomitant with the loss of GHR, PMA caused the appearance of a roughly 65-kDa anti-GHRcyt-AL47reactive protein, which we have previously described as the cytoplasmic domain-containing remnant protein [14]
Summary
Vol 280, No 19, Issue of May 13, pp. 19331–19342, 2005 Printed in U.S.A. Growth Hormone Receptor Is a Target for Presenilin-dependent ␥-Secretase Cleavage*. Treatment of a variety of cell types with the phorbol ester, PMA, or plateletderived growth factor (PDGF) results in loss of the full-length receptor and appearance of a cell-associated cytoplasmic domain-containing GHR fragment that we have termed the “remnant” protein [12,13,14,15] This cleavage yields a soluble GHR form comprised of the extracellular domain of the receptor, which is referred to as the GH-binding protein (GHBP), in correspondence with the high affinity GH-binding protein found in the circulation of many species [16]. The parallels revealed by our studies between the GHR and other ␥-secretase substrates, such as APP and Notch, suggest that a functional role(s) for metalloprotease/␥-secretase cleavage of GHR may exist
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