Growth Hormone (GH)-induced Dimerization Inhibits Phorbol Ester-stimulated GH Receptor Proteolysis

Abstract

Growth hormone (GH) initiates its cellular action by properly dimerizing GH receptor (GHR). A substantial fraction of circulating GH is complexed with a highaffinity GH-binding protein (GHBP) that in many species can be generated by GHR proteolysis and shedding of the receptor's ligand-binding extracellular domain. We previously showed that this proteolysis 1) can be acutely promoted by the phorbol ester phorbol 12-myristate 13-acetate (PMA), 2) requires a metalloprotease activity, 3) generates both shed GHBP and a membraneassociated GHR transmembrane/cytoplasmic domain remnant, and 4) results in down-regulation of GHR abundance and GH signaling. Using cell culture model systems, we now explore the effects of GH treatment on inducible GHR proteolysis and GHBP shedding. In human IM-9 lymphocytes, which endogenously express GHRs, and in Chinese hamster ovary cells heterologously expressing wild-type or cytoplasmic domain internal deletion mutant rabbit GHRs, brief exposure to GH inhibited PMA-induced GHR proteolysis (receptor loss and remnant accumulation) by 60 -93%. PMA-induced shedding of GHBP from Chinese hamster ovary transfectants was also inhibited by 70% in the presence of GH. The capacity of GH to inhibit inducible GHR cleavage did not rely on JAK2-dependent GH signaling, as evidenced by its continued protection in JAK2-deficient ␥2A rabbit GHR cells. The GH concentration dependence for inhibition of PMA-induced GHR proteolysis paralleled that for its promotion of receptor dimerization (as monitored by formation of GHR disulfide linkage). Unlike GH, the GH antagonist, G120K, which binds to but fails to properly dimerize GHRs, alone did not protect against PMA-induced GHR proteolysis; G120K did, however, antagonize the protective effect of GH. Our data suggest that GH inhibits PMAinduced GHR proteolysis and GHBP shedding by inducing GHR dimerization and that this effect does not appear to be related to GH site 1 binding, GHR internalization, or GHR signaling. The implications of these findings with regard to GH signaling and GHR down-regulation are discussed. Growth hormone (GH)1 acts on its target tissues by interacting with the transmembrane GH receptor (GHR) (1, 2). In multiple species, a substantial fraction of circulating GH is complexed with a high-affinity GH-binding protein (GHBP) that corresponds to the ligand-binding extracellular domain of the GHR (3-5). By virtue of its interaction with GH, GHBP may function as either a potentiator (by delaying GH clearance) or an inhibitor (by GH sequestration) of GH bioavailability (3, 6 -9). Whereas in some species GHBP arises as a secreted alternatively spliced form of the GHR that lacks the transmembrane and cytoplasmic domains (10 -12), GHBP in humans, rabbits, and several other species is formed posttranslationally by shedding of the proteolytically cleaved GHR extracellular domain (14). As a GHBP-generating mechanism, GHR proteolysis can also lead to down-regulation of receptor abundance and GH signaling, thus further contributing to regulation of GH bi...

Metalloprotease-mediated GH Receptor Proteolysis and GHBP Shedding

“…In cells expressing either murine or rabbit (rb) GHRs, receptor proteolysis is accompanied by a decrease in the ability of subsequent stimulation with GH to elicit JAK2 activation, suggesting that metalloprotease-mediated heterologous desensitization may be a mechanism that impacts cellular responsiveness to GH (12). Conversely, prior treatment with GH, but not with a GH antagonist, renders cells resistant to inducible GHR proteolysis (13). This suggests that GH-induced receptor dimerization or a GH-induced conformational change in the receptor's extracellular dimerization domain and/or stem region may impede access to the cleaving enzyme.…”

“…This yields a soluble receptor extracellular domain in a process termed proteolytic GHBP shedding. An obligatory byproduct of proteolytic GHBP shedding is the so-called GHR "remnant", a cytoplasmic domain-containing cell-associated fragment of the receptor that remains after the extracellular domain is shed (11)(12)(13).…”

“…We have previously shown in cell culture model systems that GHR proteolysis (receptor loss, remnant accumulation, and variable degrees of GHBP shedding) can be induced by several stimuli: 1) pharmacologic activation of protein kinase C by the phorbol ester, PMA; 2) treatment with platelet-derived growth factor (PDGF); or 3) treatment of serum-starved cells with serum (11)(12)(13)(14). In each case, the inducible proteolysis is blocked by a metalloprotease inhibitor, and genetic reconstitution studies suggest that tumor necrosis factor-␣ converting enzyme (TACE, also known as ADAM-17, Refs.…”

Dairy cows experience selective reduction of the hepatic growth hormone receptor during the periparturient period

“…To determine whether the periparturient reduction in GHR1A is sufficient to cause a reduction in GHR abundance, we measured GHR abundance with a Western immunoblotting assay based on antibodies raised against the human receptor (Zhang et al 2001). For validation of this assay, total cellular extracts were prepared from WT mouse L cells or L cells stably transfected with a vector encoding the bovine GHR cDNA, and resolved by SDS-PAGE.…”

“…The membranes were incubated for 1 h at room temperature in blocking solution (Tris-buffered saline with Tween-20 (TBST; 0·05 M Tris, pH 7·4, 0·2 M NaCl and 0·1% Tween-20) containing 5% w/v nonfat dried skimmed milk). Membranes were incubated with primary antibodies raised against the human GHR (rabbit anti-GHR cyt-AL47 , raised against a bacterially expressed, N-terminally His-tagged fusion protein incorporating GHR residues 271-620 (Zhang et al 2001)), HNF4 (rabbit anti-human HNF4, Geneka Biotechnology, Carlsbad, CA, USA) and Sp1 (rabbit anti-rat Sp1, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Primary antibodies were diluted in blocking solution (GHR and HNF-4, 1:1000; Sp1, 1:2000).…”