Tumor Necrosis Factor-α Converting Enzyme (TACE) Is a Growth Hormone Binding Protein (GHBP) Sheddase: The Metalloprotease TACE/ADAM-17 Is Critical for (PMA-Induced) GH Receptor Proteolysis and GHBP Generation*

Abstract

The GH binding protein (GHBP), which exists in many vertebrates, is a circulating high affinity binding protein corresponding to the extracellular domain of the GH receptor (GHR). In humans, rabbits, and several other species, the GHBP is generated by proteolysis of the GHR and shedding of its extracellular domain. We previously showed that GHBP shedding is inducible by the phorbol ester phorbol 12-myristate,13-acetate (PMA) and inhibited by the metalloprotease inhibitor, Immunex Corp. Compound 3 (IC3). The metzincin metalloprotease, tumor necrosis factor-alpha (TNF-alpha)-converting enzyme (TACE), catalyzes the shedding of TNF-alpha from its transmembrane precursor, a process that is also inhibitable by IC3. TACE may hence be a candidate for GHBP sheddase. In this study, we reconstitute fibroblasts derived from a TACE knockout mouse (Null cells) with either the rabbit (rb) GHR alone (Null/R) or rbGHR plus murine TACE (Null/R+T). Although GHR in both cells was expressed at similar abundance, dimerized normally and caused JAK2 activation in response to GH independent of TACE expression, PMA was unable to generate GHBP from Null/R cells. In contrast, PMA caused ample GHBP generation from TACE reconstituted (Null/R + T) cells, and this GHBP shedding was substantially inhibited by IC3 pretreatment. Corresponding to the induced shedding of GHBP from Null/R + T cells, PMA treatment caused a significant loss of immunoblottable GHR in Null/R+T, but not in Null/R cells. We conclude that TACE is an enzyme required for PMA-induced GHBP shedding and that PMA-induced down-regulation of GHR abundance may in significant measure be attributable to TACE-mediated GHR proteolysis.

Metalloprotease-mediated GH Receptor Proteolysis and GHBP Shedding

“…Resolution of proteins by SDS-PAGE, Western transfer of proteins, and blocking of Hybond-ECL (Amersham Biosciences) with 2% bovine serum albumin were performed as previously described (11)(12)(13)(14). Immunoblotting with antibodies 4G10 (1:4000), anti-GHR cyt-AL47 (1:1000), anti-Myc (1:2000), anti-pJAK2 (1:500), or anti-JAK2 AL33 (1:1000), with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (1:5000) and ECL detection reagents (all from Amersham Biosciences) and stripping and reprobing of blots were accomplished according to the manufacturer's suggestions.…”

“…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.…”

“…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. 15 and 16) can catalyze the processing (14). 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).…”

“…We previously demonstrated that fibroblasts from a mouse with a homozygous disruption of the gene encoding tumor necrosis factor-␣ converting enzyme (TACE/ADAM-17) failed to shed GHBP in response to PMA treatment and that TACE expression in these cells rescued inducible shedding (14). This implicated TACE, a transmembrane member of the ADAM (a disintegrin and metalloprotease) subfamily of the adamalysin family of metalloproteases (15,16), as a GHBP sheddase.…”

Evidence for a Critical Role of the Tumor Necrosis Factor α Convertase (TACE) in Ectodomain Shedding of the p75 Neurotrophin Receptor (p75NTR)

“…The TNF␣ convertase (TACE) is one of the first proteases shown to be involved in shedding. Its substrates include TNF␣ (28,29), TGF␣ (6,30,31), HB-EGF (9, 31), L-selectin (6), MUC1 (32), interleukin-1R II (33), amyloid precursor protein (34), HER4 (35), Notch (23), neuregulin ␣2c (36), growth hormone receptor (37), and CD30 (38). In most of these instances, TACE is required for the increased shedding seen in response to stimulation by phorbol ester, although the TACE-mediated release of erbB-4 is also enhanced by pervanadate, a tyrosine phosphatase inhibitor (35).…”