Shedding of growth hormone-binding protein is inhibited by hydroxamic acid-based protease inhibitors: proposed mechanism of activation of growth hormone-binding protein secretase

Abstract

The present study describes events postulated to be involved in the regulated mechanism of proteolytic shedding of growth hormone (GH)-binding protein (GHBP). Using Chinese hamster ovary (CHO) cell lines stably transfected either with the full-length human GH receptor (hGHR) or with the cytoplasmic domain-truncated hGHR (hGHR tr ), we show that the phorbol ester, phorbol 12-myristate 13-acetate (PMA), caused a rapid timeand dose-dependent increase in GHBP secretion, which, as expected, was matched by a corresponding decrease in cell-surface GHR. Furthermore, PMA equally enhanced GHBP release from CHO/hGHR tr cells, suggesting that the cytoplasmic domain of hGHR is not essential for PMA-induced shedding. PMA is known to specifically activate protein kinase C and, indeed, the stimulatory effects of PMA in both cell lines were completely inhibited by the protein kinase inhibitor, staurosporine (100 nM), suggesting that activation of protein kinase C (PKC) may mediate PMA-induced GHBP shedding. Since proteolytic cleavage of several cell-surface proteins was shown to be stimulated by modulators of PKC activity and inhibited by metalloprotease inhibitors, we studied the effects of two hydroxamic acid-based inhibitors of zinc-dependent metalloproteases, BB-3103 and Ro31-9790, on GHBP proteolysis. Pretreatment of CHO/hGHR cells with both these inhibitors reduced PMA-enhanced shedding of GHBP, in a dose-dependent manner, with IC 50 values of 0·41 µM for BB-3103 and 0·97 µM for Ro31-9790. In addition, these inhibitors dose-dependently reduced the shedding enhanced by the sulfhydryl alkylator, N-ethylmaleimide (NEM), with IC 50 values of 0·32 µM and 0·58 µM for BB-3103 and Ro31-9790 respectively. It was of interest to find out that Ro31-9790 acted not only to modulate PMA-or NEM-induced shedding processes, but also markedly reduced the spontaneous, time-dependent accumulation of GHBP released from CHO/hGHR cells growing in serum-containing medium. Taken together, these results suggest that one or more zinc-dependent metalloprotease(s), acting at the cell surface, may be involved in GHBP secretase activity. A scheme is proposed whereby at least part of the regulated maturation and/or activation of the protease activity may involve a cysteine-switch mechanism and/or PKCdependent phosphorylation. In the long run, specific inhibitors of these processes could be applied in the regulation of GHBP levels and, thus, of GH availability and/or activity.

Vascular Origin of a Soluble Truncated Form of the Hepatocyte Growth Factor Receptor (c-met)

“…BB-3103 has previously been used to block receptor shedding. 33 This finding is consistent with previous studies demonstrating that the sheddases or secretases that cleave membrane proteins are often metalloproteinases. 23 Currently, it is unknown whether cycloheximide blocks the synthesis of the c-met secretase or some other protein that is essential for the shedding process.…”

Metalloproteinases and the modulation of GH signaling

“…Moreover, the PMA-induced GHBP shedding could be inhibited by hydroxamate Zn chelators, such as TAPI or Immunex Compound 3 (IC3), thereby implicating metalloproteinases of the metzincin type in the shedding process (McGeehan et al 1994, Mohler et al 1994, Arribas et al 1996. Similar findings were subsequently reported by others (Amit et al 2001).…”

Platelet Receptor Proteolysis

“…20,24 MMP sheddases may also contain a regulatory Cys switch. 34 Treating human platelets with NEM has been shown to induce shedding of the Ϸ55-kDa ectodomain fragment of GPVI, 1 in the absence of GPVI ligands or platelet activation.…”