Control of ADAM17 activity by regulation of its cellular localisation

Inken Lorenzen,Ulrike Künzel,Juliane Lokau,Christoph Garbers,Stefan Düsterhöft,Yvonne Korpys,Charlotte M Flynn,Mirja Oldefest,Matthew Freeman,Joachim Grötzinger

doi:10.1038/srep35067

Abstract

An important, irreversible step in many signalling pathways is the shedding of membrane-anchored proteins. A Disintegrin And Metalloproteinase (ADAM) 17 is one of the major sheddases involved in a variety of physiological and pathophysiological processes including regeneration, differentiation, and cancer progression. This central role in signalling implies that ADAM17 activity has to be tightly regulated, including at the level of localisation. Most mature ADAM17 is localised intracellularly, with only a small amount at the cell surface. We found that ADAM17 is constitutively internalised by clathrin-coated pits and that physiological stimulators such as GPCR ligands induce ADAM17-mediated shedding, but do not alter the cell-surface abundance of the protease. In contrast, the PKC-activating phorbol ester PMA, often used as a strong inducer of ADAM17, causes not only proteolysis by ADAM17 but also a rapid increase of the mature protease at the cell surface. This is followed by internalisation and subsequent degradation of the protease. Eventually, this leads to a substantial downregulation of mature ADAM17. Our results therefore imply that physiological activation of ADAM17 does not rely on its relocalisation, but that PMA-induced PKC activity drastically dysregulates the localisation of ADAM17.

Highlights

Phorbol-12-myristat-13-acetat (PMA), a non-physiological PKC activator, is the strongest known and often used stimulator of ADAM17-mediated shedding
We found that ADAM17 undergoes constitutive clathrin-dependent internalisation and that this turnover is not linked to ADAM17 activation by physiological stimuli
This effect was not restricted to HEK293 cells, as PMA induce the downregulation of mature ADAM17 in HeLa cells (Fig. 1D)

Summary

Results

Stimulation of ADAM17-mediated shedding is not directly linked to degradation of the protease. One hour after stimulation the amount of mature ADAM17 was unchanged in TRAP-6 treated cells, whilst in PMA-treated cells the amount was decreased (Fig. 3D) This indicates that, unlike PMA stimulation, activation of ADAM17 via PAR1 does not induce a change in the cell-surface expression of the protease. Flow cytometry analysis of HEK293 and HeLa cells stimulated with PMA and treated with ikarugamycin, an inhibitor of clathrin-dependent endocytosis, revealed that ikarugamycin inhibited PMA-induced internalisation (Fig. 4A). Surface biotinylation at different time points after stimulation with PMA showed that eventually the amount of surface ADAM17 is drastically decreased with or without inhibiting clathrin-dependent endocytosis, mature ADAM17 was detected on the cell surface for a longer time with inhibitor (Fig. 4D). HEK293 cells treated with genistein showed a higher surface expression of ADAM17 90 minutes after stimulation with PMA compared to cells incubated without inhibitor This effect was not observed in HeLa cells (Fig. 4A). These results suggest that an overexpression of iRhom[1] does not influence the PMA-induced degradation or the subsequent reappearance of mature ADAM17

Discussion

Author Contributions

Additional Information