Selective roles for tumor necrosis factor alpha-converting enzyme/ADAM17 in the shedding of the epidermal growth factor receptor ligand family: the juxtamembrane stalk determines cleavage efficiency.

C Leann Hinkle,William E Russell,David C Lee,Carol E Parker,David Loiselle,Susan W Sunnarborg,Mary Stevenson

doi:10.1074/jbc.m312141200

Abstract

Epidermal growth factor (EGF) family ligands are derived by proteolytic cleavage of the ectodomains of integral membrane precursors. Previously, we established that tumor necrosis factor alpha-converting enzyme (TACE/ADAM17) is a physiologic transforming growth factor-alpha (TGF-alpha) sheddase, and we also demonstrated enhanced shedding of amphiregulin (AR) and heparin-binding (HB)-EGF upon restoration of TACE activity in TACE-deficient EC-2 fibroblasts. Here we extended these results by showing that purified soluble TACE cleaved single sites in the juxtamembrane stalks of mouse pro-HB-EGF and pro-AR ectodomains in vitro. For pro-HB-EGF, this site matched the C terminus of the purified human growth factor, and we speculate that the AR cleavage site is also physiologically relevant. In contrast, ADAM9 and -10, both implicated in HB-EGF shedding, failed to cleave the ectodomain or cleaved at a nonphysiologic site, respectively. Cotransfection of TACE in EC-2 cells enhanced phorbol myristate acetate-induced but not constitutive shedding of epiregulin and had no effect on betacellulin (BTC) processing. Additionally, soluble TACE did not cleave the juxtamembrane stalks of either pro-BTC or pro-epiregulin ectodomains in vitro. Substitution of the shorter pro-BTC juxtamembrane stalk or truncation of the pro-TGF-alpha stalk to match the pro-BTC length reduced TGF-alpha shedding from transfected cells to background levels, whereas substitution of the pro-BTC P2-P2' sequence reduced TGF-alpha shedding less dramatically. Conversely, substitution of the pro-TGF-alpha stalk or lengthening of the pro-BTC stalk, especially when combined with substitution of the pro-TGF-alpha P2-P2' sequence, markedly increased BTC shedding. These results indicate that efficient TACE cleavage is determined by a combination of stalk length and scissile bond sequence.

Highlights

Epidermal growth factor (EGF) family ligands are derived by proteolytic cleavage of the ectodomains of integral membrane precursors
We established that tumor necrosis factor ␣-converting enzyme (TACE/ ADAM17) is a physiologic transforming growth factor-␣ (TGF-␣) sheddase, and we demonstrated enhanced shedding of amphiregulin (AR) and heparin-binding (HB)-EGF upon restoration of TACE activity in TACEdeficient EC-2 fibroblasts
Variable cleavage at membrane distal sites (N-terminal cleavage) gives rise to larger soluble forms and could modulate the function of EGF family members. Whether both C- and N-terminal processing events are mediated by the same or different enzymes is presently unclear. Both integral membrane and soluble growth factors have been shown to activate EGF receptor (EGFR) [2,3,4,5] with potentially different consequences (6 –9), the observed phenotypes of EGF family knockouts are consistent with paracrine roles for these growth factors (10 –14), and some biological responses may be impaired by preventing release of soluble growth factor [7, 15,16,17,18]

Summary

THE JUXTAMEMBRANE STALK DETERMINES CLEAVAGE EFFICIENCY*

We established that tumor necrosis factor ␣-converting enzyme (TACE/ ADAM17) is a physiologic transforming growth factor-␣ (TGF-␣) sheddase, and we demonstrated enhanced shedding of amphiregulin (AR) and heparin-binding (HB)-EGF upon restoration of TACE activity in TACEdeficient EC-2 fibroblasts We extended these results by showing that purified soluble TACE cleaved single sites in the juxtamembrane stalks of mouse proHB-EGF and pro-AR ectodomains in vitro. TACE-deficient mice die soon after birth, displaying epithelial defects in lung, small intestine, stomach, thyroid, parathyroid, and salivary glands [27] These defects are broadly similar to those observed in EGFR null mice, prompting speculation that TACE is a Pan-EGF family sheddase [27, 34]. Through stalk domain swapping and mutagenesis experiments aimed at understanding TACE preference for pro-TGF-␣ versus pro-BTC, we found that in addition to scissile bond sequence, stalk length is a major determinant of which EGF family precursors are efficient TACE substrates

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION