Renal epidermal growth factor precursor: proteolytic processing in an in vitro cell-free system1Presented as a communication at the 29th Annual Scientific Meeting of the European Society for Clinical Investigation, Cambridge, UK, April 2–5, 1995. Eur. J. Clin. Invest. 25 (suppl. 2), A53, Abstract no. 305, 1995.1

Abstract

The enzymatic processing of the membrane-bound renal epidermal growth factor precursor (proEGF) could be an important step in the control of nephrogenic repair consecutive to kidney insult. The enzyme machinery responsible for that processing was examined in a cell-free system consisting of renal membranes isolated from kidney homogenates by differential centrifugation, and incubated in vitro. After a 24-h incubation at 37 degrees C, 6-14% of membrane-bound proEGF was processed and soluble products with EGF immunoreactivity were released. As revealed by HPLC and Western blotting analysis, the products of proEGF proteolysis consisted of 6 kDa EGF (the molecular weight of mature EGF) and two polypeptides with molecular weights around 45 kDa. Interestingly the 45 kDa EGF forms, like the 6 kDa EGF, exhibited mitogenic activity toward growth-arrested NRK-52E renal cell line. The kinetic study of proEGF degradation gave data consistent with the 45 kDa product(s) being processing intermediate(s) between proEGF and 6 kDa EGF. The enzymatic activity responsible for proEGF nicking was inhibited by divalent heavy metal ions (Cu2+ or Zn2+) and several protease inhibitors (aprotinin, PMSF, leupeptin, soybean trypsin inhibitor), suggesting that proEGF is processed by kallikrein-like serine proteases present in the membrane preparations. Along with previous studies, the current observations suggest that renal kallikreins might play a role in renal tubular regeneration by promoting the release of soluble EGF in renal tissue.

Regulated Cell Surface Pro-EGF Ectodomain Shedding Is a Zinc Metalloprotease-dependent Process

“…The entire ectodomain of the precursor can be shed from the cell through a proteolytic cleavage that involved a zinc-dependent metalloprotease activity that appears not to be TACE but can be activated by both PKC-dependent and -independent pathways. However, these experimental results obtained with cultured cells, are in conflict with those obtained with membrane fractions of both kidney (45,46) and mammary gland (47). Indeed, these preparations contain a membrane bound proteolytic activity that colocalizes with pro-EGF.…”

“…It has already been shown that, in vitro, mature EGF could be released from its precursor through the action of different type of proteases; acidic protease such as pepsin (36) or serine-proteases such as trypsin (22,39,41) and plasmin (44). Moreover, membrane fractions of both kidney (45,46) and mammary gland (47) contain a membrane-bound proteolytic activity that colocalized with pro-EGF and released soluble EGF from its membraneassociated precursor. Based on its sensitivity to a set of inhibitors, it was shown to belong to the serine-protease family.…”

Ectodomain cleavage of FLT1 regulates receptor activation and function and is not required for its downstream intracellular cleavage

“…The ectodomain can function in an autocrine, paracrine or endocrine fashion to antagonize or stimulate cellular events. As an example the ligand EGF is released by ectodomain cleavage from the cell surface where it is expressed as a type 1 membrane protein, proEGF (24). Similarly the intracellular fragment can traffic to the nucleus or another intracellular organelle to serve a new function.…”

Proprotein Convertase PC7 Enhances the Activation of the EGF Receptor Pathway through Processing of the EGF Precursor

“…Intriguingly, the biosynthetic pathway of pro-EGF maturation into different intermediate soluble and transmembrane forms has not been studied in detail (77)(78)(79). The cytoplasmic domain of pro-EGF has been described to have its intrinsic biological activity.…”