Role of the Juxtamembrane Domains of the Transforming Growth Factor-α Precursor and the β-Amyloid Precursor Protein in Regulated Ectodomain Shedding*

Abstract

Although regulated ectodomain shedding is a well known process that affects a large group of transmembrane molecules, it is not clear how the shedding system selects its substrates. Here we investigate the structural requirements for the regulated shedding of two substrates of the general shedding system, the transforming growth factor-α precursor, pro-TGF-α, and the β-amyloid precursor protein, β-APP. The ability of different regions of pro-TGF-α or β-APP to confer susceptibility to the shedding system was tested using as a reporter a transmembrane molecule that is not a substrate of this shedding system. For this purpose we chose the TGF-β accessory receptor, betaglycan, since genetic and biochemical evidence showed that betaglycan is not a substrate of the shedding system. We determined that replacement of the 14 extracellular amino acids adjacent to the transmembrane region of betaglycan with the corresponding regions of TGF-α or β-APP rendered betaglycan susceptible to ectodomain shedding. These domain swap constructs were cleaved in response to protein kinase C stimulation, and cleavage was prevented by the metalloprotease inhibitor TAPI, both effects being characteristic of the general shedding system. Domain swap constructs containing the transmembrane and/or the cytoplasmic domains of pro-TGF-α did not undergo regulated ectodomain cleavage. We conclude that despite a lack of sequence similarity, the extracellular regions of pro-TGF-α and β-APP immediately preceding their transmembrane domains are key determinants of ectodomain shedding.