Principal Investigator Harvey Lodish
Protease cleavage and release of the extracellular domain (ECD, "ectodomain shedding") of a multitude of transmembrane proteins has been linked to the activation of many signaling pathways including the MAPK pathway. Cleavage of the ECD is mostly carried out by metalloproteases (MMPs) of the ADAM family (“a disintegrin and metalloprotease”). ECD cleavage is often followed by and is a prerequisite for intramembranous cleavage of the intracellular domain (ICD) of the same protein by g-secretase; some of the cleaved ICDs translocate to the nucleus, where they may regulate gene transcription. Membrane-spanning pro-hormone ligands of the epidermal growth factor receptor (HER) family are well-studied examples of proteins that undergo ectodomain shedding and are physiologically important in many cellular contexts in organisms from Drosophila to mammals. But how the ectodomain cleavage machinery is regulated is largely unknown, as only a few specific stimuli that induce ectodomain shedding have been identified. Prolonged activation of the cardiac b-adrenergic receptor leads to HB-EGF-cleavage, release of soluble HB-EGF, and development of cardiac hypertrophy. Andreas Herrlich showed that another HER-ligand, neuregulin1b (NRG1b), is cleaved by an MMP in response to hypertonic stress and subsequently activates EGF- family receptors in an autocrine fashion. This signaling step leads to MAPK activation followed by enhanced expression of genes encoding water channels (aquaporins). Regulation of ectodomain cleavage could occur at least two levels - at the level of the MMP or via covalent modifications of the target protein, such as phosphorylation or ubiquitination on the cytosolic face.
Andreas, with the assistance of Eva Klinman, an MIT undergraduate, is cloning novel genes that regulate ectodomain shedding using a high-throughput expression cloning strategy. They can detect cleavage of all chosen HER-ligands either by hypertonic stress, phorbol ester addition, or stimulation with lysophosphatidic acid in a FACS-based assay using mouse or human cell lines stably expressing one of the chosen pro-hormone ligands. The ligands are tagged at the extracellular domain with one of several epitope tags; at their cytosol-facing C- termini the proteins have been fused with EGFP. The extracellular epitope of the transmembrane pro-hormone ligand is detected with a fluorochrome-coupled (red) anti-epitope antibody, while the intracellular domain of the EGFP- fusion is detected by green fluorescence. Stimulation of cleavage results in a decrease of the red to green fluorescence ratio, while inhibition of basal or induced cleavage is reflected by an increase in this ratio. Andreas’ initial studies with this system showed that, when expressed in mouse lung epithelial cells, ectodomain cleavage of these three EGF ligands is specifically triggered by different stimuli and involves different PKC isoenzymes.
Studies utilizing inhibitors of protein kinase C isoenzymes or metalloproteinase inhibition by batimastat showed that different regulatory signals are used by different stimuli and EGF substrates, suggesting differential effects that act on the substrate, the metalloproteinase, or both. Andreas and Eva are now using this assay system for a 96 well plate high-throughput shRNA gene knockdown screen. Currently they are in the early stages of testing the effect of shRNA constructs targeting about 5% of all known mammalian kinases and phosphatases on TPA-induced ectodomain cleavage of TGF-alpha. By stimulating cleavage to only about 50% of total possible cleavage they can detect both, decreases (inhibitors of cleavage) and increases (activators of cleavage) in red: green fluorescent ratio in the same screen. Once candidate genes are identified they will also be tested in the context of the other physiological cleavage stimuli, hypertonic stress and GPCR stimulation.